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5. DISCUSSION
The role of free radicals and tissue damage in diseases, such as atherosclerosis,
heart failure, neurodegenerative disorders, aging, cancer, diabetes mellitus, hypertension
and several other diseases, is gaining a lot of recognition (Flora, 2007). Both reactive
oxygen species (ROS), as well as reactive nitrogen species (RNS), are products of normal
cellular metabolism. They are well recognized as playing a dual role as both deleterious
and beneficial species, in that they can be either harmful or beneficial to living systems.
Antioxidant supplements, or foods rich in medicinal plants, may be used to help the
human body in reducing oxidative damage by free radicals and active oxygen (Valko
et al., 2004).
Currently, research interest has been focused on the role of antioxidants as well as
antioxidant enzymes, in the treatment and prevention of the diseases mentioned above.
The most commonly used antioxidants at present are vitamins, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and tert-butylhydroquinone
(TBHQ). However, they are suspected of being responsible for liver damage and acting as
carcinogens in laboratory animals (Anagnostopoulou et al., 2006). Therefore, the
development and utilization of more effective antioxidants of natural origin is desirable.
Medicinal plants that historically have been useful, are obvious choices as
potential sources of substances with significant pharmacological and biological activities.
Phytomedicines exert their beneficial effects through the additive or synergistic action of
several chemical compounds acting at single or multiple target sites associated with a
physiological process in contrast to synthetic pharmaceuticals based upon a single
chemical. This synergistic or additive pharmacological effect can be beneficial by
eliminating the problematic side effects (Raja and Pugalendi, 2010). The therapeutic
benefit of medicinal plants is often attributed to their antioxidant properties (Ljubuncic
et al., 2006).
In recent years, considerable attention has been directed towards the identification
of plants with antioxidant ability that may be used for human consumption. Therefore,
research has focused on the use of antioxidants, with particular emphasis on naturally
derived antioxidants, which may inhibit ROS production and may display protective
effects (Mruthunjaya and Hukkeri, 2008).
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Several medicinal plants have been analyzed for their biological activity and
active constituents. One such medicinal plant is Bacopa monnieri, commonly known as
Brahmi. Research has focused primarily on Bacopa’s cognitive enhancing effects,
specifically memory, learning and concentrations and the results support the traditional
Ayurvedic claims. Not many studies have been done on the antioxidant and apoptosis-
modulating effects of B. monnieri. Hence, this study mainly focused on assessing the
antioxidant and apoptosis-modulating effects of B. monnieri.
The study was carried out in four phases, as explained in the methodology section.
The results obtained are discussed below.
PHASE I
Antioxidant status of different parts of B. monnieri
In Phase I of the study, different parts of the plant, namely leaves, stolon and
roots, were analysed for their antioxidant status. The results obtained showed that all the
three parts possessed considerable quantities of enzymic and non-enzymic antioxidants.
Among the three parts tested, the leaves possessed the maximum quantity of antioxidants
analyzed, followed by the stolon and the roots.
Several plants have been studied for their antioxidant status. The leaves of three
under-exploited medicinal plants were found to exhibit moderate activities of all the
enzymic antioxidants assessed (Nirmaladevi and Padma, 2008). Increased activities of the
enzymic antioxidants have been reported in the root and leaf samples of Phalaenopsis
(Ali et al., 2005).
Chamseddine et al. (2009) reported that the leaves of Solanum locopersicon
possessed higher superoxide dismutase activity. The leaves of Prunus cerasus L.
exhibited higher catalase activity (Chatizissavidis et al., 2008). Stajner and Popovic
(2009) reported that the highest carotenoid content was observed in the leaves of Allium
ursinum and the lowest in Allium scorodoprasum. The antioxidant potential of Clitoria
ternatea L. and Eclipta prostrata L. was analysed by Bhaskar et al. (2009) by assessing
enzymic and non-enzymic antioxidants.
Patel et al. (2010) have reported that when the stem and leaf parts of Calotropis
procera Linn., Hibiscus cannabinus L., Parthenium hysterophorus L., Gemelia arborea
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Roxb. and Kigelia pinnata (jacq.) DC plants were analysed for their phenolic and
flavonoid content, the maximum amount of phenols were found in the Gemelia plant
leaves, while the maximum flavonoid content was found in Hibiscus leaf.
Triadimefon treated rosea and alba varieties of Catheranthus roseus L. G.Don.
showed variation in superoxide dismutase, ascorbate peroxidase and catalase activities
compared to untreated control plant (Jaleel et al., 2006). The antioxidant property of the
pomegranate flowers (Kaur et al., 2006) and Salix capera (Alam et al., 2006) have been
shown to be responsible for their hepatoprotective property.
The methanolic extract of cantaloupe leaf showed the highest total phenolic
content and total flavonoid content than the stem, skin, seed and flesh extracts (Ismail
et al., 2010). Among the methanolic extracts of leaf, seed and stalk of Perilla frutescens,
the methanolic extract of seed was found to have the highest phenolic content. On the
other hand, total flavonoid content of stalk was also found to be superior to the other
P. frutescens extracts (Lin et al., 2010). When analysed for the phenolic content in the
methanolic extracts of aerial flowering parts of four endemic Stachys taxa, S. anisochila
and S. beckeana extracts had the highest polyphenol content (Kukic et al., 2006).
The total phenolic content of Cyphostemma digitatum was significantly higher in
the raw material than in the processed sample, but also significantly higher in the ethanol
extracts compared to the water extracts in both raw material and processed sample
(Al-Duais et al., 2009). The fresh and dried forms of Pleurotus florida and Calocybe
indica possessed non-enzymic antioxidants such as vitamins C, E, A and GSH (Selvi
et al., 2007).
Our results show that the leaves of B. monnieri are rich sources of both enzymic
and non-enzymic antioxidants. Therefore, in all the further studies, only the leaves were
used.
In the next part of Phase I, three different extracts of B. monnieri leaves were
prepared using solvents (water, methanol and chloroform) of differing polarity. These
solvent extracts were tested for their ability to scavenge free radicals.
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DPPH RADICAL SCAVENGING EFFECTS
The inhibition of free radical DPPH is one of the oldest and most frequently used
methods for total antioxidant potential/capacity of food and biological extracts. It is based
on the ability of an antioxidant to give hydrogen radical to synthetic long-lived nitrogen
radical compound DPPH.
In the present study, the different extracts of B. monnieri leaves were assessed for
their DPPH scavenging ability. The results of both qualitative dot blot screening and
quantitative spectrophotometric assays revealed that the methanolic extract exhibited the
highest activity.
The screening of herbal extracts and their components by the DPPH scavenging
assay has become a routine parameter for testing their antioxidant efficacy (Mothana
et al., 2008).
The aqueous, methanolic and ethanolic extracts of Melissa officinalis, Matricaria
recuttia and Cymbopogan citrates were found to possess DPPH scavenging activity
(Pereira et al., 2009). The alcohol–water extract of Ichnocarpus frutescens leaves
possessed 1,1-diphenyl-2-picrylhydrazyl radical and superoxide anion radical scavenging
activity (Kumarappan and Mandal., 2008).
The methanolic extracts of leaves and flowers of Lippia alba exhibited very
significant DPPH radical scavenging activity compared to the standard antioxidant
ascorbic acid (Ara and Nur, 2009). The methanolic extract of Manikara zapota showed
strong activity on scavenging DPPH radical, which implicates an essential defence
against the free radicals (Kaneria et al., 2009).
The hot water extract of Perilla frutescens stalk showed moderate DPPH radical
scavenging abilities than the leaf and seed extracts (Chou et al., 2009). The methanol
extract of Helichrysum plicatum subsp. Plicatum, a species of the Asteraceae family and
belonging to the subtribe Gnaphaliinae, has been reported to have antioxidant activity
using two in vitro methods, namely DPPH and β-carotene linoleic acid assays (Tepe
et al., 2005).
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Kang et al. (2005) reported that tectoridin, an isoflavone compound in Puerariae
species, showed intracellular ROS scavenging activity and DPPH radical scavenging
activity. Piao et al. (2004) reported that DPPH radical-scavenging activity in
furanocoumarins correlated with the number of phenolic hydroxyl groups present in their
structures. The antioxidant capacity of Cyphostemma digitatum measured by DPPH
method was significantly higher after processing (Al-Duais et al., 2009). The essential
oils of Myrtus communis L. compound such as 1,8-cineole and methyl eugenol showed
considerable DPPH scavenging activities (Dukic et al., 2010).
In the light of the proven medicinal properties of most of these plants, the DPPH
scavenging ability of B. monnieri leaves implicate the strong medicinal properties of the
plant.
ABTS RADICAL SCAVENGING EFFECTS
Another screening method for antioxidant activity is the ABTS radical cation
decolourization assay. This assay is widely used to assess the total amount of radicals that
can be scavenged by an antioxidant, i.e., the antioxidant capacity. In the present
investigation, this method showed results quite similar to those obtained in the DPPH
reaction.
ABTS is an excellent substrate for peroxidases and is frequently used to study the
antioxidant properties of natural compounds (Reszka and Britigan, 2007). The ethyl
acetate fraction of Evax pygmaea showed strong ABTS radical scavenging and it nearly
fully scavenged ABTS+ (Boussaada et al., 2008).
Of the successively extracted Aphanamixis polystachya bark with hexane, ethyl
acetate, methanol and water, the methanolic extract possessed potent ABTS scavenging
activity (Krishnaraju et al., 2009). The acetone, ethanol, methanol and water extracts
from seed and calyx parts of persimmon (Diospyros kaki cv. Fuyu) fruit had relatively
strong ABTS radical scavenging activity, exhibiting high antioxidant capacity. The
highest ABTS activity was detected in the ethanol extract (Jang et al., 2010).
