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Chapter III
Antioxidant profile
of Liv-Pro-8
72
31 Introduction
In all living organisms production of free radicals in highly reactive oxygen
species (ROS) is balanced by antioxidant defense systems ROS in the form of
superoxide anion hydroxyl peroxyl peroxinitrite nitric oxide radicals and
hydrogen peroxide which are generated from molecular oxygen originate from
different sources such as mitochondria xanthine oxidase system (normal metabolic
processes) or from exogenous factors and agents affects DNA proteins and most
biological molecules containing a lipid component of polyunsaturated fatty acids
(Liochev amp Fridovich 1994 Halliwell amp Gutteridge 1999) Cells have
sophisticated antioxidant regulatory systems to maintain proper balance of ROS
however disruption in homeostasis can result in oxidative stress and tissue injury
(Perry et al 2002) Free radicals have been reported to play an important role in
aetiology of more than sixty different health conditions including the metabolic
syndrome aging process cancer atherosclerosis and so on (Ames et al 1993)
Therefore it is beneficial for our health to scavenge these harmful free radicals Free
radicals are known to be generated through biological processes are minimized by
the enzymatic and non-enzymatic antioxidants present in the body tissues
Inactivation and removal of reactive oxygen species depends on reactions involving
the antioxidative defense system (Ames et al 1993)
Antioxidants play an important role in inhibiting and scavenging free
radicals thus providing protection to humans against infection and degenerative
disease (Sharma amp Gupta 2008) Since dietary antioxidants have been shown to be
protective against chronic diseases Some degradation processes of aerobic living
organisms are mediated by reactive oxygen species such as superoxide anion radical
(O2bull) hydrogen peroxide radical and hydroxyl radical Particularly O2
bull has been
considered as a causative species to induce inflammation (Trenam et al 1992)
Nutritional antioxidant deficiency also leads to oxidative stress which signifies the
identification of natural antioxidative agents present in the diet consumed by the
human population There are certain naturally occurring antioxidants that can give
protection to liver from hepatotoxins Modern research is now directed towards
73
natural antioxidants originated from plants due to safe therapeutics (Sharma amp
Gupta 2008)
Recently there has been increasing interest in finding plants with high
antioxidant capacities since they can protect the human body from free radicals and
retard the progression of many chronic diseases (Miliauskas et al 2004) Plants
produce an extensive range of chemicals including bdquobdquosecondary metabolitesrdquo which
may exert beneficial health effects when consumed by man (Williamson et al
1999) Many antioxidant-based drug formulations are used for the prevention and
treatment of complex diseases (Wong et al 2006)
Antioxidant defense systems may help the body to protect itself from various
types of oxidative damage of ROS it consist of flavonoids carotenoids phenolic
compounds vitamins and antioxidant enzymes (Ozturk-Urek et al 2001) Dietary
intake of antioxidant compounds is important for health Although there are some
synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA) which are being restricted due to their carcinogenicity
(Sasaki et al 2002) Antioxidants are considered as an important nutraceuticals on
account of many health benefits (Valko et al 2007) The requirement of a standard
assay is very important 11-Diphenyl-2-picryl- hydrazyl (DPPH) is a stable free
radical which has an unpaired valance electron at one atom of nitrogen bridge
(Eklund et al 2005) Scavenging of DPPH radical is the basis of the popular DPPH
antioxidant assay (Kordali et al 2005)
The antioxidant activity or the inhibition of the generation of free radicals is
important in providing protection against hepatic damage A number of plants have
been shown to possess hepatoprotective property by improving antioxidant status
Thus the efficacy of the drug would be preventive and passive for defending against
liver damages Indian medicinal plants belonging to about 400 families were
investigated as liver protective drugs (Handa et al 1986) Nigella Sativa Entada
Pursaetha amp Ficus Glomerata is used in Siddha system of medicine as an
expectorant and in the treatment of liver amp other degenerative diseases The
botanical resources (grains vegetables fruits and medicinal plants) provides us with
74
plentiful of all sorts of antioxidants as well as incredibly diverse health benefiting
nutrition Medicinal plants have become extremely popular all over the world as
antioxidants and markets are flooded with herbal formulations
Medicinal uses and pharmacological activities of Nigella sativa seeds have
been employed for thousands of years as a spice food preservative and curative or
medicinal remedy for numerous disorders (Chopra et al 1956 Ramadan 2007)
The seeds have been used traditionally especially in the middle East and India for
the treatment of asthma cough bronchitis headache rheumatism fever (Burits and
Bucar 2000) diuretic diaphoretic stomachic liver tonic and digestive Several
members of the genus Ficus (Ficus glomerata) are being used traditionally in a wide
variety of ethnomedical remedies in all over the world (Hansson et al 2005)
Entada pursaetha is a woody climber of the legume family The seeds have various
medicinal uses including topical applications in an ointment for the treatment of
jaundice (Tapondjou et al 2005) Therefore the purpose of the present study was to
investigate amp quantify the total phenolic and flavonoid content of Liv-Pro-08 and
inorder to evaluate its free radical scavenging activity by adopting different in vitro
assay methods
Since no detailed study has been conducted on the hepatoprotective activity
of the combination of the selected (Nigella Sativa Entada Pursaetha) and (Ficus
Glomerata) samples The main objectives of the present study is to establish the
antioxidant potential of the Liv-Pro-08 by evaluating the enzymatic and non-
enzymatic antioxidants in selected plant source
75
32 Materials amp Methods
To perform the objectives various experimental protocols were deduced for
the present study entitled ldquoAntioxidant profile of Liv-Pro-08 -In vitro assayrdquo were
conducted by the following standard procedures The seeds and fruits of Nigella
Sativa Entada Pursaetha amp Ficus Glomerata were collected from Kolli Hills in
Namakkal Tamilnadu Historically plants have been used as folk medicine against
various types of disease
321 Plant Materials and preparation of extract
The seeds of Nigella sativa Entada pursaetha and fruits of Ficus glomerata
were collected from Kolli Hills in Namakkal District Tamilnadu India The
samples were shade dried at room temperature and then ground to a fine powder in a
mechanic grinder The powdered material was then extracted using solvent
extraction (ethanol and aqueous) in the ratio 110 using Soxhlet apparatus After
extracting all colouring material the solvent was removed by evaporating on water
bath which give rise to a solid mass of the extract Then the concentrated extract was
stored at 4ordmC until use
322 Determination of the antioxidant property of Liv-Pro-08 ndash In vitro study
The ethanolic and aqueous extract of the Liv-Pro-08 was subjected to various
experimental procedures enzymatic and non-enzymatic antioxidant assays
(Superoxide dismutase (SOD) Catalase (CAT) Glutathione Peroxidae (GPX)
Glucose 6 Phosphate Dehydrogenase (G6PDH) Glutathione-S-Transferase (GST)
and non-enzymatic antioxidants such as Glutathione (GSH) Vitamin-C and
Vitamin-E) Lipid Peroxidation assay and Free radical scavenging assays (11-
diphenyl-2-picrylhydrazyl [DPPH] Superoxide scavenging activity Nitric oxide
radical scavenging activity Reducing Power Hydroxy radical scavenging activity
Chelating ability 22‟-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid [ABTS]
and Ferric reducing power assay [FRAP]) as given in Table 9
76
Table 9 In Vitro Antioxidants
S No Parameters Method Appendix
no
1 Superoxide Dismutase (SOD) Kakkar et al 1984 12
2 Catalase (CAT) Luck 1974 13
3 Glutathione Peroxidae (GPX) Ellman 1959 14
4 Glutathione-S-Transferase (GST) Habig et al 1973 15
5 Glucose 6 Phosphate
Dehydrogenase (G6PDH)
Balinksy and
Bernstein 1963
16
6 Glutathione (GSH) Moron et al 1979 17
7 Vitamin-C Roe amp Keuther 1943 18
8 Vitamin-E Rosenberg 1992 19
9 Lipid peroxidation (LPO) Okhawa et al 1979 20
10 11-diphenyl-2-picrylhydrazyl
(DPPH)
Shimada et al 1992 21
11 Superoxide scavenging activity Liu et al 1997 22
12 Nitric oxide radical scavenging
activity
Madan et al 2005 23
13 Reducing power Oyaizu 1986 24
14 Hydroxy radical scavenging activity Smirnoff amp Cumbes
1989
25
15 Chelating ability Dinis et al 2000 26
16 22‟-azinobis-(3-
ethylbenzothiazoline-6-sulphonic
acid (ABTS)
Re et al 1999 27
17 Ferric reducing ability power assay
(FRAP)
Benzie and Strain
1999
28
323 Statistical analysis
All values are expressed as Mean plusmn SD The significance of differences
between the means of the tests and controls were calculated by one way ANOVA
77
33 Results and Discussion
Free radicals bear high chemical reactivity due to their unpaired electrons
Reactive oxygen species (ROS) comprise oxygen free radicals or oxygen free-
radical-generating agents such as superoxide anion (O2-bull) hydroxy radical (OH
bull) and
hydrogen peroxide (H2O2) (Valko et al 2006) Metabolic processes are usually
associated with the generation of free radicals especially oxygen-derived radicals
with the potency of oxidizing and damaging surrounding bio molecules (Valko et
al 2007)
However oxygen-consuming organisms are well protected against the free
radical damages by their own endogenous antioxidants which are either antioxidant
enzymes such as superoxide dismutase (SOD) and catalase (CAT) or organic
chemical compounds such as ascorbic acid tocopherols and glutathione (GSH)
Antioxidants inhibit the oxidation processes by blocking the oxidizing chain-
reactions Under normal physiological conditions a balance exists between the
production and consumption rates of ROS and disruption of this balance usually
leads to a state referred to as oxidative stress It is believed that oxidative stress is
the main cause of many health-associated complications such as Alzheimer
Parkinson (Rao 2009 Sagi andYoudim 2008 Zhao 2009) cancer (Hileman et al
2004) diabetes (Pan et al 2008) and cardiovascular diseases (Kurian and
Paddikkala 2009) Regarding this fact many studies have been aimed and directed
towards using natural or synthetic antioxidants with the aim of fading off the
strength of the free radical-associated damages or even preventing their effects
Aerobic organisms are protected from oxygen toxicity by a natural
antioxidant defense system involving enzymatic and non-enzymatic mechanisms
(Cotgreave et al 1988 Ames et al 1993) However in many cases such as in
unhealthy physical condition ageing or under stress environments the exogenous
antioxidants are either exhausted or insufficient to scavenge these radicals generated
which resulted in diseases associated with oxidative stress and damage Endogenous
antioxidants constitute important defense systems in cells and elicit their action by
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
72
31 Introduction
In all living organisms production of free radicals in highly reactive oxygen
species (ROS) is balanced by antioxidant defense systems ROS in the form of
superoxide anion hydroxyl peroxyl peroxinitrite nitric oxide radicals and
hydrogen peroxide which are generated from molecular oxygen originate from
different sources such as mitochondria xanthine oxidase system (normal metabolic
processes) or from exogenous factors and agents affects DNA proteins and most
biological molecules containing a lipid component of polyunsaturated fatty acids
(Liochev amp Fridovich 1994 Halliwell amp Gutteridge 1999) Cells have
sophisticated antioxidant regulatory systems to maintain proper balance of ROS
