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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-picrylhydrazyl (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


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


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


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


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)


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


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)


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


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


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


Gupta 2008)





























74

















assay methods






75




























76



no







Balinksy and

Bernstein 1963

16






(DPPH)




activity

Madan et al 2005 23



1989

25




acid (ABTS)

Re et al 1999 27


(FRAP)

Benzie and Strain

1999

28




77





bull) and




al 2007)






















78
























2008)







79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

73


Gupta 2008)





























74

















assay methods






75




























76



no







Balinksy and

Bernstein 1963

16






(DPPH)




activity

Madan et al 2005 23



1989

25




acid (ABTS)

Re et al 1999 27


(FRAP)

Benzie and Strain

1999

28




77





bull) and




al 2007)






















78
























2008)







79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

74

















assay methods






75




























76



no







Balinksy and

Bernstein 1963

16






(DPPH)




activity

Madan et al 2005 23



1989

25




acid (ABTS)

Re et al 1999 27


(FRAP)

Benzie and Strain

1999

28




77





bull) and




al 2007)






















78
























2008)







79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

75




























76



no







Balinksy and

Bernstein 1963

16






(DPPH)




activity

Madan et al 2005 23



1989

25




acid (ABTS)

Re et al 1999 27


(FRAP)

Benzie and Strain

1999

28




77





bull) and




al 2007)






















78
























2008)







79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

76



no







Balinksy and

Bernstein 1963

16






(DPPH)




activity

Madan et al 2005 23



1989

25




acid (ABTS)

Re et al 1999 27


(FRAP)

Benzie and Strain

1999

28




77





bull) and




al 2007)






















78
























2008)







79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

77





bull) and




al 2007)






















78
























2008)







79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

78
























2008)







79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

79







Pro-08 are explored






















80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

80

Catalase (CAT)
















Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)





Glucose 6 Phosphate









81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

81












potential








82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

82













1995)

Glutathione (GSH)














83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

83


Parameters

Aqueous Extract

(unitsmg protein)

Ethanol Extract

(unitsmg protein)

Glutathione





Vitamin C







bull

RO2bull RS













Vitamin E




84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

84








bull) derived from
















85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

85
















Pro-08



of inhibition





031 plusmn 0005 (62) b



038 plusmn 001 (56) c



DMRT

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

86














radicals

















87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

87
















scavenging activity







2004)








88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

88




















89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

89






























90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

90





















91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

91






























manner

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

92




















93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

93





antioxidants







Reducing power







ascorbic acid



to Fe2+










94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

94



















degradation assays

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

95











rate of reaction










96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

96


(Halliwell 1991)




ions to Fe2+

(Puppo












O2middot- and OH










middot






middot to


97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

97




















peroxidation












98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

98





In the
















99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

99































100





















101




















102









et al 2008)






















103









Liv-Pro-08

100





















101




















102









et al 2008)






















103









Liv-Pro-08

101




















102









et al 2008)






















103









Liv-Pro-08

102









et al 2008)






















103









Liv-Pro-08

103









Liv-Pro-08

chapter iii - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/17409/8/09... · 2015-12-04 ·...

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