Loizzo et al. (2009) reported that the Diospyros lotus extract tested in different
in vitro systems (DPPH, ABTS, FRAP and Fe2+ chelating activity assays) showed
significant antioxidant activity. An aqueous extract of Crataegi folium (Hawthorn) leaves
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exhibited the highest ABTS scavenging activity, followed by the acetone and chloroform
extracts, when compared to flower extracts (Demiray et al., 2009). In another study, 95%
ethanol extract of Agrimonia pilosa exhibited high ABTS radical activity (He et al.,
2009).
Gulcin et al. (2008) have shown that Ligustroside and Oleuropein, isolated from
the methanolic extracts of the root bark of Chinonanthus virginicus exhibited good ABTS
scavenging activity. Lopez-Laredo et al. (2009) reported that Tecoma stans possessed
strong ABTS scavenging activity and the activity was attributed to its phenolic and
flavonoid content.
With the support of the above studies, the ability of B. monnieri leaf extracts in
effectively scavenging ABTS radicals reveals the strong radical scavenging potential of
the leaves.
HYDROGEN PEROXIDE SCAVENGING EFFECTS
Hydrogen peroxide is a normal cellular metabolite that is continuously generated
and maintained at low concentration (Jones et al., 2008). Excessive production of this
oxidant has been associated with cell dysfunction and generation of diseases (Gonzalez
et al., 2005).
The ability of B. monnieri leaf extracts to scavenge hydrogen peroxide in an
in vitro system was carried out in the present study and the results revealed that the
methanolic extract exhibited a strong scavenging effect against hydrogen peroxide.
Raja and Pugalendi (2010) showed that the aqueous extract of Melothria
maderaspatana was capable of scavenging H2O2 in a dose-dependent manner. The grape
seed extracts possessed strong antioxidant activity by scavenging hydrogen peroxide
when compared to bagasse extract (Baydar et al., 2007). Gulcin et al. (2007) have
reported that the water and ethanolic extracts of Ocimum basilicum had strong antioxidant
activity and were effective in scavenging H2O2.
The H2O2 scavenging activity of the aqueous extract of Strychnos henningsii Gilg.
indicated a concentration-dependent activity against H2O2 (Oyedemi et al., 2010). The
petroleum ether fraction of Coccinia grandis showed strong H2O2 scavenging activity
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followed by chloroform and ethyl acetate fractions (Umamaheswari and Chatterjee,
2008). The enzymatic extracts from seven species of brown seaweeds exhibited more
prominent effects in hydrogen peroxide scavenging activity (Heo et al., 2005a).
Ak and Gulcin (2008) reported that curcumin (diferuoyl methane), a phenolic
compound and a major component of Curcuma longa L. had an effective hydrogen
peroxide scavenging activity. Resveratrol, a natural phenolic product, was found to
scavenge hydrogen peroxide effectively (Gulcin, 2010).
From the observations made from the study, it is clear that B. monnieri leaf
extracts can effectively scavenge H2O2, which shows the strong antioxidant activity of the
leaves.
HYDROXYL RADICAL SCAVENGING ACTIVITY
Hydroxyl radical is the most reactive among the ROS. It has the shortest life
compared with other ROS and is considered to be responsible for much of the biological
damage in free radical pathology. The deoxyribose degradation is the common method
for determining the rate constant of hydroxyl radical reactions (Jelili et al., 2010). It is
difficult to directly determine the hydroxyl radical. The scavenging activity is measured
as OH� induced TBA-reactive substance (TBARS) formation. The rate of TBARS
formation is dependent on the reaction of deoxyribose with hydroxyl radical.
The effect of B. monnieri leaf extracts on hydroxyl radical-induced damage to
deoxy ribose was analysed. It was found that all the three extracts exhibited strong
protection against hydroxyl radical, with the methanolic extract faring better than the
other two.
There are many literature reports to support this observation. The methanol extract
of Lagerstroema speciosa L. showed higher hydroxyl radical scavenging activity when
compared to the ethyl acetate, ethanol and water extracts (Priya et al., 2008). The
methanolic extract of Picrasma quassiades (Yin et al., 2009), the ethanol extract of the
leaves of Stachytarpheta angustifolia (Awah et al., 2010) and the aqueous extract of
Wagatea spicata flowers (Samak et al., 2009) efficiently inhibited hydroxyl radicals.
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Inula viscose was also found to scavenge hydroxyl radical (Danino et al., 2009).
Esmaeili and Sonboli (2010) showed that when an endemic Salvia species (Salvia
brachyantha (Bordz)) was assessed in vitro for its radical scavenging activity, the plant
exhibited antioxidant activity. Heo et al. (2005b) demonstrated positive effects against
hydroxyl radicals using Ecklonia cava enzymatic extract.
Pan et al. (2009) reported that a novel red pigment isolated from Osmanthus
fragrans seeds exhibited a strong concentration-dependent inhibition of hydroxyl radical
at low concentrations compared with ascorbic acid and quercetin. Siddhuraju and Becker
(2007) showed the hydroxyl radical scavenging activity of dry heated samples of light
brown and dark brown seeds of cowpea (Vigna unguiculata). Sakanaka and Ishihara
(2008) reported the hydroxyl radical-scavenging activity for vinegar made from
persimmon Saijyo varieties, and unpolished rice vinegar.
Anthocyanins from litchi fruit pericarp strongly exhibited a dose-dependent
hydroxyl radical-scavenging activity (Duan et al. 2007). The in vitro antioxidant activity
of liquor from fermented shrimp biowaste showed strong hydroxyl radical scavenging
activity (Sachindra and Bhaskar, 2008). Embelin (from Embelia ribes), a component of
herbal drugs inhibited hydroxyl radical induced deoxyribose degradation (Joshi et al.,
2007).
From the results obtained, it is evident that B. monnieri leaf extracts possess very
good hydroxyl radical scavenging activity. The available literature lend credibility to this
observation as a strong indicator of the antioxidant potential of B. monnieri leaves.
SUPEROXIDE RADICAL SCAVENGING EFFECTS
Superoxide radicals are one of the most important reactive oxygen free radicals
constantly produced in living cells (Naik et al., 2006). It has relatively weak chemical
reactivity because it cannot penetrate lipid membranes and it is rapidly converted into
H2O2 by superoxide dismutase (Valko et al., 2007).
The ability of B. monnieri leaf extracts to inhibit the in vitro generation of SO●
was tested. The results of the study revealed that the methanolic extract exhibited the
highest SO● scavenging activity. Many reports in the literature associate the SO
●
scavenging of plants and their components with strong antioxidant activity.
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Anandjiwala (2007) reported that the methanolic extract of Bergia suffruticosa
showed inhibition of superoxide generation. Ogunlana et al. (2008) showed that the crude
extract of Newboudia leaves exhibited very high inhibition of superoxide generation.
The aqueous and ethanolic extracts of Iris germanica was found to exhibit strong
SO� scavenging activity (Nadaroglu et al., 2007). The methanol extracts of bark and
leaves of Cassia siamea and Cassia javanica plants showed effective superoxide anion
radical scavenging activity (Kaur and Arora, 2009).
Gymnema sylvestre extract showed antioxidant activity by inhibiting DPPH,
scavenging superoxide, as well as hydrogen peroxide (Rachh et al., 2009). Chloroform
and methanol extracts of the roots of Rhubarb (Rheum ribes L.) showed more potent
superoxide anion radical scavenging activity than BHT, and was comparable with well
known radical scavenger L-ascorbic acid (Ozturk et al., 2007).
Saito et al. (2008) reported that the extracts from Punica granatum (peel),
Syzygium aromaticum (bud), Mangifera indica (kernel) and Phyllanthus emblica (fruit)
scavenged superoxide anions, which was comparable to that of L-ascorbic acid. Kim
et al. (2009b) reported that α-chymotrypsin extract from the center of the root of Korean
Elk velvet antler showed high superoxide scavenging activity. The crude extract and
fractions of Vaccinium uliginosum L. containing polyphenol and pigment exhibited
superoxide scavenging activity (Kim et al., 2009c).
Li et al. (2009b) showed that Lysimachia foenum-graecum Hance exhibited SO●
radical-scavenging activities. Salvia brachyantha (Bordz) exhibited superoxide radical
scavenging activity at different magnitudes of potency (Esmaeili and Sonboli, 2010).
Inoue et al. (2005) reported that manuka honey had specific scavenging activity
for superoxide anion radicals. Caffeic acid, a major hydroxycinnamic acid present in
wine, was found to possess effective superoxide anion radical scavenging activity
(Gulcin, 2006). The superoxide anion radical scavenging activity was found to be
significantly higher in the raw and dry heated seed extracts than the hydrothermally
processed seed samples of cowpea (Vigna unguiculata) (Siddhuraju and Becker, 2007).
Yang et al. (2010a) reported that the methylated polysaccharides of longan fruit pericarp
resulted in a decrease in the superoxide anion radical scavenging activity with increasing
degree of methylation.
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In the backdrop of the above studies, it is clear that the leaves of B. monnieri also
possess strong SO� scavenging moieties as evidenced by our results. These observations
reflect the radical scavenging and antioxidative effects of B. monnieri.
NITRIC OXIDE SCAVENGING EFFECTS
Nitric oxide radicals play an important role in inducing inflammatory response
and their toxicity multiplies only when they react with O2�― radicals to form
peroxynitrite, which damage biomolecules like protein, lipids and nucleic acids
(Saraswathi and Mary, 2009).
The leaf extracts of B. monnieri, when analysed for their effect on the inhibition of
NO generation, showed that the methanolic extract exhibited maximum inhibition. The
determination of the extent of inhibition of NO generation adds credibility to the
antioxidant activity of extracts and compounds, as is evident from several reports in the
published literature.
The aqueous extract of Wasabia japonica showed antioxidant property by strongly
scavenging NO in a cell free system (Lee et al., 2010). The aqueous extract of Strychnos
henningsii caused a moderate dose-dependent inhibition of nitric oxide generation
(Oyedemi et al., 2010). The crude ethanolic bark extract of Terminalia arjuna was found
to possess high phenolics, high reducing power and high free radical scavenging activity
including nitric oxide radicals (Sree et al. 2007).