however disruption in homeostasis can result in oxidative stress and tissue injury
(Perry et al 2002) Free radicals have been reported to play an important role in
aetiology of more than sixty different health conditions including the metabolic
syndrome aging process cancer atherosclerosis and so on (Ames et al 1993)
Therefore it is beneficial for our health to scavenge these harmful free radicals Free
radicals are known to be generated through biological processes are minimized by
the enzymatic and non-enzymatic antioxidants present in the body tissues
Inactivation and removal of reactive oxygen species depends on reactions involving
the antioxidative defense system (Ames et al 1993)
Antioxidants play an important role in inhibiting and scavenging free
radicals thus providing protection to humans against infection and degenerative
disease (Sharma amp Gupta 2008) Since dietary antioxidants have been shown to be
protective against chronic diseases Some degradation processes of aerobic living
organisms are mediated by reactive oxygen species such as superoxide anion radical
(O2bull) hydrogen peroxide radical and hydroxyl radical Particularly O2
bull has been
considered as a causative species to induce inflammation (Trenam et al 1992)
Nutritional antioxidant deficiency also leads to oxidative stress which signifies the
identification of natural antioxidative agents present in the diet consumed by the
human population There are certain naturally occurring antioxidants that can give
protection to liver from hepatotoxins Modern research is now directed towards
73
natural antioxidants originated from plants due to safe therapeutics (Sharma amp
Gupta 2008)
Recently there has been increasing interest in finding plants with high
antioxidant capacities since they can protect the human body from free radicals and
retard the progression of many chronic diseases (Miliauskas et al 2004) Plants
produce an extensive range of chemicals including bdquobdquosecondary metabolitesrdquo which
may exert beneficial health effects when consumed by man (Williamson et al
1999) Many antioxidant-based drug formulations are used for the prevention and
treatment of complex diseases (Wong et al 2006)
Antioxidant defense systems may help the body to protect itself from various
types of oxidative damage of ROS it consist of flavonoids carotenoids phenolic
compounds vitamins and antioxidant enzymes (Ozturk-Urek et al 2001) Dietary
intake of antioxidant compounds is important for health Although there are some
synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA) which are being restricted due to their carcinogenicity
(Sasaki et al 2002) Antioxidants are considered as an important nutraceuticals on
account of many health benefits (Valko et al 2007) The requirement of a standard
assay is very important 11-Diphenyl-2-picryl- hydrazyl (DPPH) is a stable free
radical which has an unpaired valance electron at one atom of nitrogen bridge
(Eklund et al 2005) Scavenging of DPPH radical is the basis of the popular DPPH
antioxidant assay (Kordali et al 2005)
The antioxidant activity or the inhibition of the generation of free radicals is
important in providing protection against hepatic damage A number of plants have
been shown to possess hepatoprotective property by improving antioxidant status
Thus the efficacy of the drug would be preventive and passive for defending against
liver damages Indian medicinal plants belonging to about 400 families were
investigated as liver protective drugs (Handa et al 1986) Nigella Sativa Entada
Pursaetha amp Ficus Glomerata is used in Siddha system of medicine as an
expectorant and in the treatment of liver amp other degenerative diseases The
botanical resources (grains vegetables fruits and medicinal plants) provides us with
74
plentiful of all sorts of antioxidants as well as incredibly diverse health benefiting
nutrition Medicinal plants have become extremely popular all over the world as
antioxidants and markets are flooded with herbal formulations
Medicinal uses and pharmacological activities of Nigella sativa seeds have
been employed for thousands of years as a spice food preservative and curative or
medicinal remedy for numerous disorders (Chopra et al 1956 Ramadan 2007)
The seeds have been used traditionally especially in the middle East and India for
the treatment of asthma cough bronchitis headache rheumatism fever (Burits and
Bucar 2000) diuretic diaphoretic stomachic liver tonic and digestive Several
members of the genus Ficus (Ficus glomerata) are being used traditionally in a wide
variety of ethnomedical remedies in all over the world (Hansson et al 2005)
Entada pursaetha is a woody climber of the legume family The seeds have various
medicinal uses including topical applications in an ointment for the treatment of
jaundice (Tapondjou et al 2005) Therefore the purpose of the present study was to
investigate amp quantify the total phenolic and flavonoid content of Liv-Pro-08 and
inorder to evaluate its free radical scavenging activity by adopting different in vitro
assay methods
Since no detailed study has been conducted on the hepatoprotective activity
of the combination of the selected (Nigella Sativa Entada Pursaetha) and (Ficus
Glomerata) samples The main objectives of the present study is to establish the
antioxidant potential of the Liv-Pro-08 by evaluating the enzymatic and non-
enzymatic antioxidants in selected plant source
75
32 Materials amp Methods
To perform the objectives various experimental protocols were deduced for
the present study entitled ldquoAntioxidant profile of Liv-Pro-08 -In vitro assayrdquo were
conducted by the following standard procedures The seeds and fruits of Nigella
Sativa Entada Pursaetha amp Ficus Glomerata were collected from Kolli Hills in
Namakkal Tamilnadu Historically plants have been used as folk medicine against
various types of disease
321 Plant Materials and preparation of extract
The seeds of Nigella sativa Entada pursaetha and fruits of Ficus glomerata
were collected from Kolli Hills in Namakkal District Tamilnadu India The
samples were shade dried at room temperature and then ground to a fine powder in a
mechanic grinder The powdered material was then extracted using solvent
extraction (ethanol and aqueous) in the ratio 110 using Soxhlet apparatus After
extracting all colouring material the solvent was removed by evaporating on water
bath which give rise to a solid mass of the extract Then the concentrated extract was
stored at 4ordmC until use
322 Determination of the antioxidant property of Liv-Pro-08 ndash In vitro study
The ethanolic and aqueous extract of the Liv-Pro-08 was subjected to various
experimental procedures enzymatic and non-enzymatic antioxidant assays
(Superoxide dismutase (SOD) Catalase (CAT) Glutathione Peroxidae (GPX)
Glucose 6 Phosphate Dehydrogenase (G6PDH) Glutathione-S-Transferase (GST)
and non-enzymatic antioxidants such as Glutathione (GSH) Vitamin-C and
Vitamin-E) Lipid Peroxidation assay and Free radical scavenging assays (11-
diphenyl-2-picrylhydrazyl [DPPH] Superoxide scavenging activity Nitric oxide
radical scavenging activity Reducing Power Hydroxy radical scavenging activity
Chelating ability 22‟-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid [ABTS]
and Ferric reducing power assay [FRAP]) as given in Table 9
76
Table 9 In Vitro Antioxidants
S No Parameters Method Appendix
no
1 Superoxide Dismutase (SOD) Kakkar et al 1984 12
2 Catalase (CAT) Luck 1974 13
3 Glutathione Peroxidae (GPX) Ellman 1959 14
4 Glutathione-S-Transferase (GST) Habig et al 1973 15
5 Glucose 6 Phosphate
Dehydrogenase (G6PDH)
Balinksy and
Bernstein 1963
16
6 Glutathione (GSH) Moron et al 1979 17
7 Vitamin-C Roe amp Keuther 1943 18
8 Vitamin-E Rosenberg 1992 19
9 Lipid peroxidation (LPO) Okhawa et al 1979 20
10 11-diphenyl-2-picrylhydrazyl
(DPPH)
Shimada et al 1992 21
11 Superoxide scavenging activity Liu et al 1997 22
12 Nitric oxide radical scavenging
activity
Madan et al 2005 23
13 Reducing power Oyaizu 1986 24
14 Hydroxy radical scavenging activity Smirnoff amp Cumbes
1989
25
15 Chelating ability Dinis et al 2000 26
16 22‟-azinobis-(3-
ethylbenzothiazoline-6-sulphonic
acid (ABTS)
Re et al 1999 27
17 Ferric reducing ability power assay
(FRAP)
Benzie and Strain
1999
28
323 Statistical analysis
All values are expressed as Mean plusmn SD The significance of differences
between the means of the tests and controls were calculated by one way ANOVA
77
33 Results and Discussion
Free radicals bear high chemical reactivity due to their unpaired electrons
Reactive oxygen species (ROS) comprise oxygen free radicals or oxygen free-
radical-generating agents such as superoxide anion (O2-bull) hydroxy radical (OH
bull) and
hydrogen peroxide (H2O2) (Valko et al 2006) Metabolic processes are usually
associated with the generation of free radicals especially oxygen-derived radicals
with the potency of oxidizing and damaging surrounding bio molecules (Valko et
al 2007)
However oxygen-consuming organisms are well protected against the free
radical damages by their own endogenous antioxidants which are either antioxidant
enzymes such as superoxide dismutase (SOD) and catalase (CAT) or organic
chemical compounds such as ascorbic acid tocopherols and glutathione (GSH)
Antioxidants inhibit the oxidation processes by blocking the oxidizing chain-
reactions Under normal physiological conditions a balance exists between the
production and consumption rates of ROS and disruption of this balance usually
leads to a state referred to as oxidative stress It is believed that oxidative stress is
the main cause of many health-associated complications such as Alzheimer
Parkinson (Rao 2009 Sagi andYoudim 2008 Zhao 2009) cancer (Hileman et al
2004) diabetes (Pan et al 2008) and cardiovascular diseases (Kurian and
Paddikkala 2009) Regarding this fact many studies have been aimed and directed
towards using natural or synthetic antioxidants with the aim of fading off the
strength of the free radical-associated damages or even preventing their effects
Aerobic organisms are protected from oxygen toxicity by a natural
antioxidant defense system involving enzymatic and non-enzymatic mechanisms
(Cotgreave et al 1988 Ames et al 1993) However in many cases such as in
unhealthy physical condition ageing or under stress environments the exogenous
antioxidants are either exhausted or insufficient to scavenge these radicals generated
which resulted in diseases associated with oxidative stress and damage Endogenous
antioxidants constitute important defense systems in cells and elicit their action by
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
73
natural antioxidants originated from plants due to safe therapeutics (Sharma amp
Gupta 2008)
Recently there has been increasing interest in finding plants with high
antioxidant capacities since they can protect the human body from free radicals and
retard the progression of many chronic diseases (Miliauskas et al 2004) Plants
produce an extensive range of chemicals including bdquobdquosecondary metabolitesrdquo which
may exert beneficial health effects when consumed by man (Williamson et al
1999) Many antioxidant-based drug formulations are used for the prevention and
treatment of complex diseases (Wong et al 2006)
Antioxidant defense systems may help the body to protect itself from various
types of oxidative damage of ROS it consist of flavonoids carotenoids phenolic
compounds vitamins and antioxidant enzymes (Ozturk-Urek et al 2001) Dietary
intake of antioxidant compounds is important for health Although there are some
synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated
hydroxyanisole (BHA) which are being restricted due to their carcinogenicity
(Sasaki et al 2002) Antioxidants are considered as an important nutraceuticals on
account of many health benefits (Valko et al 2007) The requirement of a standard
assay is very important 11-Diphenyl-2-picryl- hydrazyl (DPPH) is a stable free
radical which has an unpaired valance electron at one atom of nitrogen bridge
(Eklund et al 2005) Scavenging of DPPH radical is the basis of the popular DPPH
antioxidant assay (Kordali et al 2005)