The hydroalcoholic extract of Alocasia indica possessed potent nitric oxide radical
scavenging activity (Mulla et al., 2010). The hydromethanolic extract of the Mikai
scandens wild leaves exhibited inhibition of nitric oxide radical (Hasan et al., 2009). High
nitric oxide inhibiting activity by the methanolic extract of Enicostemma axillarae was
shown by Vaijinathappa et al. (2008).
Razali et al. (2008) showed that when the methanol, hexane and ethyl acetate
extracts of the shoots of Anacardium occidentale were measured for their antioxidant
activities, the methanolic extract exhibited the maximum scavenging of superoxide anion
and nitric oxide radicals. Wu et al. (2010) reported that among the extracts and four
fractions of Geranium sibiricum, the ethyl acetate fraction showed the highest nitric oxide
scavenging activity.
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The strong inhibition of nitric oxide generation by Citrullus colocynthus fruit
extract indicated its strong antioxidant property (Kumar et al., 2008a). The roots of
Baliospermum montanum Muell-Arg (Desai et al., 2010) showed NO scavenging effects
in a dose-dependent fashion. Rao et al. (2010) reported that the Njavara rice bran extract
showed the highest nitric oxide scavenging activity compared to the methanolic extracts
of Jyothi, Yamini and Vasumathi rice bran.
In the light of these literature, it is clear that B. monnieri leaf extracts exhibited
good nitric oxide scavenging activity, which reiterated the strong antioxidant potential of
the leaves.
Thus, the present study found that B. monnieri leaves have effective in vitro
antioxidant and radical scavenging activity, implying their use in pharmacological and
food industries due to its antioxidant properties.
PHASE II
In this phase, the leaf extracts were analyzed for their biomolecular-protective
effects in cell-free systems and intact cells using the aqueous, methanol and chloroform
extracts. Since the methanolic extract of the leaves exhibited the maximum protection, the
same was used to study the effect on different cells subjected to oxidative stress. The
results obtained are discussed below.
EFFECTS OF THE B. monnieri LEAF EXTRACTS ON OXIDANT-INDUCED
DAMAGE TO BIOMOLECULES
Free radicals such as ROS and RNS produced during the normal metabolism can
damage the cells, resulting in lipid peroxidation and alteration of protein and DNA
structures (Ajith, 2010). When ROS attack cells, the lipids, which are the major
components of the membranes, take the immediate brunt of the attack. However, the
ultimate targets of these attacks, which lead to sustained damage, are the DNA molecules.
Hence, in the present study, the effects of the extracts of B. monnieri were studied on both
lipids and DNA subjected to oxidative stress.
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EFFECTS OF B. monnieri LEAF EXTRACTS ON OXIDATIVE DAMAGE TO
LIPIDS
ROS have the ability to degrade macromolecules such as lipids, nucleic acids,
proteins and pigments, finally leading to cell death. The primary targets of oxidative
damage are the lipid molecules, which, if left unrepaired, may have deleterious effects.
In the present study, the effect of the extracts in inhibiting LPO was analysed in
three different membrane models. All the three extracts showed the maximum inhibition
of LPO in all the membrane models. The extent of protection was found to be much better
in the liver homogenate, followed by the RBC ghosts and liver slices.
The literature is very rich with studies and reports assessing the ability of plants
and herbs in inhibiting LPO in different membrane lipid sources and these studies support
our findings.
Moringa oleifera leaf extract inhibited the amount of MDA generated (and thus
LPO) in liver homogenate (Sreelatha and Padma, 2009). A high degree of inhibition of
LPO was shown by the polar and non-polar extracts of Cyanthillium cinereum (Less.) H.
Rob (Guha et al., 2009). Lou et al. (2010a) reported that the burdock leaves fraction in
combination with tertiary butylhydroquinone exhibited high LPO inhibitory activity.
The methanol extracts of Cocculus hirsutus (Palsamy and Malathi, 2007), Rhus
coriaria L. fruits (Chandan and Sokeman, 2004) and Triumfetta rhomboidea (Sivakumar
et al., 2008) showed protection against LPO in vitro. The aqueous and methanolic
extracts of the tuberous roots of Decalepis hamiltonii inhibited microsomal LPO
(Srivastava et al., 2006).
The administration of methanol extracts of the lichen species Peltigera rufescans
(weis.) Humb, diclofenac and indomethacin reduced the LPO in the paw tissues in acute
and chronic inflammation models (Tanas et al., 2010). Bavarva and Narasimhacharya
(2010) reported that the ethanol extract of the leaves of Leucas cephalotes decreased LPO
in diabetic rats.
The methanolic extract of Aphanes arvensis extract significantly reduced TBARS
formation, indicating significant anti-lipid peroxidation in a concentration dependent
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manner and their inhibitory effects were comparable to the reference compound, Trolox
(Hamad et al., 2010). The methanolic extract of the plant Hedyotis corymbosa
significantly reduced the accumulation of lipid peroxides in vitro in a dose-dependent
manner in rat liver homogenate (Sadasivan et al., 2006). The flowers of the plant
Dendrobium nobile inhibited the in vitro LPO significantly in both liver homogenate and
RBC ghosts (Devi et al., 2009).
Resveratrol strongly inhibited the LPO of linoleic acid emulsion (Gulcin, 2010).
Embelin (from Embelia ribes), a component of herbal drugs was found to inhibit LPO
and restore impaired Mn-superoxide dismutase in rat liver mitochondria (Joshi et al.,
2007). Quercitrin, a glycoside form of quercetin, was able to prevent the formation of
TBARS induced by pro-oxidant agents (Wagner et al., 2006).
These reports support our findings, wherein the leaf extracts were very effective in
inhibiting LPO in the different membrane systems. Since all the three systems comprise
of plasma membrane lipids, the observation that a better protection occurs in homogenate
than in plasma membrane and intact cell, make it clear that some endogenous component
in the cells is involved. It implies that an endogenous factor in the cells, interacts
synergistically with the leaf components to render better protection against lipid damage.
Thus, our results clearly demonstrate that the extracts of B. monnieri leaves are
very effective in protecting membrane lipids against oxidative damage.
EFFECTS OF B. monnieri LEAF EXTRACTS ON OXIDANT-INDUCED
DAMAGE TO DNA
The protective effect of B. monnieri leaf extracts (aqueous, methanol and
chloroform) to DNA was studied using DNA from different sources, i.e., commercially
available DNA and DNA from intact cells. In the commercial sources, DNA from
different sources namely λ DNA, pUC18 DNA, herring sperm DNA and calf thymus
DNA were used. The results showed that all the three extracts were able to revert the
damage caused by H2O2.
Many studies have reported the use of herbal extracts or products in rendering
protection to the damaged DNA.
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Aphanes arvensis aqueous and methanolic extracts showed inhibitory effect on
DNA oxidation (Hamad et al., 2010). The aqueous extract of Cyanthillium cinereum
(Less.) H. Rob. evidently demonstrated the protective effect on pBR322 plasmid DNA
against oxidative breakdown (Guha et al., 2009).
The methanolic extract of Nelumbo nucifera flowers rendered protection to
pUC18 DNA exposed to H2O2 (Wong et al., 2005). Karawita et al. (2007) showed that
the enzymatic extracts from seven species of microalgae yielded promising DNA damage
inhibitory properties on mouse lymphoma L 5178 cells treated with H2O2.
Bitter cumin extract offered complete protection to DNA damage induced in calf
thymus DNA and reduced uncoiling or open circular form of pUC18 DNA (Ani et al.,
2006). Acorus calamus extract effectively reduced the disappearance of the covalently
closed circular form of plasmid DNA (pBR322) following exposure to γ-radiation
(Sandeep and Nair, 2010). Treatment with Azadirachta indica significantly mitigated
H2O2-induced oxidative damage to pBR322 DNA (Manikandan et al., 2009).
Kumar and Chattopadhyay (2007) showed that the n-butanol soluble fraction
derived from the methanol extract of Mentha spicata Linn. exhibited significant
protecting activity against DNA strand scission. Prakash et al. (2007) reported that some
of the food and medicinal plants were found to be effective in protecting plasmid DNA
nicking induced by hydroxyl radicals generated by Fenton's reaction.
The addition of Bambusar caulis in Liquamen, provided a protective barrier
against hydroxyl radical-induced damage to pBR322 supercoiled DNA in a dose-
dependent manner (Je et al., 2009). The supplementation of water cress in the diet
reduced lymphocyte DNA damage in healthy adults (Gill et al., 2007). Hydroxylated-4-
thiaflavans, a group of antioxidants, showed protection against oxidative DNA damage in
herring sperm DNA induced by cumene hydroperoxide (Lodovici et al., 2006).
In the present study, the extracts of B. monnieri leaves were found to offer very
significant protection against oxidative damage in purified DNA samples under
physiological conditions. Following this, the extent of DNA damage in the presence and
the absence of the leaf extracts was followed in intact cells using the comet assay.
15
Among the methods to measure oxidative damage to DNA, the comet assay has
been shown to be the most accurate method for measuring DNA oxidation (Stoyanova
et al., 2010).
In the present study, untransformed cells (peripheral blood lymphocytes) were
used to assess the oxidant-induced damage in intact cells using the comet assay. The leaf
extracts were very effective in bringing down the extent of oxidative DNA damage. The
damage caused by the oxidant was completely nullified on exposure to the extracts.
The results obtained with the purified DNA preparations showed that the damage
could not be completely reverted by the leaf extracts. The fact that the same dose of leaf
extracts could completely revert the damage to basal levels in the intact cells suggests the
possible involvement of some endogenous cellular component that works in conjunction
with the leaf components to protect DNA. A similar indication has also been obtained
from our results with the inhibition of LPO in different membrane preparations.