The antioxidant activity or the inhibition of the generation of free radicals is
important in providing protection against hepatic damage A number of plants have
been shown to possess hepatoprotective property by improving antioxidant status
Thus the efficacy of the drug would be preventive and passive for defending against
liver damages Indian medicinal plants belonging to about 400 families were
investigated as liver protective drugs (Handa et al 1986) Nigella Sativa Entada
Pursaetha amp Ficus Glomerata is used in Siddha system of medicine as an
expectorant and in the treatment of liver amp other degenerative diseases The
botanical resources (grains vegetables fruits and medicinal plants) provides us with
74
plentiful of all sorts of antioxidants as well as incredibly diverse health benefiting
nutrition Medicinal plants have become extremely popular all over the world as
antioxidants and markets are flooded with herbal formulations
Medicinal uses and pharmacological activities of Nigella sativa seeds have
been employed for thousands of years as a spice food preservative and curative or
medicinal remedy for numerous disorders (Chopra et al 1956 Ramadan 2007)
The seeds have been used traditionally especially in the middle East and India for
the treatment of asthma cough bronchitis headache rheumatism fever (Burits and
Bucar 2000) diuretic diaphoretic stomachic liver tonic and digestive Several
members of the genus Ficus (Ficus glomerata) are being used traditionally in a wide
variety of ethnomedical remedies in all over the world (Hansson et al 2005)
Entada pursaetha is a woody climber of the legume family The seeds have various
medicinal uses including topical applications in an ointment for the treatment of
jaundice (Tapondjou et al 2005) Therefore the purpose of the present study was to
investigate amp quantify the total phenolic and flavonoid content of Liv-Pro-08 and
inorder to evaluate its free radical scavenging activity by adopting different in vitro
assay methods
Since no detailed study has been conducted on the hepatoprotective activity
of the combination of the selected (Nigella Sativa Entada Pursaetha) and (Ficus
Glomerata) samples The main objectives of the present study is to establish the
antioxidant potential of the Liv-Pro-08 by evaluating the enzymatic and non-
enzymatic antioxidants in selected plant source
75
32 Materials amp Methods
To perform the objectives various experimental protocols were deduced for
the present study entitled ldquoAntioxidant profile of Liv-Pro-08 -In vitro assayrdquo were
conducted by the following standard procedures The seeds and fruits of Nigella
Sativa Entada Pursaetha amp Ficus Glomerata were collected from Kolli Hills in
Namakkal Tamilnadu Historically plants have been used as folk medicine against
various types of disease
321 Plant Materials and preparation of extract
The seeds of Nigella sativa Entada pursaetha and fruits of Ficus glomerata
were collected from Kolli Hills in Namakkal District Tamilnadu India The
samples were shade dried at room temperature and then ground to a fine powder in a
mechanic grinder The powdered material was then extracted using solvent
extraction (ethanol and aqueous) in the ratio 110 using Soxhlet apparatus After
extracting all colouring material the solvent was removed by evaporating on water
bath which give rise to a solid mass of the extract Then the concentrated extract was
stored at 4ordmC until use
322 Determination of the antioxidant property of Liv-Pro-08 ndash In vitro study
The ethanolic and aqueous extract of the Liv-Pro-08 was subjected to various
experimental procedures enzymatic and non-enzymatic antioxidant assays
(Superoxide dismutase (SOD) Catalase (CAT) Glutathione Peroxidae (GPX)
Glucose 6 Phosphate Dehydrogenase (G6PDH) Glutathione-S-Transferase (GST)
and non-enzymatic antioxidants such as Glutathione (GSH) Vitamin-C and
Vitamin-E) Lipid Peroxidation assay and Free radical scavenging assays (11-
diphenyl-2-picrylhydrazyl [DPPH] Superoxide scavenging activity Nitric oxide
radical scavenging activity Reducing Power Hydroxy radical scavenging activity
Chelating ability 22‟-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid [ABTS]
and Ferric reducing power assay [FRAP]) as given in Table 9
76
Table 9 In Vitro Antioxidants
S No Parameters Method Appendix
no
1 Superoxide Dismutase (SOD) Kakkar et al 1984 12
2 Catalase (CAT) Luck 1974 13
3 Glutathione Peroxidae (GPX) Ellman 1959 14
4 Glutathione-S-Transferase (GST) Habig et al 1973 15
5 Glucose 6 Phosphate
Dehydrogenase (G6PDH)
Balinksy and
Bernstein 1963
16
6 Glutathione (GSH) Moron et al 1979 17
7 Vitamin-C Roe amp Keuther 1943 18
8 Vitamin-E Rosenberg 1992 19
9 Lipid peroxidation (LPO) Okhawa et al 1979 20
10 11-diphenyl-2-picrylhydrazyl
(DPPH)
Shimada et al 1992 21
11 Superoxide scavenging activity Liu et al 1997 22
12 Nitric oxide radical scavenging
activity
Madan et al 2005 23
13 Reducing power Oyaizu 1986 24
14 Hydroxy radical scavenging activity Smirnoff amp Cumbes
1989
25
15 Chelating ability Dinis et al 2000 26
16 22‟-azinobis-(3-
ethylbenzothiazoline-6-sulphonic
acid (ABTS)
Re et al 1999 27
17 Ferric reducing ability power assay
(FRAP)
Benzie and Strain
1999
28
323 Statistical analysis
All values are expressed as Mean plusmn SD The significance of differences
between the means of the tests and controls were calculated by one way ANOVA
77
33 Results and Discussion
Free radicals bear high chemical reactivity due to their unpaired electrons
Reactive oxygen species (ROS) comprise oxygen free radicals or oxygen free-
radical-generating agents such as superoxide anion (O2-bull) hydroxy radical (OH
bull) and
hydrogen peroxide (H2O2) (Valko et al 2006) Metabolic processes are usually
associated with the generation of free radicals especially oxygen-derived radicals
with the potency of oxidizing and damaging surrounding bio molecules (Valko et
al 2007)
However oxygen-consuming organisms are well protected against the free
radical damages by their own endogenous antioxidants which are either antioxidant
enzymes such as superoxide dismutase (SOD) and catalase (CAT) or organic
chemical compounds such as ascorbic acid tocopherols and glutathione (GSH)
Antioxidants inhibit the oxidation processes by blocking the oxidizing chain-
reactions Under normal physiological conditions a balance exists between the
production and consumption rates of ROS and disruption of this balance usually
leads to a state referred to as oxidative stress It is believed that oxidative stress is
the main cause of many health-associated complications such as Alzheimer
Parkinson (Rao 2009 Sagi andYoudim 2008 Zhao 2009) cancer (Hileman et al
2004) diabetes (Pan et al 2008) and cardiovascular diseases (Kurian and
Paddikkala 2009) Regarding this fact many studies have been aimed and directed
towards using natural or synthetic antioxidants with the aim of fading off the
strength of the free radical-associated damages or even preventing their effects
Aerobic organisms are protected from oxygen toxicity by a natural
antioxidant defense system involving enzymatic and non-enzymatic mechanisms
(Cotgreave et al 1988 Ames et al 1993) However in many cases such as in
unhealthy physical condition ageing or under stress environments the exogenous
antioxidants are either exhausted or insufficient to scavenge these radicals generated
which resulted in diseases associated with oxidative stress and damage Endogenous
antioxidants constitute important defense systems in cells and elicit their action by
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
74
plentiful of all sorts of antioxidants as well as incredibly diverse health benefiting
nutrition Medicinal plants have become extremely popular all over the world as
antioxidants and markets are flooded with herbal formulations
Medicinal uses and pharmacological activities of Nigella sativa seeds have
been employed for thousands of years as a spice food preservative and curative or
medicinal remedy for numerous disorders (Chopra et al 1956 Ramadan 2007)
The seeds have been used traditionally especially in the middle East and India for
the treatment of asthma cough bronchitis headache rheumatism fever (Burits and
Bucar 2000) diuretic diaphoretic stomachic liver tonic and digestive Several
members of the genus Ficus (Ficus glomerata) are being used traditionally in a wide
variety of ethnomedical remedies in all over the world (Hansson et al 2005)
Entada pursaetha is a woody climber of the legume family The seeds have various
medicinal uses including topical applications in an ointment for the treatment of
jaundice (Tapondjou et al 2005) Therefore the purpose of the present study was to
investigate amp quantify the total phenolic and flavonoid content of Liv-Pro-08 and
inorder to evaluate its free radical scavenging activity by adopting different in vitro
assay methods
Since no detailed study has been conducted on the hepatoprotective activity
of the combination of the selected (Nigella Sativa Entada Pursaetha) and (Ficus
Glomerata) samples The main objectives of the present study is to establish the
antioxidant potential of the Liv-Pro-08 by evaluating the enzymatic and non-
enzymatic antioxidants in selected plant source
75
32 Materials amp Methods
To perform the objectives various experimental protocols were deduced for
the present study entitled ldquoAntioxidant profile of Liv-Pro-08 -In vitro assayrdquo were
conducted by the following standard procedures The seeds and fruits of Nigella
Sativa Entada Pursaetha amp Ficus Glomerata were collected from Kolli Hills in
Namakkal Tamilnadu Historically plants have been used as folk medicine against
various types of disease
321 Plant Materials and preparation of extract
The seeds of Nigella sativa Entada pursaetha and fruits of Ficus glomerata
were collected from Kolli Hills in Namakkal District Tamilnadu India The
samples were shade dried at room temperature and then ground to a fine powder in a
mechanic grinder The powdered material was then extracted using solvent
extraction (ethanol and aqueous) in the ratio 110 using Soxhlet apparatus After
extracting all colouring material the solvent was removed by evaporating on water
bath which give rise to a solid mass of the extract Then the concentrated extract was
stored at 4ordmC until use
322 Determination of the antioxidant property of Liv-Pro-08 ndash In vitro study
The ethanolic and aqueous extract of the Liv-Pro-08 was subjected to various
experimental procedures enzymatic and non-enzymatic antioxidant assays
(Superoxide dismutase (SOD) Catalase (CAT) Glutathione Peroxidae (GPX)
Glucose 6 Phosphate Dehydrogenase (G6PDH) Glutathione-S-Transferase (GST)
and non-enzymatic antioxidants such as Glutathione (GSH) Vitamin-C and
Vitamin-E) Lipid Peroxidation assay and Free radical scavenging assays (11-
diphenyl-2-picrylhydrazyl [DPPH] Superoxide scavenging activity Nitric oxide
radical scavenging activity Reducing Power Hydroxy radical scavenging activity
Chelating ability 22‟-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid [ABTS]
and Ferric reducing power assay [FRAP]) as given in Table 9
76
Table 9 In Vitro Antioxidants
S No Parameters Method Appendix
no
1 Superoxide Dismutase (SOD) Kakkar et al 1984 12
2 Catalase (CAT) Luck 1974 13
3 Glutathione Peroxidae (GPX) Ellman 1959 14
4 Glutathione-S-Transferase (GST) Habig et al 1973 15
5 Glucose 6 Phosphate
Dehydrogenase (G6PDH)
Balinksy and
Bernstein 1963
16
6 Glutathione (GSH) Moron et al 1979 17
7 Vitamin-C Roe amp Keuther 1943 18
8 Vitamin-E Rosenberg 1992 19
9 Lipid peroxidation (LPO) Okhawa et al 1979 20
10 11-diphenyl-2-picrylhydrazyl
(DPPH)
Shimada et al 1992 21
11 Superoxide scavenging activity Liu et al 1997 22
12 Nitric oxide radical scavenging
activity
Madan et al 2005 23
13 Reducing power Oyaizu 1986 24
14 Hydroxy radical scavenging activity Smirnoff amp Cumbes
1989
25
15 Chelating ability Dinis et al 2000 26
16 22‟-azinobis-(3-
ethylbenzothiazoline-6-sulphonic
acid (ABTS)
Re et al 1999 27
17 Ferric reducing ability power assay
(FRAP)
Benzie and Strain
1999
28
323 Statistical analysis
All values are expressed as Mean plusmn SD The significance of differences