Jang et al. (2010) have shown that the solvents (acetone, ethanol and methanol)
extracts of calyx and seed of Diospyros kaki exhibited the greatest protective effect on
H2O2 induced DNA damage in human leukocytes. The protective effect of Cnidium
officinale extract against H2O2-induced oxidative damage in the human skin fibroblasts
was revealed by Jeong et al. (2009). Acorus calamus extract effectively protected DNA
from radiation induced strand breaks and enhanced the DNA repair process in murine
cells and human peripheral blood leukocytes (Sandeep and Nair, 2010).
Diphlorethohydroxycarmalol isolated from Ishige okamurae exerts profound
antioxidant effects against H2O2-mediated cell damage and significantly enhanced cell
viability against H2O2-induced oxidative damage (Heo and Jeon, 2009). Garlic extracts
efficiently enhanced the ability of normal human leukocytes to resist hydrogen peroxide
and 4-hydroxynonenal induced oxidative damage (Park et al., 2009).
Ecbalium elaterium fruit juice significantly reduced the frequency of comet
bearing cells in human lymphocytes (Rencuzogullari et al., 2006). A red mixed berry
juice and a corresponding polyphenol-depleted juice, serving as control, revealed a
decrease of oxidative DNA damage (Weisel et al., 2006). Fabiani et al. (2008) showed
that phenolic compounds, when used both as purified compounds and in complex crude
extracts in olive oil, prevented hydrogen peroxide-induced DNA damage.
16
Miranda et al. (2008) reported that ingestion of mate tea (Ilex paraguariensis)
increased the resistance of DNA to H2O2-induced DNA strand breaks and improved the
DNA repair after H2O2 challenge in liver cells, irrespective of the dose ingested. Miorelli
et al. (2008) revealed that ebselen at a concentration of 5-10 µM diminished the extent of
the DNA damage induced by hydrogen peroxide.
The cytoprotective effects of the Kojizyme extract against H2O2-induced DNA
damage increased significantly with increasing extract concentrations in comet assay tests
(Heo and Jeon, 2008). Athukorala et al. (2005) also reported that antioxidant compounds
from seaweed extracts exhibited superior ROS scavenging activity and cytoprotective
effects against H2O2-induced DNA damage.
The results of the present study are in agreement with the above reports. Our
results of the assays probing the extent of protection rendered by B. monnieri leaf extracts
to lipids and DNA strongly suggest the synergistic involvement of some, as yet
unidentified, endogenous factor in the target cells, that acts in conjunction with the plant
components to greatly reduce the extent of oxidative damage. Thus, our study shows that
B. monnieri leaves are an excellent source of naturally occurring antioxidant compounds
with potent lipid and DNA damage inhibition potential.
EFFECTS OF B. monnieri LEAF EXTRACTS ON OXIDATIVE STRESS-
INDUCED APOPTOTIC CHANGES
Damage to cells and tissues involve the generation of ROS and RNS, followed by
alterations in lipids, DNA and proteins, which eventually lead to cellular dysfunction and
cell death. The consequent role of free radicals in the mechanism of cell death, especially
in the induction of apoptotic death has been receiving growing attention in the field of
cancer therapy (Mishra, 2004).
With this backdrop, in the present study, the effect of B. monnieri leaf extract on
the oxidative stress-induced apoptosis was analysed in untransformed (S. cerevisiae and
chick embryo fibroblasts) and transformed (Hep2 laryngeal carcinoma) cells. The
oxidants used were H2O2 (in S. cerevisiae cells) and etoposide, a standard
chemotherapeutic drug (in chick embryo fibroblasts and Hep2 cells).
17
EFFECT OF B. monnieri LEAF EXTRACT ON H2O2-INDUCED DEATH IN
S. cerevisiae CELLS
The results obtained clearly indicated that the exposure to H2O2 resulted in a steep
rise in the number of cells undergoing apoptosis. B. monnieri leaf extract, by itself, did
not cause an increase in the extent of apoptosis. When co-administered along with H2O2,
the plant extract resulted in a markedly decreased number of apoptotic cells. Thus, it is
evident that the leaf extract protect the yeast cells from oxidative stress-induced death.
EFFECT OF B. monnieri LEAF EXTRACT ON ETOPOSIDE-INDUCED DEATH
IN UNTRANSFORMED AND TRANSFORMED CELLS
The results of the present study revealed that on exposure to B. monnieri leaf
extract, a differential response was evoked in the untransformed and transformed cells.
On exposure to etoposide, the number of cells undergoing apoptosis was significantly
increased in both the cell types. The leaf extract, by itself, did not induce apoptosis in
normal cells, but apoptosis was induced in cancer cells. This indicated the anticancer
activity of the B. monnieri leaf extract.
The administration of the leaf extract effectively counteracted the apoptosis-
inducing effect in primary cells, whereas, the apoptosis-inducing effect of etoposide was
further augmented in Hep2 cells in the presence of the extract.
This observation has a very significant bearing. It is deducible from our results
that the B. monnieri leaf extract protects the normal cells from oxidative death, while
rendering the cancer cells more susceptible to the cytotoxic action of the
chemotherapeutic drug etoposide. Thus, based on our results, it can be suggested that
B. monnieri leaves can be administered as a supportive therapy during cancer
chemotherapy, to minimize the toxic side effects to non-cancerous cells and to maximize
the anticancer drug action.
The above results were confirmed by a battery of tests, namely MTT assay, SRB
assay, staining procedures (with Giemsa, PI, EtBr and DAPI) and DNA fragmentation.
All these are highly reliable assays as is evident from the rich literature support, which is
discussed below.
18
MTT VIABILITY ASSAY
The viability of both untransformed and transformed cells was significantly
decreased on exposure to the oxidant H2O2/etoposide. B. monnieri leaf extract increased
the viability of the untransformed cells, whereas the viability was further increased in
cancer cells.
MTT is considered to be a reliable assay to determine the extent of cell viability,
as observable from the vast number of studies reported in the literature.
The ethanolic extract of Pleurotus ostreatus inhibited the cell proliferation in a
dose-dependent manner in HL-60 leukemia cells (Venkatakrishnan et al., 2010). The
methanolic extract of Piper sarmentosum possessed anticarcinogenic properties in HepG2
cells (Ariffin et al., 2009). An aqueous extract of several Asteraceae species exhibited its
antiproliferative activity against HeLa (cervix epithelial adenocarcinoma), A431 (skin
epidermoid carcinoma) and MCF-7 (breast epithelial adenocarcinoma) cells, in the MTT
assay (Rthy et al., 2007).
The ethanolic extract of Celastrus orbiculatus exhibited a cytotoxic effect on
human melanoma A375-S2 and human cervical carcinoma HeLa cell lines (Xu et al.,
2008). Je et al. (2009) reported that Bambusae caulis in Liquamen significantly protected
against hydrogen peroxide-induced apoptosis in PC-12 cells. MTT assay in U87 and
U251n glioma cells treated with various concentrations of gallic acid showed decreased
cell viability (Lu et al., 2010a).
Honokiol, an active component isolated and purified from the Magnolia induced
cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes (Chen
et al., 2010b). Phloroglucinol derivative (2,4-bis(2-fluorophenylacetyl) phloroglucinol)
induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but
not in primary chondrocytes (Liu et al., 2010b).
Treatment with celecoxib resulted in a concentration-dependent decrease of
cellular viability in U87-MG, U251 and A172 (Gaiser et al., 2008). Withaferin A, a major
constituent of the dietary component Withania somnifera, induced Par-4-dependent
apoptosis in androgen-refractory prostate cancer cells (Srinivasan et al., 2007).
19
The cell viability estimated by the MTT assay showed that Unearia rhynchophylla
induced cytotoxicity in HT-29 (human colon carcinoma) cells (Jo et al., 2008). Similarly,
Zhang and Poporich (2008) estimated, using the MTT assay, the inhibition of cell
proliferation in Hep G2 (liver carcinoma) cells by soya saponins, which was found to be
dose-dependent. Koon et al. (2006) investigated the effect of curcumin on the viability of
nasopharyngeal carcinoma cells using MTT.
Resveratrol rendered protection against arsenic trioxide induced cardiotoxicity
in vitro and in vivo. Pretreatment with resveratrol dose-dependently attenuated the As2O3-
induced reduction in cell viability (Zhao et al., 2008a). Wali et al. (2009) analysed the
cell viability in neoplastic mouse +SA mammary epithelial cells by the MTT assay, and
reported that the cell viability was significantly decreased by α-tocotrienol in a dose- and
time-dependent manner.
Peng et al. (2007) determined the cytotoxic activity of the root of Spiranthes
australis on A549 (lung carcinoma), BEL-7402 (hepatoma cell line), SGC-7901 (gastric
cancer), MCF-7 (breast cancer), HT-29 (colon cancer), K562 (leukemia) and A498 (renal
cancer) cell lines by MTT assay, where the cell viability was reduced by the plant extract.
Human hepatoma SMMC-7721 cells treated with the Rosa roxburghii extract showed a
decrease in the cell viability (Yu et al., 2007). The extract of Linum persicum and
Euphorbia cheiradenia induced the reduction of K562 cell (leukemia cell lines) viability
through apoptosis, which was quantified by the MTT assay (Amirghofran et al., 2006).
From the results of the MTT assay in the present study, it can be inferred that
B. monnieri leaf extract was potent in inducing apoptosis in cancerous cells and
augmenting it in the presence of an oxidant. The extract effectively counteracted the toxic
effect of the oxidant in non-cancerous cells.
SRB ASSAY
The results of the SRB assay also followed the same trend as the MTT assay in the
present study. Several studies have used the SRB assay to ascertain the cytotoxic effect of
phytocomponents against cancer cells.
The phenolic ethanolic extract of Clitocybe alexandri inhibited the growth of four
different tumor cell lines (lung, breast, colon and gastric cancer) (Vaz et al., 2010).
20
Primary rat hepatic stellate cells and the human hepatic stellate cell line LX-2 treated with
Withagulatin A showed a dose-dependent decrease in cell viability as determined by SRB
assay (Liu et al., 2010a). The extracts of Phyllanthus emblica and Terminalia bellerica
are reported to have high cytotoxic effects against the human hepatocellular carcinoma
(HepG2) and lung carcinoma (A549) cells (Pinmai et al., 2008).