between the means of the tests and controls were calculated by one way ANOVA
77
33 Results and Discussion
Free radicals bear high chemical reactivity due to their unpaired electrons
Reactive oxygen species (ROS) comprise oxygen free radicals or oxygen free-
radical-generating agents such as superoxide anion (O2-bull) hydroxy radical (OH
bull) and
hydrogen peroxide (H2O2) (Valko et al 2006) Metabolic processes are usually
associated with the generation of free radicals especially oxygen-derived radicals
with the potency of oxidizing and damaging surrounding bio molecules (Valko et
al 2007)
However oxygen-consuming organisms are well protected against the free
radical damages by their own endogenous antioxidants which are either antioxidant
enzymes such as superoxide dismutase (SOD) and catalase (CAT) or organic
chemical compounds such as ascorbic acid tocopherols and glutathione (GSH)
Antioxidants inhibit the oxidation processes by blocking the oxidizing chain-
reactions Under normal physiological conditions a balance exists between the
production and consumption rates of ROS and disruption of this balance usually
leads to a state referred to as oxidative stress It is believed that oxidative stress is
the main cause of many health-associated complications such as Alzheimer
Parkinson (Rao 2009 Sagi andYoudim 2008 Zhao 2009) cancer (Hileman et al
2004) diabetes (Pan et al 2008) and cardiovascular diseases (Kurian and
Paddikkala 2009) Regarding this fact many studies have been aimed and directed
towards using natural or synthetic antioxidants with the aim of fading off the
strength of the free radical-associated damages or even preventing their effects
Aerobic organisms are protected from oxygen toxicity by a natural
antioxidant defense system involving enzymatic and non-enzymatic mechanisms
(Cotgreave et al 1988 Ames et al 1993) However in many cases such as in
unhealthy physical condition ageing or under stress environments the exogenous
antioxidants are either exhausted or insufficient to scavenge these radicals generated
which resulted in diseases associated with oxidative stress and damage Endogenous
antioxidants constitute important defense systems in cells and elicit their action by
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
75
32 Materials amp Methods
To perform the objectives various experimental protocols were deduced for
the present study entitled ldquoAntioxidant profile of Liv-Pro-08 -In vitro assayrdquo were
conducted by the following standard procedures The seeds and fruits of Nigella
Sativa Entada Pursaetha amp Ficus Glomerata were collected from Kolli Hills in
Namakkal Tamilnadu Historically plants have been used as folk medicine against
various types of disease
321 Plant Materials and preparation of extract
The seeds of Nigella sativa Entada pursaetha and fruits of Ficus glomerata
were collected from Kolli Hills in Namakkal District Tamilnadu India The
samples were shade dried at room temperature and then ground to a fine powder in a
mechanic grinder The powdered material was then extracted using solvent
extraction (ethanol and aqueous) in the ratio 110 using Soxhlet apparatus After
extracting all colouring material the solvent was removed by evaporating on water
bath which give rise to a solid mass of the extract Then the concentrated extract was
stored at 4ordmC until use
322 Determination of the antioxidant property of Liv-Pro-08 ndash In vitro study
The ethanolic and aqueous extract of the Liv-Pro-08 was subjected to various
experimental procedures enzymatic and non-enzymatic antioxidant assays
(Superoxide dismutase (SOD) Catalase (CAT) Glutathione Peroxidae (GPX)
Glucose 6 Phosphate Dehydrogenase (G6PDH) Glutathione-S-Transferase (GST)
and non-enzymatic antioxidants such as Glutathione (GSH) Vitamin-C and
Vitamin-E) Lipid Peroxidation assay and Free radical scavenging assays (11-
diphenyl-2-picrylhydrazyl [DPPH] Superoxide scavenging activity Nitric oxide
radical scavenging activity Reducing Power Hydroxy radical scavenging activity
Chelating ability 22‟-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid [ABTS]
and Ferric reducing power assay [FRAP]) as given in Table 9
76
Table 9 In Vitro Antioxidants
S No Parameters Method Appendix
no
1 Superoxide Dismutase (SOD) Kakkar et al 1984 12
2 Catalase (CAT) Luck 1974 13
3 Glutathione Peroxidae (GPX) Ellman 1959 14
4 Glutathione-S-Transferase (GST) Habig et al 1973 15
5 Glucose 6 Phosphate
Dehydrogenase (G6PDH)
Balinksy and
Bernstein 1963
16
6 Glutathione (GSH) Moron et al 1979 17
7 Vitamin-C Roe amp Keuther 1943 18
8 Vitamin-E Rosenberg 1992 19
9 Lipid peroxidation (LPO) Okhawa et al 1979 20
10 11-diphenyl-2-picrylhydrazyl
(DPPH)
Shimada et al 1992 21
11 Superoxide scavenging activity Liu et al 1997 22
12 Nitric oxide radical scavenging
activity
Madan et al 2005 23
13 Reducing power Oyaizu 1986 24
14 Hydroxy radical scavenging activity Smirnoff amp Cumbes
1989
25
15 Chelating ability Dinis et al 2000 26
16 22‟-azinobis-(3-
ethylbenzothiazoline-6-sulphonic
acid (ABTS)
Re et al 1999 27
17 Ferric reducing ability power assay
(FRAP)
Benzie and Strain
1999
28
323 Statistical analysis
All values are expressed as Mean plusmn SD The significance of differences
between the means of the tests and controls were calculated by one way ANOVA
77
33 Results and Discussion
Free radicals bear high chemical reactivity due to their unpaired electrons
Reactive oxygen species (ROS) comprise oxygen free radicals or oxygen free-
radical-generating agents such as superoxide anion (O2-bull) hydroxy radical (OH
bull) and
hydrogen peroxide (H2O2) (Valko et al 2006) Metabolic processes are usually
associated with the generation of free radicals especially oxygen-derived radicals
with the potency of oxidizing and damaging surrounding bio molecules (Valko et
al 2007)
However oxygen-consuming organisms are well protected against the free
radical damages by their own endogenous antioxidants which are either antioxidant
enzymes such as superoxide dismutase (SOD) and catalase (CAT) or organic
chemical compounds such as ascorbic acid tocopherols and glutathione (GSH)
Antioxidants inhibit the oxidation processes by blocking the oxidizing chain-
reactions Under normal physiological conditions a balance exists between the
production and consumption rates of ROS and disruption of this balance usually
leads to a state referred to as oxidative stress It is believed that oxidative stress is
the main cause of many health-associated complications such as Alzheimer
Parkinson (Rao 2009 Sagi andYoudim 2008 Zhao 2009) cancer (Hileman et al
2004) diabetes (Pan et al 2008) and cardiovascular diseases (Kurian and
Paddikkala 2009) Regarding this fact many studies have been aimed and directed
towards using natural or synthetic antioxidants with the aim of fading off the
strength of the free radical-associated damages or even preventing their effects
Aerobic organisms are protected from oxygen toxicity by a natural
antioxidant defense system involving enzymatic and non-enzymatic mechanisms
(Cotgreave et al 1988 Ames et al 1993) However in many cases such as in
unhealthy physical condition ageing or under stress environments the exogenous
antioxidants are either exhausted or insufficient to scavenge these radicals generated
which resulted in diseases associated with oxidative stress and damage Endogenous
antioxidants constitute important defense systems in cells and elicit their action by
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
76
Table 9 In Vitro Antioxidants
S No Parameters Method Appendix
no
1 Superoxide Dismutase (SOD) Kakkar et al 1984 12
2 Catalase (CAT) Luck 1974 13
3 Glutathione Peroxidae (GPX) Ellman 1959 14
4 Glutathione-S-Transferase (GST) Habig et al 1973 15
5 Glucose 6 Phosphate
Dehydrogenase (G6PDH)
Balinksy and
Bernstein 1963
16
6 Glutathione (GSH) Moron et al 1979 17
7 Vitamin-C Roe amp Keuther 1943 18
8 Vitamin-E Rosenberg 1992 19
9 Lipid peroxidation (LPO) Okhawa et al 1979 20
10 11-diphenyl-2-picrylhydrazyl
(DPPH)
Shimada et al 1992 21
11 Superoxide scavenging activity Liu et al 1997 22
12 Nitric oxide radical scavenging
activity
Madan et al 2005 23
13 Reducing power Oyaizu 1986 24
14 Hydroxy radical scavenging activity Smirnoff amp Cumbes
1989
25
15 Chelating ability Dinis et al 2000 26
16 22‟-azinobis-(3-
ethylbenzothiazoline-6-sulphonic
acid (ABTS)
Re et al 1999 27
17 Ferric reducing ability power assay
(FRAP)
Benzie and Strain
1999
28
323 Statistical analysis
All values are expressed as Mean plusmn SD The significance of differences
between the means of the tests and controls were calculated by one way ANOVA
77
33 Results and Discussion
Free radicals bear high chemical reactivity due to their unpaired electrons
Reactive oxygen species (ROS) comprise oxygen free radicals or oxygen free-
radical-generating agents such as superoxide anion (O2-bull) hydroxy radical (OH
bull) and
hydrogen peroxide (H2O2) (Valko et al 2006) Metabolic processes are usually
associated with the generation of free radicals especially oxygen-derived radicals
with the potency of oxidizing and damaging surrounding bio molecules (Valko et
al 2007)
However oxygen-consuming organisms are well protected against the free
radical damages by their own endogenous antioxidants which are either antioxidant
enzymes such as superoxide dismutase (SOD) and catalase (CAT) or organic
chemical compounds such as ascorbic acid tocopherols and glutathione (GSH)
Antioxidants inhibit the oxidation processes by blocking the oxidizing chain-
reactions Under normal physiological conditions a balance exists between the
production and consumption rates of ROS and disruption of this balance usually
leads to a state referred to as oxidative stress It is believed that oxidative stress is
the main cause of many health-associated complications such as Alzheimer
Parkinson (Rao 2009 Sagi andYoudim 2008 Zhao 2009) cancer (Hileman et al
2004) diabetes (Pan et al 2008) and cardiovascular diseases (Kurian and
Paddikkala 2009) Regarding this fact many studies have been aimed and directed
towards using natural or synthetic antioxidants with the aim of fading off the
strength of the free radical-associated damages or even preventing their effects
Aerobic organisms are protected from oxygen toxicity by a natural
antioxidant defense system involving enzymatic and non-enzymatic mechanisms
(Cotgreave et al 1988 Ames et al 1993) However in many cases such as in
unhealthy physical condition ageing or under stress environments the exogenous
antioxidants are either exhausted or insufficient to scavenge these radicals generated
which resulted in diseases associated with oxidative stress and damage Endogenous
antioxidants constitute important defense systems in cells and elicit their action by
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
77
33 Results and Discussion
Free radicals bear high chemical reactivity due to their unpaired electrons
Reactive oxygen species (ROS) comprise oxygen free radicals or oxygen free-
radical-generating agents such as superoxide anion (O2-bull) hydroxy radical (OH
bull) and
hydrogen peroxide (H2O2) (Valko et al 2006) Metabolic processes are usually
associated with the generation of free radicals especially oxygen-derived radicals
with the potency of oxidizing and damaging surrounding bio molecules (Valko et
al 2007)
However oxygen-consuming organisms are well protected against the free
radical damages by their own endogenous antioxidants which are either antioxidant
enzymes such as superoxide dismutase (SOD) and catalase (CAT) or organic
chemical compounds such as ascorbic acid tocopherols and glutathione (GSH)
Antioxidants inhibit the oxidation processes by blocking the oxidizing chain-
reactions Under normal physiological conditions a balance exists between the
production and consumption rates of ROS and disruption of this balance usually
leads to a state referred to as oxidative stress It is believed that oxidative stress is
the main cause of many health-associated complications such as Alzheimer
Parkinson (Rao 2009 Sagi andYoudim 2008 Zhao 2009) cancer (Hileman et al
2004) diabetes (Pan et al 2008) and cardiovascular diseases (Kurian and
Paddikkala 2009) Regarding this fact many studies have been aimed and directed
towards using natural or synthetic antioxidants with the aim of fading off the
strength of the free radical-associated damages or even preventing their effects
Aerobic organisms are protected from oxygen toxicity by a natural
antioxidant defense system involving enzymatic and non-enzymatic mechanisms
(Cotgreave et al 1988 Ames et al 1993) However in many cases such as in
unhealthy physical condition ageing or under stress environments the exogenous
antioxidants are either exhausted or insufficient to scavenge these radicals generated
which resulted in diseases associated with oxidative stress and damage Endogenous
antioxidants constitute important defense systems in cells and elicit their action by
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
78
suppressing the formation of ROS their scavenging or by repairing the damage
caused (Kamat et al 2000 Devasagayam et al 2001)
Enzymatic antioxidants defense include SOD GPx CAT etc and non-
enzymatic antioxidants are ascorbic acid (vitamin C) α-tocopherol (vitamin E)
glutathione (GSH) carotenoids flavonoids etc All these act by one or more of the
mechanisms like reducing activity free radical-scavenging potential complexing of
pro-oxidant metals and quenching of singlet oxygen (Stanner et al 2004) Since
antioxidants are capable of preventing oxidative damage the wide use of natural
antioxidants as a replacement of conventional synthetic antioxidants in food and
food supplements has been employed owing to the fact that natural products are
considered to be promising and safe source Plants have evolved complex
antioxidant system to avoid the harmful effects of ROS (Arnon and Sairam 2002
Sharma and Dubey 2005) Antioxidants like SOD GPx and GR have a protective
role in plant cells (Arnon and Sairam 2002 Asada 1992) Natural antioxidant
derived from plant products such as herbs was reported to prevent oxidative stress
Under normal conditions the total amount of ROS formed in the plant was
determined by the balance between the multiple ROS producing pathways and the
ability of the enzymatic and non-enzymatic mechanisms to deal with them
(Yannerelli et al 2006)
Screening of plant material on the basis of their antioxidant potency seems to
be central importance in order to identify extracts of fractions possessing the ability
either in scavenging both free radical and chain reactions initiation or in binding
which catalyse the oxidative reactions such as some metal ions (Nsimba et al
2008)
331 In vitro antioxidant status
The energetic benefit of aerobic metabolism is associated with the generation
of ROS which are implicated in variety of diseased conditions (Broadhurst et al
2000) Diet contains several substances that are capable of scavenging ROS directly
or indirectly by promoting mechanism which enhance detoxification (Osawa 1994)
Strong evidence suggests that consumption of fruits and vegetables results in
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
79
decreased incidence of all types of disease (Huang et al 2005) They are known to
contain variety of non-enzymatic antioxidants namely carotenoids tocopherols
ascorbic acid and plant polyphenols which exert their antimutagenic activity even
after subjected to the cooking process In spite of its rich antioxidant store The
combination of these plants source (Nigella Sativa Entada Pursaetha ampFicus
Glomerata) are rarely included in the diet hence the antioxidant properties of Liv-
Pro-08 are explored
3311 Enzymatic antioxidants
The in vitro antioxidant status of Liv-Pro-08 oral ayurvedic formulation is
presented in the table 10 and figure 16 The activities of SOD CAT GPx GST and
G6PD were examined The plants are susceptible to damage caused by the active
oxygen and thus develop numerous antioxidant defence system resulting in
formation of numerous potent antioxidants
Superoxide dismutase (SOD)
The level of antioxidant enzymes assessed in ethanolic and aqueous extract
of Liv-Pro-08 collectively presented in table 10 The highest activity of SOD was
noted in the ethanolic extract (84 plusmn 058) compared to the aqueous extract (30 plusmn
016) SOD is a family of metallo enzymes catalyse the decomposition of O2bull to O2
and H2O2 It prevents the formation of OHbull and hence been implicated as an
essential defense against the potential toxicity of oxygen The ROS scavenging
activity of SOD is effective only when it is followed by the actions of CAT and
GPx because the dismutase activity of SOD generates H2O2 which needs to be
further scavenged by CAT and GPx (Lee et al 2003) This enzyme is present in all
aerobic organisms and in all subcellular compartments susceptible to oxidative stress
(Bowler et al 1992) The result of SOD clearly shows that the plant possess
significant amount of SOD which could exert a beneficial action against
pathological alteration caused by the presence of O2 and bullOH hence it can be able to
counteract the toxicity of oxygen
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
80
Catalase (CAT)
The significant amount of catalase in ethanolic and aqueous extract of Liv-
Pro-08 was inferred as 170 plusmn 014 amp 80 plusmn 014 unitsmg protein respectively
Highest activity of Catalase observed in ethanolic extract of Liv-Pro-08 coincides
very well with the highest activity of SOD noted in the same extract indicating that
the H2O2 formed by SOD is effectively removed by the Catalase CAT is a
tetrahedrical protein constituted by four heme groups which catalyse the
dismutation of hydrogen peroxide in water and oxygen (Scandalios 1987) CAT is
the most efficient antioxidant enzymes which protects plants by scavenging free
radicals and H2O2 (Vichnvetskaia and Roy 2001) This protein is localized to
peroxisomes in most eukaryotic cells It protects the cellular constituents from the
oxidative damage by highly reactive hydroxyl radicals (Klichko et al 2004) Thus
the plant promotes the scavenging activity by scavenging the free radicals
Significant proportion was found in this Liv-Pro-08 especially ethanolic extract
holds effective antioxidant system
Table 10 Enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Superoxide dismutase 30 plusmn 016 84 plusmn 015
Catalase 80 plusmn 014 170 plusmn 014
Glutathione-S-transferase 03 plusmn 015 46 plusmn 015
Glutathione peroxidase 08 plusmn 001 18 plusmn 017
Glucose 6 Phosphate
Dehydrogenase 06 plusmn 014 07 plusmn 006
Values are expressed as a mean plusmn SD of three observations
Glutathione-S-transferase (GST)
Table 10 demonstrates the in vitro level of GST in Liv-Pro-08 GST offers
protection against LPO by promoting the conjugation of toxic electrophoresis with
GSH (Jakoby 1998) GST consists a family of multifunctional enzymes that
metabolise wide variety of electrophilic compounds via glutathione conjunction
Hence this reveals that ethanolic extract of Liv-Pro-08 promotes protection against
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
81
LPO Glutathione peroxidise activity is high in ethanolic extract (460 plusmn 015) than
the aqueous extract (03 plusmn 015) Both catalase amp glutathione peroxidase activities
are reasonably indicating their scavenging role in peroxide removal
Glutathione peroxidase (GPx)
The value of GPx 08 plusmn 001 amp 18 plusmn 017 unitsmg protein in aqueous and
ethanolic extract of Liv-Pro-08 was depicted in the table 10 GPx is a selenium-
containing enzyme which scavenges other peroxides as well as hydrogen peroxide
(Blake et al 1987) It catalyses the decomposition of both H2O2 and organic
peroxides (ROOH) at the expense of reduced glutathione with the formation of
glutathione disulphide (GSSG) water and organic alcohol (Vural et al 2004) The
inference of GPx shows that the Liv-Pro-08 emphasis significant scavenging
potential
Glucose 6 Phosphate Dehydrogenase (G6PDH)
G6PDH is a cytosolic NADP dependent enzyme This generates NADPH
which is necessary for the regeneration of reduced glutathione from oxidized GSH
Maintenance of GSH in the reduced state is an important function of G6PDH The
activity of G6PDH level in aqueous extract and ethanolic extract of Liv-Pro-08 is
06 plusmn 014 amp 07 plusmn 006 respectively Both ethanolic and aqueous extracts possess
equal level of G6PDH
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
82
3312 Non-enzymatic antioxidants
Non-enzymatic antioxidants (Glutathione Vitamin C ampVitamin E) are
important in curtailing the ROS in cellular system The levels of these antioxidants
are assessed and the results are tabulated in Table 11 and figure 17 The ethanolic
extract of Liv-Pro-08 possesses a very high store of glutathione (2190 plusmn 125) The
Vitamin C level of the ethanolic extract (392 plusmn 2164) has found to be markedly
higher than the aqueous extract (144 plusmn 635) The remarkable elevation in the
Vitamin E level was noted in ethanolic extract of Liv-Pro-08 The antioxidant
property amp the rich tocopherol content in ethanolic extract of Liv-Pro-08 have the
ability to quench both singlet oxygen amp peroxides (Fryer 1992) Besides various
available antioxidants but within the membrane tocopherol is the only protective
agent that can act against the toxic effect of oxygen radicals (Suntress amp Sheck
1995)
Glutathione (GSH)
GSH is known to be widely distributed in plant cells and is the major free
thiol in many higher plants (McCay 1985 Rennberg 1987) It is a vital substance in
detoxification and cell physiology GSH reduces the formation of toxic peroxides on
biological system by acting as substrate for GPx (Banumathi 1992) It scavenges
cytotoxic H2O2 and reacts non-enzymatically with other ROS singlet oxygen
superoxide radical and hydroxyl radical (Larson 1988) The central role of GSH in
the antioxidative defense is due to its ability to regenerate another powerful water
soluble antioxidant ascorbic acid via the ascorbate-glutathione cycle (Foyer and
Halliwell 1976 Noctor and Foyer 1998)
The value of GSH was identified as 261 plusmn 070 amp 2190 plusmn 180 unitsmg
protein in aqueous and ethanolic extract of Liv-Pro-08 Since ethanolic extract of
Liv-Pro-08 produces high levels of GSH activity it counteracts the formation of
toxic peroxides on biological system
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
83
Table 11 Non-enzymatic Antioxidant Profiles of Liv-Pro-08
Parameters
Aqueous Extract
(unitsmg protein)
Ethanol Extract
(unitsmg protein)
Glutathione
261 plusmn 070 2190 plusmn 180
Vitamin C 413 plusmn 041 380 plusmn 083
Vitamin E 1410 plusmn 040 1800 plusmn 060
Values are expressed as a mean plusmn SD of three observations
Vitamin C
Table 11 reveals the amount of vitamin C in ethanolic and aqueous extract of
Liv-Pro-08 and it was deduced as 413 plusmn 041amp 380 plusmn 083 microgmg protein Vitamin
C also known as ascorbic acid is considered as the most important water soluble
antioxidant in both extracellular and intracellular fluids as it is capable of
neutralizing ROS in the aqueous phase before lipid peroxidation is initiated (Jacob
1995) Vitamin C is an effective scavenger of free radicals which include O2bull HO2
bull
RO2bull RS
bull and other sulphur and nitrogen radicals It has been detected in the
majority of plant cell types organelles and in the apoplast Vitamin C along with
GSH and vitamin E play a key role in protecting cells against oxidative damage due
to its ability to interact with a variety of oxygen species (Rojas et a l 1996)
Vitamin C is an excellent source of electrons therefore it can donate electrons to
free radicals such as hydroxyl and superoxide radicals In addition to its direct
antioxidants effects ascorbic acid is also a substrate for the antioxidant enzyme
ascorbate peroxidises a function that is particularly important in stress resistance in
plants (Shigeoka et al 2002) It is present at high levels in all parts of plants and can
reach a concentration of 20 millimolar in chloroplasts (Smirnoff 2001) Ethanolic
extract exhibited high levels of ascorbic acid and hence protects cells against
oxidative damage by interacting with a variety of oxygen
Vitamin E