The ethylacetate extract of Ageratum conyzoides exhibited high cytotoxic activity
on A-549 and P-388 cancer cells (Adebayo et al., 2010). Cistus incanus L. and
Cistus monspeliensis L. extracts treatment on prostate cell lines resulted in a significant
decrease in cell viability (Vitali et al., 2010). The potential antiproliferative activity of the
Ailanthus excelsa extract and isolated flavonoids against five human cancer cell lines
(ACHN, COR-L23, A375, C32, and A549) was investigated in vitro by the SRB assay in
comparison with one normal cell line, 142BR. The extract exhibited the highest inhibitory
activity against C32 cells (Said et al., 2010).
An anthocyanin extract from blueberry and an anthocyanin-pyruvic acid adduct
extract significantly reduced cell proliferation in two breast cancer cell lines (MDA-MB-
231 and MCF7) as assessed by the SRB assay (Faria et al., 2010). A new diterpene
alkaloid named delphatisine isolated from the aerial parts of Delphinium chrysotrichum,
showed significant cytotoxic activity against the A549 cell line (He et al., 2010).
Andrographolide, a diterpenoid lactone isolated from a traditional herb
Andrographis paniculata, has been reported to inhibit prostate cancer (PC-3) cells in a
dose-dependent manner, as determined by sulphorhodamine B assay (Zhao et al., 2008b).
The cytotoxic effect of 7-O-methylaromadendrin and tectorigenin against MCF-7 (breast
cancer) and C32 amelanotic melanoma cell line was determined by the SRB assay (Fang
et al., 2008).
The cytotoxicities of the 14 alkaloids isolated from Corydalis ternata were
evaluated on several human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15)
using the SRB assay (Kim et al., 2010b). Abu-Dahab and Afifi (2007) determined the
antiproliferative activity of several medicinal plants against a breast adenocarcinoma cell
line by SRB analysis and observed that the plant extracts exhibited high cytotoxicity
against MCF-7 (breast cancer) cells. Patel et al. (2009) reported that the methanolic
21
extract of Solanum nigrum fruits, when screened for its cytotoxicity against HeLa and
Vero cell lines at different concentrations, exhibited significant toxicity on HeLa cell line.
Fibrarecisin, a novel triterpenoid, isolated from the chloroform extract of the stem
bark of Fibraurea recisa Pierre showed cytotoxicity against A549 (lung cancer) cells (Fu
et al., 2007). Mucronulatol from Caribbean propolis was found to possess cytotoxic
effect against cancer cells (Carballo et al., 2008). The cytotoxic effect of etoposide,
vinblastine sulfate and dacarbazime against B16–F10 and HMEC-1 cells was determined
by SRB assay (Dandamudi and Campbell, 2007).
From the observations made from the cell viability assays in the present study, it
can be inferred that oxidative stress caused significant death in both untransformed and
transformed cells. B. monnieri leaves showed their protective effect against oxidative
stress in untransformed cells, while enhancing the cytotoxic effect of etoposide in cancer
cells.
MORPHOLOGICAL CHANGES ASSOCIATED WITH APOPTOSIS
The morphological changes associated with apoptosis are membrane blebbing,
cell shrinkage, membrane-bound apoptotic bodies and chromatin condensation. In the
present study, a marked increase in the number of apoptosing cells in the cancer (Hep2)
cells exposed to methanolic extract of B. monnieri leaves was observed. The oxidative
stress-induced cellular death in normal cells was effectively counteracted by the presence
of B. monnieri leaf extract.
Staining with giemsa has been reported by many researchers characterizing the
effect of herbal components on apoptosis in different cells.
Apoptotic cell death was observed in WM793B cell line as evident from the
morphological changes (Lesiak et al., 2010). Morphological changes of L02 cells
observed by Giemsa staining showed that Polygonum multiflorum could induce L02 cell
apoptosis (Zhang et al., 2010). Studies have demonstrated that wogonin, a monoflavonoid
extracted from the root of Scutellaria baicalensis, could effectively inhibit the
proliferation of several cancer cell lines as determined by Giemsa staining (Zhang et al.,
2008b).
22
Clitocine, a natural biologically active substance isolated from the mushroom
Leucopaxillus giganteus, induced cell death as characterized with the changes in cell
morphology (Ren et al., 2008). The julibroside J8 isolated from the Albizia julibrisin,
inhibited the growth in the BGC-823, BEL-7402 and HeLa cell lines (Zheng et al., 2006).
Zhou et al. (2007) reported that oridonin, a diterpenoid extracted from medicinal herbs,
had potent antitumor activity with low adverse effects on U937 cells.
Artesunate induced apoptosis of tumor cells in vitro, as observed from the
morphological changes of Raji and Jurkat cells under light microscopy after Wright-
Giemsa dyeing (Zeng et al., 2009). Apoptosis in ECV-304 cell line supplemented with
sodium morrhuate and liposodium morrhuate was also demonstrated by Giemsa staining
(Tu et al., 2006).
Gao et al. (2007) investigated the effect of lidamycin in human hepatoma BEL-
7402 cells, showing cell multinucleation by Giemsa staining. Histone deacetylase
inhibitor showed visible morphological changes of U266 cells when subjected to Wright-
Giemsa staining (Ma et al., 2009). Morphological changes were observed in human
leukemia U937 and KG1 cells treated with guanosine 5'-triphosphate (Yazdanparast
et al., 2006).
Rosiglitazone treated cells displayed morphological characteristics of cell
differentiation, and more evident signs of differentiation were observed when combined
with all trans-retinoic acid (Huang et al., 2009a). Trans-resveratrol, a compound found in
grapes, berries, peanuts and red wine, exerted anticancer roles in T47D cells and caused
apoptosis, as demonstrated by Giemsa staining (Alkhalaf, 2007a).
The effects of arsenic trioxide and/or transforming growth factor-beta1 (TGF-β1)
of NB4 cells showed apoptotic morphological changes when stained with Giemsa (Liang
et al., 2009). Song and Zhang (2006) showed that parthenolide inhibited the proliferation
of human hepatocellular carcinoma cell line BEL-7402. The apoptotic effect of Berberine
on K562 cells showed inhibition of proliferation of K562 cells in a dose- and time-
dependent manner (Jin et al., 2009).
The results of the present study revealed that the methanolic extract of B. monnieri
leaves can exert a differential response against the oxidative stress-induced apoptosis in
different types of cells.
23
NUCLEAR CHANGES ASSOCIATED WITH APOPTOSIS
Apoptotic nuclei undergo typical changes including chromatin condensation,
peripheral marginalization, nuclear shrinkage and subsequent fragmentation (Cohen et al.,
2003).
The characteristic nuclear changes associated with apoptosis were quantified by
EtBr, PI and DAPI staining. The results of the study revealed that the number of cells
undergoing apoptosis increased in both untransformed and transformed cells subjected to
oxidative stress. The administration of B. monnieri leaf extract exerted no cytotoxic effect
in normal cells, but significant cytotoxicity was observed with cancer cells. These results
show that the leaf extract exhibited anticancer potential.
PROPIDIUM IODIDE STAINING
In this study, staining with propidium iodide showed that the methanolic extract of
B. monnieri leaves reduced apoptosis in untransformed cells, and not in cancer cells,
indicating the anti-apoptotic and anticancer property of the plant.
Propidium iodide intercalates into double-stranded nucleic acids. It is excluded by
viable cells but can penetrate cell membranes of dying or dead cells. Propidium iodide
staining is a routine parameter in most studies centering on apoptosis.
An ethanolic extract of Serenoae repentis fructus induced apoptosis in MDA
MB231 prostate, breast carcinoma, renal Caki-1, urinary bladder J82, colon HCT 116 and
lung A549 cancer cells, which was assessed by PI staining (Hostanska et al., 2007). The
methanolic and methane dichloride extract of the aerial parts of Larrea divariculata
exerted cytotoxic activity on MCF-7 cell line (Bongiovanni et al., 2008).
Platycodon grandiflorum extract induced cell apoptosis in SKOV3 ovarian cancer
cells as observed by PI staining (Hu et al., 2010). Panax quinquefolins leaf extract was
tested for cytotoxic activity in THP-1 leukemia cells using PI and showed significant
increase in apoptosis (Kitts et al., 2007). Huang et al. (2009b) showed that Antrodia
camphorate induced apoptosis in a concentration-dependent manner on human oral
cancer cells.
24
Some Chinese herbs, screened for antiproliferative properties in human HaCaT
keratinocytes model, induced cell arrest and death as assayed by PI (Tse et al., 2007).
Fenugreek seed polyphenolic extract treatment showed an increased hepatocyte viability
and reduced apoptotic nuclei in ethanol-induced hepatic injury in rats (Kaviarasan and
Anuradha, 2007). The apoptosis-inducing effects of Iris tectorum Maxim on MCF-7 and
C32 cell lines was shown by Fang et al. (2008).
An aqueous extract of Rhodiola imbricata rhizome significantly decreased the
proliferation of k-562 cells, as determined by PI staining (Mishra et al., 2007). In HepG2
(hepatocellular carcinoma), PI staining displayed the apoptotic cells when treated with
Trichosanthes kirilowii tuber extract (Shin et al., 2008). Ma et al. (2010) indicated that
Pinus massoniana bark extract, a mixture of flavonoids, selectively induced apoptosis in
HepG2 cells through caspase-dependent pathways.
Propidium iodide staining revealed that bovine aortic endothelial cells exposed to
methylglyoxal after DL-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione
biosynthesis, displayed features characteristic of apoptosis (Takahashi et al., 2010).
Gambogic acid showed significant effects in inducing apoptosis as assessed by PI staining
(Wang et al., 2010a). 4',7-diacetoxyapigenin increased apoptotic population as observed
by PI staining (Xu et al., 2010).
Brucea javanica oil induced apoptosis of T24 cells, which was analyzed by PI
staining (Lou et al., 2010b). Imatinib treatment resulted in an increased number of
apoptotic cells in a time- and dose-dependent manner on rat C6 glioma cell (Yang et al.,
2010b).
Liu et al. (2010c) reported that aeoniflorin reduced the rate of neuronal apoptosis
in corticosterone-induced primary cultures of rat cortical neurons as measured by PI
staining. Cai et al. (2008) examined the cytotoxic effect of emodin against human
pancreatic adenocarcinoma cell lines Mia Paca-2, BxPC-3, Panc-1, and L3 6pl. WST-1.
In the light of these reports, the results of PI staining in the present study are
highly validated.
25
ETHIDIUM BROMIDE STAINING
EtBr, which is an intercalating agent, can be used to visualize the changes that
occur in the nucleus during apoptosis. Apart from EtBr alone, the treatment with a
combination of acridine orange and ethidium bromide has been used as a reliable index of
cellular degeneration (Campos-Dapaz et al., 2008).
The ethanolic extract of Curcuma aromatica induced apoptosis and inhibited
angiogenesis in Ehrlich Ascites Tumor cells as assessed by EtBr staining (Thippeswamy
and Salimath, 2006). Acridine orange / ethidium bromide (AO/EB) staining indicated that
the cytotoxicity induced by 2',4'-dihydroxychalcone, one of the main components in
Herba oxytropis was mediated by apoptosis in human gastric cancer MGC-803 cells (Lou
et al., 2009). Analysis of the acridine orange / ethidium bromide staining revealed that the
crude extract of Solanum hydratum reduced cell proliferation and induced apoptosis in a
time- and dose-dependent manner in human colon cancer Colo205 cells (Hsu et al.,
2008).
Sanguinarine, a benzophenanthridine alkaloid derived from the root of
Sanguinaria canadensis, induced apoptosis in human cancer cells, which was assessed by
AO/EB staining (Hau et al., 2008). Sanguinarine, a benzophenanthridine alkaloid derived
from the root of Sanguinaria canadensis, induced apoptosis in human cancer cells, which
was assessed by AO/EB staining (Han et al., 2008). Hahnvajanawong et al. (2010)
reported the growth suppression by caged xanthones from Garcinia hanburyi in
cholangiocarcinoma KKU-100 and KKU-M156 cells.
The percentage of norepinephrine-induced apoptosis in the primary cultured rat
cardiac myocytes was decreased, as observed by EtBr staining assay, after pretreatment
with crocetin (Shen et al., 2009). The cytotoxicity of Pristimerin isolated from Maytenus
ilicifolia was evaluated in human tumor cell lines and in human peripheral blood
mononuclear cells. The cells stained with AO/EB, showed the occurrence of necrosis and
apoptosis in a concentration-dependent manner in HL-60 cell lines (Costa et al., 2008).
Cells treated with different concentrations of beta-sitosterol showed
morphological changes by acridine orange / ethidium bromide double staining in SGC-
7901 human stomach cancer cells (Zhao et al., 2009a). Fluorescence microscopy using
AO/EB staining indicated that kauren-19-oic acid induced apoptosis in HL-60 cell
26
cultures (Cavalcanti et al., 2009). The apoptotic morphology induced by cycloheximide in
human leukocytes was detected by AO/EB staining (Baskic et al., 2006).
In the present study, EtBr staining also confirmed that the methanolic extract of
B. monnieri leaves significantly protected the yeast and primary (untransformed) cells
from oxidant-induced damage. In the case of Hep2 (transformed) cells, the extract caused
an increase in the number of cells undergoing apoptosis.
DAPI STAINING
The nuclear changes that are characteristic of apoptosis can be monitored by
fluorescence microscope using DAPI staining. Epigallocatechin gallate, the major
component of polyphenols in green tea protected H9c2 cardiomyoblasts against hydrogen
dioxides-induced apoptosis as analysed by DAPI staining (Sheng et al., 2010).
The cell death in human hepatocellular carcinoma Hep 3B cells treated with the
ethanolic extracts of Euchresta formosana radix was identified as apoptosis using DAPI
staining (Hsu et al., 2007). Human breast cancer cells treated with the extracts of
Astrodaucus persicus also showed potential decrease in the cell proliferation by staining
with DAPI (Abdolmohammadi et al., 2008).
Phyllanthus niruri treatment, counteracted the changes and maintained normalcy
in hepatocytes against tertiary butyl hydroperoxide-induced apoptosis, as evidenced by
DAPI staining (Sarkar and Sil, 2010). Gallic acid induced apoptosis via caspase-3 and
mitochondrion-dependent pathways in vitro and suppressed lung xenograft tumor growth
in vivo in NCI-H460 cells as examined by DAPI staining (Ji et al., 2009).
Studies with kaempferol, a natural flavonoid, have reported that kaempferol had
anti-proliferation activities and induced apoptosis in human osteosarcoma cells (Huang
et al., 2010b). The effect of curcumin on apoptosis in Sk-hep-1 cells was investigated by
DAPI staining and the results indicated that curcumin was able to inhibit proliferation and
induce apoptosis in Sk-hep-1 cells (Wang et al., 2010b).
Aiyar et al. (2010) reported that 2,4,3',5'-tetramethoxystilbene, a chemically
modified herbal derivative of resveratrol, induced cell death by targeting Bax, as
evidenced by DAPI staining. Epigallocatechin-3-gallate effectively inhibited cellular
27
proliferation and induced apoptosis of the synovial sarcoma cells, as indicated by DAPI
staining (Sun et al., 2010).
Rajabalan (2008) observed that anticancer drugs caused leukemic lines to undergo
apoptosis, using DAPI staining. DAPI staining confirmed apigenin-induced DNA
condensation and damage in A549 cells (Lu et al., 2010b).
The results of DAPI staining in our studies indicate that the methanolic extract of
B. monnieri leaves brought the proportion of apoptosing cells down in untransformed
cells, while causing an increase in the number of apoptosing cells in transformed cells.
DNA FRAGMENTATION
Apoptosis is the predominant form of programmed cell death and occurs under a
variety of physiological and pathological conditions. One of the most prominent
biological features of apoptosis is nucleosomal DNA fragmentation. DNA fragments are
considered to be the hallmark of apoptosis and have been followed by many researchers
(Cheah et al., 2007). It is acknowledged to be a late event in apoptosis.
In the present study, in the yeast cells, eventhough apoptosis was observable by
both the assays of viability, as well as all the staining procedures, the characteristic DNA
fragmentation could not be observed.
It has been reported that the DNA laddering during apoptosis has been observed in
most, but not all, apoptotic systems (Cornillon et al., 1994). Several researchers have
reported that DNA fragmentation did not occur in S. cerevisiae cells undergoing apoptotic
cell death (Madeo et al., 2002; Mazzoni et al., 2003; Lundin et al., 2005).
DNA fragmentation is a late event in apoptosis. Large fragments of 50-300kb are
observed in the early stages and only later do they get further fragmented to 200bp
fragments or its multiples (Schliephacke et al., 2004). Jeon et al. (2002) have shown that
the process of apoptosis in yeast cells can be triggered by oxidative stress much earlier
than DNA fragmentation, which took over four hours to manifest. Similar observations
have been reported by Collins et al. (1997).
Methyl methane sulfonate did not cause DNA fragmentation upto 90 minutes of
exposure in rad52 mutant cells. In our study, an exposure interval of one hour was used,
28
which is the probable reason for the lack of observation of DNA fragmentation. This
observation of lack of DNA fragmentation in yeasts can also be explained by the fact that
S. cerevisiae chromatin structure has short or no linker DNA between nucleosomes
(Lowary and Widom, 1989; Madeo et al., 1999).
While DNA fragmentation was not observable in the S. cerevisiae cells,
significant DNA damage was observed in the primary cells and Hep2 cancer cells, which
was altered by the methanolic extract of B. monnieri, which results supported the trend
observed by the earlier assays. This observation further reiterated the fact that yeast cells
are unique in their DNA fragmentation pattern. On the other hand, it is also possible that
the DNA damage is observable in the primary and cancer cells, as they were exposed to
the oxidant and the leaf extract for 24 hours as against one hour for the yeast cells.
A methanolic extract of Oroxylum indicum induced DNA fragmentation in HL60
(promyelocytic leukemia) cells (Roy et al., 2007). A549 human non-small cell lung
cancer cells exposed to ethanol extract of Dunaliella salina showed significant DNA
fragmentation (Shen et al., 2008).
Inagaki et al. (2007) demonstrated that a compound purified from the ethyl acetate
extract of black soybean vinegar induced DNA fragmentation and the development of
apoptotic bodies in U937 cancer cells. Duchesnea indica phenolic fraction significantly
inhibited SKOV-3 cell proliferation and markedly induced apoptosis by characteristic
nuclear DNA fragmentation (Peng et al., 2008).
Clitocine, a natural biologically active substance isolated from the mushroom
Leucopaxillus giganteus, induced DNA fragmentation (Ren et al., 2008). Agarwala et al.
(2010) demonstrated the cytoprotective potential of mangiferin, against mercury chloride-
induced toxicity in HepG2 cell line using DNA fragmentation as an index. A progressive
increase in fragmented DNA was also observed in esophageal cancer cells (TE-2) treated
with the natural antioxidant gallic acid, which was isolated from the fruits of a medicinal
Indonesian plant (Faried et al., 2007).
DNA fragmentation was observed in human breast cancer cells treated with
cajanol, a novel anticancer agent from pigeonpea [Cajanus cajan (L.) Millsp.] roots (Luo
et al., 2010). The fragmented DNA ladder in d-gal-treated mice was inhibited by
troxerutin, a naturally occurring bioflavonoid (Liu et al., 2010d). Ethanolic extracts of
29
Sophora moorcroftiana seeds significantly inhibited SGC-7901 (gastric cancer) cell
proliferation and induced apoptosis by characteristic nuclear DNA fragmentation (Ma
et al., 2006).