Level of vitamin E was depicted in table 11 Vitamin E resides in the lipid
bilayer of the cell membrane It can transfer its phenolic hydrogen to a peroxyl free
radical of a peroxidised PUFA in cellular and sub cellular membrane phospholipids
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
84
During these reactions vitamin E is consumed and converted to the radical form
(vitamin Ebull) but vitamin E
bull can abstract hydrogen atoms from membrane lipids
because its unpaired electron is energetically stable Vitamin Ebull is reduced back to
vitamin E by ascorbic acid (Shimizu et al 2004) It reacts with alkoxy radicals
(LObull) lipid peroxyl radicals (LOO
bull) and with alkyl radicals (L
bull) derived from
PUFA oxidation (Kamal-Eldin and Appelqvist 1996 Buettner 1993)
Verma et al (2010) reported that FGlomerata fruits are rich in antioxidant
(carotenoids amp vitamin C) and nutritional content (sugars and proteins) Since
vitamin C and E are synergistic antioxidants Liv-Pro-08 protect the cells against
free radical mediated oxidative damage The level of antioxidative response depends
on the species the development and the metabolic state of the plant as well as the
duration and intensity of the stress (Reddy et al 2004) Aqueous extract of Liv-Pro-
08 showed higher scavenging activity it may be due to the presence of hydroxyl
groups existing in the phenolic and flavonoid compounds chemical configuration
that can provide the essential constituents as a radical scavenger Free radical
mediated processes have been implicated in the pathogenesis of most of the diseases
(Gyamfi et al 1999) The present study demonstrated that the Liv-Pro-08 extract
has exerted a significant extent of antioxidant and free radical scavenging activities
The results may be concluded that the ethanolic extract possesses promising sources
of non-toxic natural antioxidants
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
85
332 Effect of Liv-Pro-08 extracts on Lipid peroxidation of liver mitochondria
The effect of aqueous and ethanolic extracts of Liv-Pro-08 on in vitro lipid
peroxidation of liver mitochondria was assessed by estimating TBARS The results
are represented in table 12
Lipid peroxidation (LPO) was induced in the model system by incubating the
liver mitochondria in the presence of FeSO4 for 60 min with or without Liv-Pro-08
extracts LPO has been implicated in the pathogenesis of hepatic injury by
compounds like FeSO4 and CCl4 and is responsible for cell membrane alterations In
the present study significantly elevated level of LPO was observed in FeSO4
induced in group II indicated excessive formation of free radicals amp activation of
LPO system resulting in hepatic damage The significant decline in the LPO level in
the liver mitochondria of FeSO4 + Liv-Pro-08 treated group III amp IV indicated the
antilipid peroxidative effect of Liv-Pro-08 From this two extracts used the
ethanolic extract is exhibiting LPO than aqueous extract Collectively states that the
ethanolic extract provides to be effective in extending the antioxidant activity in Liv-
Pro-08
Table 12 Effects of Liv-Pro-08 extracts on Lipid Peroxidation
Experimental group TBARS (μMmg protein)
of inhibition
Liver mitochondria (group I) 025 plusmn 0005 a
Liver mitochondria + FeSO4 (group II) 046 plusmn 0015 d
Liver mitochondria + FeSO4 + ethanol
extract of Liv-Pro-08 (group III)
031 plusmn 0005 (62) b
Liver mitochondria + FeSO4 + aqueous
extract of Liv-Pro-08 (group IV)
038 plusmn 001 (56) c
(Values are means of 3 replicates of inhibition of lipid peroxidation) Means
followed by a common letter are not significantly different at the 5 level by
DMRT
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
86
In states of oxidative stress reduced glutathione (GSH) is converted to
oxidized glutathione (GSSG) and depletion in it leads to LPO Therefore the role of
GSH as a reasonable marker for evaluation of oxidative stress is important as it acts
as an antioxidant both extra and intracellularly and is produced in the liver
(Recknagel et al 1982)
Increased level of LPO is due to increase in generation of reactive oxygen
species during stress leading to oxidative damage SOD converts the reactive
superoxide radical to H2O2 which if not scavenge by CAT can by itself cause LPO
by generation of hydroxyl radicals Hence decrease in CAT levels has led to increase
in accumulation of these reactive oxygen species amp thus has caused increased LPO
amp tissue damage (Sairam et al 2002) Liv-Pro-08 significantly reversed these
oxidative changes induced by FeSO4 Liv-Pro-08 also inhibited the oxidation of
reduced glutathione GSH is a non-enzymatic mode of defense against the free
radicals
Peroxyl radicals are important agents that mediate LPO thereby damaging
cell membranes (Boik 1996) In order to determine if the extracts were capable of
reducing in vitro oxidative stress LPO was assessed by means of an assay that
determines the production of malondialdehyde TBARS are produced as byproducts
of LPO that occurs in the hydrophobic core of bio membranes (Fraga et al 1987a)
The results obtained are shown in table 12 the extracts tested are effective in
reducing the production of TBARS in a single dose Ethanolic extract of Liv-Pro-08
was found to be more effective in curtailing by 62 while the extent of inhibition
was around 55 for aqueous extract The inhibition of in vitro LPO by the Liv-Pro-
08 extracts observed in the present study can be attributed due to the presence of
non-enzymatic antioxidants like Vitamin C E glutathione and also the various
enzymatic antioxidants like SOD CAT in Liv-Pro-08 extracts
333 Free radical scavenging activity
Polyphenols particularly flavonoids which are widely distributed in the
plant kingdom and are present in considerable amounts in fruits vegetables spices
medicinal herbs and beverages (Broadhurst et al 2000) The biological activities of
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
87
these polyphenols in different systems are believed to be due their redox properties
which can play an important role in absorbing and neutralizing free radicals
quenching singlet and triplet oxygen or decomposing peroxides (Osawa 1994)
Recent studies have begun to demonstrate that a number of vegetable juices display
high total antioxidant capacity when quantified using biochemical assays
(Giovanelli amp Pagliarini 2009 Lichtenthaler amp Marx 2005)
Free radicals are involved in the normal physiology of living organisms
Under certain conditions the excess of free radicals and reactive oxygen species
have been proposed to induce cellular damage and to be involved in several human
diseases such as cancer arteriosclerosis inflammatory disorders as well as in
ageing process In recent past several dietary and herbal formulation which
have free radical scavenging potential have gained important in treating such
chronic diseases (Wang et al 2005 Tiwari amp Tripathi 2007) In this study different
free radicals were used to assay the free radical scavenging activity of the extract
Diphenyl-picrylhydrazyl radical (DPPH)
scavenging activity
The Diphenyl-picrylhydrazyl radical (DPPHbull) assay is based on the normal
hydrogen atom transfer (HAT) reaction that occurs between antioxidants and the
peroxyl radical Instead of peroxyl radicals more stable and less transient nitrogen
radicals are created with which some antioxidants react more slowly than they
would with the peroxyl radical in a biological system Additionally the reaction then
proceeds in a manner which bears more similarity to a SET reaction (Foti et al
2004)
DPPH radical bleaching is one of the strategies used to evaluate the
antioxidant properties of herbal extracts this method has shown to be rapid and
simple and it measures the capacity of herbal extract to bleach the DPPH radical a
nitrogen-centred free radical (Antolovich et al 2002) The structural changes that
this radical provokes on herbal principles as well as the involved mechanism
however are not clear yet (Wang and Zhang 2003) In foods antioxidants have
been defined as substances that in small quantity are able to prevent or greatly retard
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
88
the oxidation of easily oxidable materials such as fats (Chipault 1962) In the
present experiment the mode of antioxidant capacity of the ethanolic and aqueous
extract of Liv-Pro-08 was probed
Values are expressed as a mean plusmn SD of three observations
Figure 18 shows the ethanolic extract exhibited good DPPH scavenging
potential with IC50 values of 50 microgml which were significantly lower than the IC50
of ascorbic acid (IC50 of 70 microgml) and aqueous extract indicating that this ethanolic
extract has a good potential as free radical scavengers Among the two extracts
ethanol showed the highest DPPH scavenging activity as evident from the lower
IC50 value than the rest of the extract
The herbal extract considered as a mixture of antioxidant principles
prevented the oxidative changes induced by oxygen free radicals however it did not
prevent and neither reversed the reduction of microsomal thiol content provoked by
DPPH radical By exchange herbal extract prevents the oxygen free radicals
modifications on biomolecules through the similar mechanisms used by the
organism ie polyphenols could act as hydrogen donators to oxygen free radicals
so stabilizing it Therefore antioxidants are considered important nutraceuticals on
account of their many health benefits and they are widely used in the food industry
as potential inhibitors of LPO (Scherer amp Godoy 2009) However it has been
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
89
demonstrated that synthetic antioxidants can accumulate in the body which can
result in liver damage and carcinogenesis These problems are not seen when natural
antioxidants extracted from herbs and spices with high antioxidant activity are used
in food applications (Deng et al 2011) These extracts are safe potentially
nutritional and have therapeutic effects
The disappearance of DPPH is directly proportional to the amount of
antioxidant present in the reaction mixture (antioxidant react with stable free radical
ie aa-diphenyl-b-picrylhydrazyl and convert it to aa-diphenyl-b-picrylhydrazine)
The transfers of hydrogen or electron from antioxidant to DPPH occur at different
redox potentials and also depend on the structure of the antioxidant (Yu et al 2002
Romero et al 2004) High phenolic compounds may cause the antioxidative
activities of this plant These antioxidants donate hydrogen to free radicals leading
to non-toxic species and therefore to inhibition of the propagation of lipid oxidation
(Lugasi et al 1998) The activities of both extracts increased with increasing
concentration based on total phenol and flavonoid contents
Verma et al (2010) noted that highest free radical scavenging activity of the
extract and fractions of Ficus glomerata Recent study shows the DPPH values for
investigated different fractions of black cumin seedcake showed potent free radical
scavenging activity on DPPH (Mariod et al 2009) Recently Nzowa reported that
two triterpenoid saponins have been isolated from the seed kernals of Entada rheedii
and an effective antioxidant activity was found for these compounds which
exhibited good scavenging activity
Superoxide scavenging capacity
Superoxide the one-electron reduced form of molecular oxygen is a
precursor of other ROS such as hydrogen peroxide hydroxyl radical and singlet
oxygen that have the potential of reacting with biological macromolecules and
thereby inducing tissue damages (Aruoma 1998) and also it has been implicated in
initiating oxidation reactions associated with aging (Wickens 2001) Figure 19
shows the superoxide radical scavenging capacity of the extracts of Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
90
measured by the PMS-NADH superoxide generating system The extracts
demonstrated a dose dependent inhibition of the superoxide anion radicals Both
extracts have quite similar superoxide scavenging capacity (1913 plusmn 010 to 5857 plusmn0
17 amp 1814 plusmn 009 to 5383 plusmn 004) at 20 to 100 microgml of the concentration
Superoxide scavenging capacity of ethanolic and aqueous extracts of Liv-Pro-08
showing high reactive than ascorbic acid (1516 plusmn 007 to 5215 plusmn 009) The present
results also supported by Bourgou et al (2008) observed that methanolic extract of
Nigella sativa shoots and roots exhibited significant superoxide anion scavenging
capacity Nzowa et al (2010) measured superoxide anion radical scavenging activity
of Entada rheedii the results are effective antioxidant activity was found which
exhibited superoxide anion radical scavenging activity
Superoxide radical is produced in vivo by activated phagocytes by electron
leakage from the mitochondrial electron transport chain (Halliwell 1991) and in the
conversion of xanthine to uric acid (Bast et al 1991) The reactivity of this radical
is limited but it is considered to be toxic In fact much of the molecular damage that
can be done by superoxide radical is due to its conversion into much more reactive
species namely hydroxyl radical and peroxynitrite (Halliwell et al 1995) which
initiate peroxidation of lipids Compared to other oxygen radicals O2˙- anions have
longer half-lives with potential damaging effects on biomolecules mainly lipids
Values are expressed as a mean plusmn SD of three observations
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
91
In cellular oxidation reactions superoxide radical is normally formed first
and its effects can be magnified because it produces other types of cell-damaging
free radicals and oxidizing agents which has been implicated in the initiating
oxidation reactions associated with aging (Liu amp Ng 2000) It has been reported that
antioxidant properties of some flavonoids are effective mainly via scavenging of
superoxide anion radicals (Robak amp Gryglewski 1988) Verma et al (2010)
observed ethyl acetate fraction of Ficus glomrata significantly inhibited the activity
of superoxide radical and shows highest free radical scavenging activity The present
results clearly indicated that ethanolic extract of Liv-Pro-08 is a potent scavenger of
superoxide radicals in a dose-dependent manner
Results showed that ethanolic extract significantly inhibited the activity of
O2˙- and showed highest free radical scavenging activity Further antioxidant
activity of ethanolic extract in the above assays may be mediated through direct
trapping of the free radicals through transfers of hydrogen or electron The
compounds such as flavonoids and phenols which contain hydroxyl functional
groups are responsible for antioxidant effect in the plants (Younes 1981) These
results indicated that the tested extract had a notable effect on scavenging of
superoxide when compared with ascorbic acid which was used as positive control
Under the assay conditions all the samples were able to scavenge O2˙- in a
concentration dependent way (Fig 19) among this ethanolic extract of Liv-Pro-08
showed the strongest scavenging activity
Nitric oxide radical scavenging activity
The ethanolic amp aqueous extract of Liv-Pro-08 showed the high nitric oxide
scavenging ability compared to the standard At 20-100 microgml the nitric
oxide radical scavenging activity of ethanol amp aqueous extracts of Liv-Pro-08
were 2815 to 8439 and 2790 to7217 respectively A significant difference in
nitric oxide radical scavenging activity was observed with different concentration
and two different extracts showed in figure 20 The extract also showed a moderate
nitric oxide-scavenging activity between 20 to 100 microgml in a dose dependent
manner
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
92
In addition to reactive oxygen species nitric oxide is also implicated in
inflammation cancer and other pathological conditions (Moncada et al 1991) The
plantplant products may have the property to counteract the effect of NO formation
and in turn may be of considerable interest in preventing the ill effects of excessive
NO generation in the human body Further the scavenging activity may also help to
arrest the chain of reactions initiated by excess generation of NO that are detrimental
to human health (Sreejayan amp Rao 1997) The extract showed a moderate nitric
oxide-scavenging activity The inhibition was increased with increasing
concentration of the extract
Values are expressed as a mean plusmn SD of three observations
Despite the possible beneficial effects of NO its contribution to oxidative
damage is increasingly becoming evident This is due to the fact that NO can
react with superoxide to form the peroxynitrite anion which is a potential
strong oxidant that can decompose to produce OH and NO2 (Pacher et al 2007)
NO released from SNP has a strong NO+ character which can alter the structure
and function of many cellular components Our study showed that Liv-Pro-08
in SNP solution decreased levels of nitrite a stable oxidation product of NO
liberated from SNP (Fig 20) The Liv-Pro-08 exhibited strong NO radical
scavenging activity leading to the reduction of the nitrite concentration in the
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
93
assay medium a possible protective effect against oxidative damage Nigella
sativa and Ficus glomerata has also been shown to possess antioxidant effects
inhibiting O2˙- and NO amp DPPH (Verma et al 2010 Juntachote and Berghofer
2005) These results indicate that the plants could be an important source of natural
antioxidants
Although this scavenging effect was lower than that of BHT it was stronger
than the antiradical activity reported in the seeds of the same species from India
(Thippeswamy and Naidu 2005) which showed an IC50 value of 1240 μg mlminus1
These results suggest that N sativa shoots were electron donors reacting with free
radicals to convert them into more stable products and to terminate radical chain
reactions as described by (Shimada et al 1992)
Reducing power
The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity The two different extracts from
Liv-Pro-08 exhibited a dose dependent reducing power activity at various
concentrations The reducing power of ethanolic and aqueous extract from
Liv-Pro-08 was 058 to 270 and 025 to 084 at 20 to 100 microgml respectively
Figure 21 shows the reductive capabilities of the Liv-Pro-08 extracts compared to
ascorbic acid
In the reducing power assay the presents of reductant in the samples would
result in the reducing of Fe3+
to Fe2+
by donating an electron Increasing absorbance
at 700nm indicates an increase in reductive ability It was found that the reducing
powers of both extracts also increased with the increase of their concentrations (fig
21) The reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity (Moncada et al 1991) The antioxidant activity has
been attributed to various mechanisms which are the prevention of chain initiation
the binding of transition metal ion catalysts decomposition of peroxides the
prevention of continued hydrogen abstraction the reductive capacity and radical
scavenging (Hebbel et al 1990)
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
94
Humans are unable to eliminate the iron released from the breakdown of
transfused red blood cells and the excess iron is deposited as hemosiderin and
ferritin in the liver spleen endocrine organs and myocardium The accumulation of
toxic quantities of iron causes tissue damage and leads to complications such as
heart failure endocrine abnormalities like diabetes hypothyroidism liver failure and
ultimately early death (Taher et al 2006) The reducing power property indicates
that these secondary compounds are electron donors and can reduce the oxidised
intermediates of lipid peroxidation processes so they can act as primary and
secondary antioxidants (Yen and Chen 1995)
Values are expressed as a mean plusmn SD of three observations
The antioxidant mechanisms of polyphenols like flavonoids can be due to
their hydrogen or electron donating ability and the ability of the polyphenol-derived
radical in chain-breaking function (free radical scavenging) metal chelation
modulation of enzymatic activity and alteration of signal transduction pathways
(Itagaki et al 2009) In the present study the hydrogen or electron donating ability
of the extract was confirmed in reducing power and DPPH radical scavenging
assays The other important mechanism of the plant extract could be due to its direct
radical scavenging ability which was shown in lipid peroxidation and deoxyribose
degradation assays
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
95
Hydroxy radical scavenging activity
The results presented in figure 22 clearly indicate the dose dependent
hydroxyl radical scavenging activity of the ethanolic amp aqueous extract of Liv-Pro-
08 Assay is based on Fenton reaction The value of ethanolic aqueous extract and
standard were found to be 2846 plusmn 019 to 6477 plusmn 346 1419 plusmn 015 to 5415 plusmn 012
amp 1524 plusmn 015 to 5515 plusmn 009 at various concentrations (20-100 microgml) Hydroxyl
radical scavenging capacity of an extract is directly related to its antioxidant activity
(Babu et al 2001) The ability of extracts to quench hydroxyl radicals seems to be
directly related to the prevention of propagation of the process of lipid peroxidation
and they seem to be good scavengers of active oxygen species thus reducing the
rate of reaction
Values are expressed as a mean plusmn SD of three observations
Hydroxyl radical is the most reactive radical known it can attack and
damage almost every molecule found in living cells Reactions of bullOH include its
ability to interact with the purine and pyrimidine bases of DNA It can also abstract
hydrogen atoms from biological molecules including thiols leading to the
formation of sulfur radicals able to combine with oxygen to generate oxysulfur
radicals a number of which damage biological molecules (Halliwell 1991) The
best-characterised biological damage caused by hydroxyl radical is its capacity to
stimulate lipid peroxidation which occurs when bullOH is generated close to
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
96
membranes and attacks the fatty acid side chains of the membrane phospholipids
(Halliwell 1991)
Hydroxy radicals are produced in vivo by Fenton type reactions in which
transition metals (eg iron) reduce hydrogen peroxide Reducing agents such as
ascorbic acid can accelerate OH formation by reducing Fe3+
ions to Fe2+
(Puppo
1992) Deoxyribose is degraded into malonaldehyde on exposure to hydroxyl
radicals generated by Fenton systems If the resulting mixture is heated under acid
conditions malonaldehyde may be detected by its ability to react with thiobarbituric
acid to form a pink chromogen (Halliwell et al 1987) In the work herein Liv-Pro-
08 extracts (ethanolic and aqueous) exhibited scavenging activity for hydroxyl
radical in a concentration dependent manner (Fig 22) from this ethanolic extract
possess the highest capacity compared to aqueous extract and standard Previous
studies determined the antioxidant activities of saponins from different plants
towards different possible antioxidant effects as effective free radical scavenging
activities superoxide anion radical scavenging amp hydroxyl radical scavenging
potential (Gulcin et al 2004 2006)
O2middot- and OH
middot are two kinds of free radicals that spontaneously produce when
the organism is under stress and are quite harmful to biological molecules O2middot is the
product of biological metabolism in the presence of oxygen and is quite toxic which
is closely related to the generation of a variety of inflammatory diseases including
those in liver O2middot- played a potential deleterious role in NAFLD (Laurent et al
2004) OHmiddot is the most active and harmful radicals in the organism that can react
with a wide range of molecules to induce large damage to DNA lipids and proteins
Hydroxyl radical scavenger had been proved to protect liver from oxidative injury
(Ninomiya et al 2004) The scavenging activities of CSGS on O2middot- and OH
middot
indicated that it may have potential abilities in vivo The direct reflection of
organism under oxidative stress is to increase neural excitability During this process
catecholamine increases significantly which generates O2middot- by automatic oxidation