When MDA-MB-231 breast cancer cells were treated with resveratrol, DNA
fragmentation was found to occur in a dose-dependent manner (Alkhalaf, 2007b). The
exposure of doxorubicin caused DNA fragmentation in cardiomyocytes (Du and Lou,
2008). DNA fragmentation was also observed in melanoma cells (B16F1) treated with
laurinterol (Kim et al., 2008b). A progressive increase in fragmented DNA was observed
in gastric cancer cells (NCL-N87) and bile duct cancer cells (YGIC-6B) treated with
paclitaxel combined with photodynamic therapy (Park et al., 2008).
Thus, from the results obtained, it can be inferred that B. monnieri leaves, alone or
in combination with chemotherapy drugs, could inhibit the proliferation of cancer cell
lines in vitro, through the mechanism of apoptosis, which suggest that B. monnieri is
likely to be a potential drug in the treatment of cancer.
PHASE III
EFFECT OF B. monnieri LEAF EXTRACT ON THE ANTIOXIDANT STATUS
OF GOAT LIVER SLICES SUBJECTED TO OXIDATIVE STRESS
The results obtained so far revealed that the leaves of B. monnieri were very
effective in protecting cellular biomolecules from oxidative damage. The extract was also
found to alleviate the oxidative stress imposed on untransformed cells, while causing an
increase in the extent of death in the transformed cells. As the next step, the influence of
the leaf extract was tested on the antioxidant status of cells maintained in their tissue
architecture. For this, thin slices of liver tissue exposed to the oxidant in the presence and
the absence of the extract were used.
Liver slices are an intermediate between liver cells and isolated organs. A major
advantage of hepatic slices compared to isolated hepatocytes is the lack of disruption of
cell-to-cell contacts as occurring during the hepatocyte isolation procedure. With liver
slices, the normal tissue architecture, cell heterogeneity and cell-cell interactions are
maintained; the native cell types and integrity of the organ remain intact (Scott, 2005).
30
In the present study, the protective effects of the B. monnieri leaf extract in vitro
against hydrogen peroxide-induced oxidative stress were evaluated. The enzymic and
non-enzymic antioxidants were analyzed in the slices, and the results are discussed below.
ENZYMIC ANTIOXIDANTS
The enzymic antioxidants analysed were SOD, CAT, POD and GST.
SUPEROXIDE DISMUTASE
SOD is considered as the first line of defense against the deleterious effects of oxy
radicals in the cell by catalyzing the dismutation of superoxide radicals to H2O2 (Chandra
et al., 2007).
The activities of SOD in the slices subjected to oxidative stress decreased
significantly. The exposure to the extract did not improve the values. The extract, by
itself, did not decrease the activity of SOD, when compared to the control group.
Several studies in the literature have reported a modulation in SOD activity after
exposure to herbal extracts and components.
Intragastric administration of a polyphenolic extract from Pinus koraiensis seed
increased the levels of SOD in vivo (Su et al., 2009). The activity of SOD increased
significantly in the liver of mice in Kigelia africana, Calotropis procera, Hibiscus
sabdariffa and Alchornea cordifolia extract treated groups and counteracted the
paracetamol-induced oxidative stress (Olalye and Rocha, 2008).
Flavonoids extracts from Inula britannica inhibited oxidative stress in rat aorta
after balloon injury and the activity of SOD was increased significantly in both serum and
vascular tissues (Zhang et al., 2009). An increased level of SOD was reported in a
glycoportein isolated from Rhus verniciflua in the liver of hyperlipidemic mice (Oh et al.,
2006).
The green pods of Acacia nilotica caused a significant increase in the levels of
SOD in the liver, lungs, kidneys and blood in CCl4 treated rats (Singh et al., 2009b).
Preincubation of primary cultured rat cardiac myocytes with crocetin remarkably
prevented the decrease in SOD activity (Shen et al., 2009).
31
In the present study however, B. monnieri leaf extract failed to improve the
activities of superoxide dismutase. The reason for this non-response is presently unclear.
CATALASE
By catalyzing the conversion of H2O2 to O2 and H2O, catalase has been shown to
shift the redox balance towards antioxidant and leading to increased antioxidant capacity,
which may alleviate the detrimental effect of H2O2 (Ren et al., 2004).
In the present study, the activity of catalase was found to decrease on exposure of
the slices to the oxidant hydrogen peroxide. This may be due to the utilization of the
enzyme in the detoxification of hydrogen peroxide. However, on administration of the
extract, the activity of the enzyme was found to increase significantly.
Catalase decomposes hydrogen peroxide and helps to protect from highly reactive
hydroxyl radicals. Wang et al. (2008a) showed that, in Douchi extracts treated,
cholesterol-fed rats, CAT activity in the liver, increased significantly compared to the
negative control group. Aphanamixis polystachya bark extract supplementation resulted in
dose-dependent and significant improvement in hepatic catalase level in rats (Krishnaraju
et al., 2009).
Sumnath and Rana (2006) reported that the pretreatment with hydroalcoholic
extracts of Tarxacum officinale roots improved the levels of catalase and peroxidase in
the liver of rats intoxicated with CCl4. The ethanol:water (7:3) extract of Coriandrum
sativum increased the activities of SOD, CAT and GPx in the liver of CCl4 treated rats
(Sreelatha et al., 2009).
Our results clearly show that B. monnieri leaf extract increased the activities of
both CAT and SOD, which will bring about effective and complete scavenging of
superoxide-peroxide oxidants.
PEROXIDASE
The treatment with the oxidant decreased the activity of peroxidase in the liver
slices. The activity was reversed by the administration of the leaf extract.
32
The antioxidant enzymes CAT and GPx have been reported to protect SOD
against inactivation by H2O2 (Parthasarathy et al., 2006). The oral administration of
methanolic extract of Phyllanthus significantly elevated the activity of hepatic glutathione
peroxidase during CCl4 stress in rats (Lee et al., 2006). Glutathione peroxidase activity in
the kidney of cholesterol-fed rats increased significantly compared with the negative
control group when treated with Douchi extract (Wang et al., 2008a).
Li et al. (2010b) reported that saponins from Panax japonicus protected against
alcohol-induced hepatic injury in mice by up-regulating the expression of GPx, and SOD.
The statistically significant losses in the activities of SOD, CAT and Px in
acetaminophen-induced liver damage in mice was counteracted by all the tested plants
(Kigelia africana, Calotropis procera, Hibiscus sabdariffa and Alchornea cordifolia)
except Calotropis procera as reported by Olalye and Rocha (2008).
The oral administration of Coscinium fnestratum stem extract in graded doses
caused a significant increase in peroxidase activity in the liver and kidney of
streptozotocin-nicotinamide induced type II diabetic rats (Punitha et al., 2005). The
treatment with the alcoholic extract of Momordica charantia increased the activity of
peroxidase in the liver and brain tissues of ammonium chloride treated rats (Thenmozhi
and Subramanian, 2010).
From the available literature, it is obvious that the treatment with the extract
significantly increased the activities of SOD, CAT and POD, which form the first line of
defense against ROS.
GLUTATHIONE S-TRANSFERASE
Glutathione S-transferases are a group of enzymes that catalyse the conjugation of
GSH to a variety of electrophiles, thereby decreasing its reactivity with cellular
components (Bickers and Athar, 2006).
The activities of GST were slightly decreased on exposure to H2O2 but the leaf
extract did not alter the activity of GST in the oxidant exposed slices.
The ethanolic extract of Hibiscus sabdariffa increased the activity of hepatic GST
in sodium arsenate-induced oxidative stress in rats (Usoh et al., 2005). Lima et al. (2005)
33
reported a significant increase of liver GST activity in rats and mice of sage drinking
groups.
Lee et al. (2007a) showed that pre-treatment of rats with tea seed oil (Camellia
oleifera Abel.) could increase the activities of glutathione peroxidase, glutathione
reductase and glutathione S-transferase in liver when compared with CCl4-treated group.
In our study, however, the activities of GST in the various treatment groups did not vary
significantly in the goat liver slices.
Our results show that on exposure to the oxidant H2O2, the activities of the major
enzymic antioxidants were significantly affected, which was improved by the
administration of B. monnieri leaf extract.
NON-ENZYMIC ANTIOXIDANTS
ROS are continuously produced in the cells, which are regulated by a variety of
cellular defenses consisting of enzymic and non-enzymic antioxidants. The non-enzymic
antioxidants analysed were vitamins C, E, A and reduced glutathione. The results
obtained are discussed below.
VITAMIN C
Vitamin C, a major ubiquitous non-enzymic antioxidant, has a crucial role in
scavenging several reactive oxygen species. At physiological concentrations, vitamin C is
a potent free radical scavenger in the plasma, protecting cells against oxidative damage
caused by ROS (Pavana et al., 2007).
In the present study, the levels of vitamin C decreased in the goat liver slices
exposed to the oxidant H2O2, which was effectively improved by the leaf extract.
Sumathi and Padma (2008) reported that Withania somnifera extracts effectively
counteracted the damage produced by H2O2 and replenished vitamin C levels in hydrogen
peroxide induced goat liver slices. Jia et al. (2009) reported a significant increase in the
plasma and liver antioxidant status in streptozotocin-induced diabetic rats when treated
with the polysaccharides from Ganoderma lucidum root extract.
34
Asparagus racemosus root powder administration significantly elevated the
decreased activities of hepatic SOD, CAT and vitamin C levels in hypercholesterolemic
rats (Visavadiya and Narasimhacharya, 2009). Exposure to the oxidant CCl4 significantly
depleted vitamin C, which was counteracted effectively by the oral administration of
curcumin (Kamalakkannan et al., 2005).
In the present study also, the extract of B. monnieri leaves effectively counteracted
the damage produced by H2O2 to liver slices and replenished the vitamin C levels.
VITAMIN E
Vitamin E is one of the major chain breaking lipophilic antioxidant, within the cell
membrane, where it protects membrane fatty acids from peroxidation. Vitamin E has been
effective in blocking peroxyl mediated chain reaction and in combination with ascorbate,
it scavenges SO� in the lipid membrane (Manikandan and Devi, 2005).