Then H2O2 is formed by catalysed reaction of O2middot- to further generate more active
OHmiddot Polyunsaturated fatty acids in cell membrane are prone to react with OH
middot to
produce lipid peroxides which cause damage to cell and tissue (Yao et al 2005) and
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
97
then decompose to cytotoxic substances such as malonaldehyde (MDA) So MDA
content can quantitatively reflect the degree of lipid peroxidation caused by
oxidative damage (Richter 1987)
Hydroxyl radical is an extremely reactive oxygen species capable of
modifying almost every molecule in the living cells Moreover hydroxyl radicals
are capable of the quick initiation of lipid peroxidation process as by abstracting
hydrogen atoms from unsaturated fatty acids (Aruoma 1998 Kappus 1991) Due to
this high reactivity of hydroxyl radicals measurements based on scavenging
hydroxyl radicals such as the nonsite-specific method are not accurate
measurement of oxidative protection of an antioxidant molecule in vivo (Halliwell amp
Gutteridge 1999) This is because the radical is more likely to be scavenged by
direct reaction with other surrounding molecules before it can attack its target
molecule Deoxyribose assay when performed in the absence of EDTA (site-specific
model) forms hydroxyl radicals on the surface of the ribose substrate in the
presence of H2O2 and ascorbic acid In this model the only substances that inhibit
deoxyribose degradation are those that bind iron ions strongly enough to remove
them from deoxyribose and form complexes less reactive in generating hydroxyl
radicals (Aruoma et al 1987) The ability of Liv-Pro-08 to quench hydroxyl
radicals seems to be directly related to the prevention of propagation of lipid
peroxidation
Iron chelating radical scavenging activity
Metal ions cause lipid peroxidation that can produce free radicals and lipid
peroxides (Koksal et al 2009) Therefore metal chelating activity indicates
antioxidant and antiradical properties Decreased absorbance of the reaction mixture
indicates higher metal chelating capability EDTA was used as a standard metal
chelating agent According to the results the order of increasing chelating ability of
the ethanol standard (EDTA) and aqueous extracts was 20-100 microgml The chelating
ability values were 2558 plusmn 015 to 6223 plusmn 009 (ethanol extract) 2381 plusmn 009 to
5825 plusmn 020 (EDTA) amp 2313 plusmn 10 to 5978 plusmn 015 (aqueous extract) respectively
The ethanolic extract of the Liv-Pro-08 has the highest chelating ability at 100 microg
with 6223 when compared with a standard EDTA amp aqueous extract of Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
98
Antioxidant activity of the standard ethanolic and aqueous extract of the Liv-Pro-08
against various concentrations as shown in figure 23
The chelating of ferrous ions by the extract was estimated by the method of
(Dinis et al 1994) Ferrozine can quantitatively form complexes with Fe2+
In the
presence of other chelating agents the complex formation is disrupted with the
result that the red colour of the complexes decreases Measurement of the rate of
colour reduction therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al 2000) In this assay both extract and EDTA
interfered with the formation of ferrous and ferrozine complex suggesting that it has
chelating activity and captures ferrous ion before ferrozine The absorbance of Fe2+
ferrozine complex was decreased dose-dependently otherwise the activity was
increased on increasing concentration from 20 to 100 microgml Metal chelating
capacity was significant since the extract reduced the concentration of the catalysing
transition metal in lipid peroxidation (Duh amp Tu 1999)
Values are expressed as a mean plusmn SD of three observations
Chelation of metal ions and quenching of singlet oxygen are the major
characteristics of antioxidant activity Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals (Michalak 2006) Phenolics possess
hydroxyl group able to particularly iron and copper Chelating property may afford
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
99
protection against oxidative damage and iron-overload (Lai et al 2001) Chelating
ability of the plant extract investigate in the present work is dose-dependent
Chelating ability of plant extract provides a strategy to avoid free-radical generation
and iron-overload by chelation of metal ion (Robak et al 1985) Antioxidants are
widely applied to foods and medicines because they can counteract cellular free
radicals and reduce metal ion to interrupt the oxidizing chain reaction before they
cause damages Therefore it can protect our body against many health problems
(Halliwel and Gutteridge 1999 Venkat Ratnam et al 2007)
From the above results the antioxidant activity of the Liv-Pro-08 based on
DPPH scavenging activity and phosphomolybdenum methods are attributed to the
presence of phenolic compounds as major components in these species Plant
phenolics constitute one of the major groups of compounds acting as primary
antioxidant or free radical terminators (Cao et al 1997) Phenolic compounds such
as flavonoids phenolic acids and tannins are considered to be the major contributor
to the antioxidant activity of vegetables fruits or medicinal plants The antioxidant
activity of the phenolic compounds were attributed to its redox properties which
allow them to act as reducing agents hydrogen donators singlet oxygen quenchers
and have also metal chelating properties (Rice-Evans et al 1996) High amounts of
phenolic compounds indicate high antioxidant capabilities of samples Many studies
showed that the phenolic contents in plants have some antioxidant properties
(Kumar et al 2010 Viuda-Martos et al 2010)
ABTS radical scavenging activity
Trolox equivalent antioxidant capacity (TEAC) assay is one of the most
commonly employed methods for determining antioxidant capacity TEAC assay
measures the ability of a compound to scavenge ABTSbull+
radicals and is widely used
to screen antioxidant activity of fruits vegetables foods and plants and is
applicable to both lipophilic and hydrophilic antioxidants (Van den Berg et al
1999)Proton radical scavenging is an important attribute of antioxidants ABTS a
protonated radical has characteristic absorbance maxima at 734 nm which decreases
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
100
with the scavenging of the proton radicals (Re et al 1999) Indicating that they may
be useful therapeutic agents for treating radical-related pathological damage
Values are expressed as a mean plusmn SD of three observations
Figure 24 shows the ethanolic amp aqueous extract of Liv-Pro-08 lowest
radical scavenging activities at various (20 ndash 100 microgml) concentration when reacted
with the ABTS radicals approximately 5 folds lower than the standard (6414 plusmn 006
to 9797 plusmn 006) BHT Firstly the ABTSbull+
assay is an electron transfer end-point
assay whereby different antioxidant compounds donate one or two electrons to
reduce the radical cation Regardless of the donating potential of individual
antioxidants they all have time to react fully giving an accurate measurement of
TAC at the end-point of the assay (Huang et al 2005) Sahreen et al reported that
the chloroform and aqueous fractions of Carissa opaca fruit have strong antioxidant
(DPPH suproxide anion radical H2O2 and ABTS) activities (Sahreen et al 2010)
Firstly the ABTS+bull
assay is an electron transfer end-point assay whereby
different antioxidant compounds donate one or two electrons to reduce the radical
cation Regardless of the donating potential of individual antioxidants they all have
time to react fully giving an accurate measurement of TAC at the end-point of the
assay (Huang et al 2005) In particular it is recommended to be used for plant
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
101
extracts because the long wavelength absorption maximum at 734 nm eliminates
colour interference in plant extracts
Ferric Reducing Power Assay
Absorbance of ferric reducing power assay of ethanolic amp aqueous extract of
Liv-Pro-08 were 028 to 064 amp 013 to 047 respectively The FRAP of Liv-Pro-08
was higher in ethanolic extract as compared to aqueous extract and standard as
shown in figure 25 It is found to be effective in ethanolic extract with different
sample concentration
Values are expressed as a mean plusmn SD of three observations
Antioxidant capacities of Liv-Pro-08 were also evaluated using the Ferric
reducing antioxidant power (FRAP) assay In this assay the antioxidant capacity is
measured on the basis of the ability to reduce ferric (III) ions to ferrous (II) ions
The FRAP assay is a simple method and can be applied to both aqueous and
ethanolic extracts of Liv-Pro-08 Comparisons were also drawn between the results
obtained by the FRAP assay and those obtained for total polyphenols by FCR Both
of these assays utilise similar SET reaction kinetics and are essentially measures of
reducing capacity Where FRAP measures the reducing capacity of all the
antioxidants in the mixture FCR records only those classed as polyphenolic
compounds (Huang et al 2005)
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
102
Epidemiological evidence suggests that diet and nutrition may have a
significant effect in the prevention of serious diseases and potentially a very
significant effect on wider public health (Agarwal amp Rao 2000 Asplund 2002
Johnson 2004) Previously a number of in vitro methods have been used to
determine the bioaccessibility andor bioavailability of individual antioxidant
compounds such as carotenoids tocopherols or polyphenols in order to isolate
compounds which remain stable and active throughout the digestion and absorption
processes (McDougall et al 2005 OConnell et al 2007 Paetau et al 1998a Ryan
et al 2008)
Bourgou et al 2008 reported that the methanolic extracts of Nigella sativa
shoots amp roots showed significant antioxidant capacity in different assays in vitro
and confirms that these extracts represent a significant source of phenolic
compounds Abdel-Hameed studied six different ficus species have radical
scavenging activity and antioxidant capacity Phenolic compounds are the major
components of these species and the antioxidants properties were attributed to them
Therefore these species may have great relevance in the prevention and therapies of
diseases in which oxidants or free radicals are implicated after more in vitro studies
Naturally there is dynamic balance between the amount of free radicals
produced in the body and antioxidants to scavenge or quench them to protect the
body against deleterious effects The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated Therefore it is obvious to enrich our diet with antioxidants to protect
against harmful diseases Hence there has been an increased interest in the food
industry and in preventive medicine in the development of ldquoNatural antioxidantsrdquo
from plant materials The presence of antioxidants in Liv-Pro-08 ethanolic extract is
generally depends on the presence of reductant which have been exhibited
antioxidant potential by breaking the free radical chains
The results of the present study exhibited antioxidant activity of Liv-Pro-08
showed a higher potency than ascorbic acid BHA and EDTA in scavenging free
radical This could be due to the presence of enormous amount of phenolic
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
103
compounds which are responsible for the immense antioxidant property and also
revealed the possible antioxidant mechanism of the extract such as hydrogen or
electron donating ability and direct free radical scavenging properties The high
scavenging property of ethanolic extract of Liv-Pro-08 may be due to hydroxyl
groups existing in the phenolic compounds that can scavenge the free radicals The
results of the present study would certainly help to ascertain the potency of the crude
ethanolic extract of Liv-Pro-08 as a potential source of natural antioxidants
However further research is required to identify anti-steatotic (NAFLD) effects of
Liv-Pro-08
top related