A significant reduction in the levels of vitamin E was observed in the slices
exposed to the oxidant, which were improved on exposing the oxidant exposed slices with
the B. monnieri leaf extract.
The levels of vitamin E in the liver and kidney of streptozotocin-induced type II
diabetic rats improved by the administration of alcoholic extract of Coscinium
fenestratum (Punitha et al., 2005). The co-administration of vitamin E with the extract of
Withania somnifera, Ocimum sanctum and Zingiber officinalis elevated the antioxidant
status in the cardiac, skeletal, hepatic tissues, cerebrum and cerebellum of oxidative stress
imposed rats (Misra et al., 2005).
The effect of atrazine in the liver of atrazine-induced male Wistar rats was
ameliorated by the administration of vitamin E (Singh et al., 2010). Thus, it is clear from
the present study that the increase in the vitamin E levels by the leaf extract of
B. monnieri proves its antioxidant potential.
VITAMIN A
The exposure of the slices to the oxidant decreased the levels of vitamin A. On
administration of B. monnieri leaf extract the levels increased significantly when
compared to the control.
35
Ragavan and Kumari (2006b) reported that the oral administration of Trigonella
arjuna reversed the levels of vitamin A to normal in the liver and kidney of diabetic rats.
Vitamin A administration has been reported to prevent hepatic injury caused by CCl4
treatment in rats (Noyan et al., 2006).
The chemopreventive potential of vitamin A and β-carotene during early
hepatocarcinogenensis induced by 5-azacytidine in Wistar rats has been demonstrated
(Sampaio et al., 2007). Consumption of water cress (cruciferous vegetable) can decrease
the damage to DNA and cause a modification in the antioxidant status by increasing
carotenoid concentration in healthy adults (Gill et al., 2007).
Thus, it is perceivable from these studies, that an increase in the levels of vitamin
A by the leaf extract proves the ability of the extract to increase the antioxidant status in
the tissues.
REDUCED GLUTATHIONE
GSH is an important intracellular defense against damage by ROS. The reduced
form of glutathione is necessary to maintain the normal reduced state of cells so as to
alleviate all the injurious effects of oxidative stress (Ahmad et al., 2009).
In the present investigation, a marked depletion in the levels of GSH was observed
when the slices were exposed to the oxidant. The levels improved when the slices were
co-treated with the extract.
Administration of chloroform and methanol extracts of Ichnocarpus frutescens
significantly increased the level of glutathione in the liver of paracetamol-treated rats in a
dose-dependent manner (Dash et al., 2007). Aqueous extract of Phyllanthus amarus
treated rats showed a significant decrease in plasma LPO and a significant increase in
plasma vitamin C, uric acid and GSH levels, and GPx, CAT and SOD activities (Karuna
et al., 2009).
Treatment with Ocimum basilicum and Trigonella foenum-graecum showed
significant increase in reduced glutathione levels in the H2O2 and CCl4 intoxicated goat
liver (Meera et al., 2009). In the present investigation, treatment with the extract
significantly increased the activities of GSH-dependent enzymes like GST and POD,
36
which further strengthens the fact that the extract improves the antioxidant status of the
liver slices exposed to oxidative stress.
Thus, the results obtained revealed that the methanolic extract of B. monnieri
leaves influences favourably the antioxidant status of oxidatively stressed goat liver
slices.
EFFECT OF B. monnieri LEAF EXTRACT ON THE ANTIOXIDANT STATUS
OF EXPERIMENTAL ANIMALS SUBJECTED TO OXIDATIVE STRESS
The in vivo assessment of drug metabolism in experimental animals provides the
most physiologically relevant test system and gives information that relates to the
complexity of sequential and parallel routes of metabolic clearance, not only by multiple
enzymes, but also by multiple organs. The results obtained in the in vitro studies showed
that B. monnieri leaves possessed antioxidant and hepatoprotective activity. Many factors
such as signaling pathways involving hormones, regulatory substances and other
molecules, physiological nature and multiple organ systems play an important role in
influencing the in vivo environment. To confirm the results obtained in vitro, in vivo
studies were carried out using male Wistar rats. Oxidative stress was induced in the
animals by the administration of CCl4 following the induction of CYP2E1 with alcohol
pre-treatment.
Ethanol-induced oxidative stress plays a major role in the mechanisms by which
ethanol produces liver injury (Cederbaum et al., 2009). CCl4, a widely used
hepatotoxicant, is biotransformed by cytochrome P450 systems to produce the
trichloromethyl and trichloromethyl peroxy radicals that causes lipid peroxidation and
thereby, produce liver damage (Sisodia and Bhatnagar, 2010).
Silymarin, an antioxidant flavonoid complex, has long been used in the treatment
of liver diseases. It has been demonstrated that silymarin acts as an antioxidant, reducing
free radical-mediated damage in tissues and inhibiting lipid peroxidation. It has been
reported as having multiple pharmacological activities including antioxidant,
hepatoprotectant, anti-inflammatory, antibacterial, antiallergic, antiviral and
antineoplastic effects (Upadhyay et al., 2007).
37
In the present investigation, male Wistar rats were exposed to oxidative stress and
the effect of the leaf extract on the stress was studied. Liver and kidney were used to
assess the antioxidant potential of B. monnieri. Liver is one of the tissues showing a high
rate of free radical generation with high metabolic capacity and detoxifying capacity. The
other organ, kidney is an excretory organ and is relevant for measuring biochemical
changes and oxidative damage. Thus, in the present study, the oxidative status of both the
organs was studied in rats exposed to CCl4. Ethanol pretreatment sensitizes the liver to
toxic challenge by CCl4 so as to potentiate the liver damage.
The extent of liver damage in different treatment groups were evaluated by the
activities of the liver marker enzymes and the lipid profile in the serum. The results
obtained are discussed below.
SERUM MARKER ENZYMES
Cellular damage exhibits good correlation with enzyme leakage (Sherawat and
Sultana, 2006). Serum AST, ALT, ALP and γ-GT are the most sensitive markers
employed in the diagnosis of hepatic damage. The increase in the activities of these
enzymes in serum and subsequent fall in the tissue might be due to the leakage of these
cytosolic enzymes into the circulatory system, resulting from hepatocellular damage
during ethanol administration. This is indicative of the onset of hepatocellular damage
due to liver dysfunction and disturbance of the biosynthesis of these enzymes, with
alteration in the permeability of liver membrane (Pradeep et al., 2010).
The serum markers analyzed were aspartate transaminase (AST), alanine
transaminase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT).
A significant increase in the activities of the serum markers were observed in the groups
treated with ethanol both with and without CCl4.
Opoku et al. (2007) showed that the administration of Rhoicissus tridentata
extracts after CCl4 intoxication in rats resulted in significantly reduced concentrations of
ALT and ASP. CCl4-induced hepatotoxicity in rats, as judged by the raised serum
enzymes, was prevented by pretreatment with the aqueous and methanolic extracts of
Phyllanthus niruri, demonstrating their hepatoprotective action (Harish and
Shivanandappa, 2006).
38
The partially purified petroleum ether extractable fraction of the whole plant
Aerva lanata restored the elevated activities of liver marker enzymes against liver damage
induced by carbon tetrachloride (CCl4) in Sprague Dawley rats (Nevin and Vijayammal,
2005). Oral pretreatment with the chloroform extracts of Terminalia catappa significantly
reduced the increased serum AST and ALT activities in CCl4 treated rats (Tang et al.,
2006).
Oral administration of aqueous suspension of dried latex of Calotropis procera
produced a dose-dependent reduction in the serum levels of liver enzymes AST and ALT
against CCl4 induced hepatotoxicity in rats (Padhy et al., 2007). The treatment with the
alkaloid fraction of Hygrophila auriculata leaves showed a significant decrease in AST,
ALT and ALP activities in serum when compared with CCl4 treated rats (Raj et al.,
2010).
Pretreatment with Wild Panax ginseng C.A. Meyer for 4 weeks completely
abrogated increases in the ALT, AST and LPO levels in the rats, when challenged with
benzo[α]pyrene (Gum et al., 2007). Shim et al. (2010) have reported that ginsan, a
polysaccharide extracted from Panax ginseng, on CCl4-induced liver injury in rats,
markedly suppressed the serum ALT and AST levels.
Pre-treatment and post-treatment with dehydrocavidine, a main active ingredient
of Corydalis saxicola Bunting, significantly prevented increases in serum enzymatic
activities of ALT, AST and ALP in the liver of CCl4 intoxicated rats (Wang et al., 2008b).
Oral administration of curcumin and BDMC-A [bis-1,7-(2-hydroxyphenyl)-hepta-1,6-
diene-3,5-dione] to CCl4-induced rats for a period of three months significantly decreased
the levels of serum marker enzymes AST, ALP and γ-GT (Kamalakkannan et al., 2005).
Resveratrol treatment was able to mitigate hepatic damage induced by acute
intoxication of CCl4 and showed pronounced curative effect and deviated serum
enzymatic variables in rats (Fan et al., 2009). Kim et al. (2005) have reported that the
pretreatment of momordin Ic and oleanolic acid obtained from Kochiae fructus fruit
significantly lowered the increased activities of AST, ALT and γ-GT in CCl4 treated rats.
Rutin prevents changes in the activities of ALT and AST in the serum, liver and heart,
indicating the protective effect of rutin against the hepatic and cardiac toxicity caused by
streptozotocin (Fernandes et al., 2010).
39
The hepatoprotective effects of kahweol and cafestol, coffee-specific diterpenes,
on CCl4-induced liver damage revealed an increase in the serum levels of hepatic enzyme
markers ALT and AST in a dose-dependent manner (Lee et al., 2007b). Yan et al. (2006)
showed that administration of chitosan oligosaccharide, d-glucosamine and N-acetyl-d-
glucosamine on CCl4-induced hepatotoxicity in mice, induced marked change in serum
AST and ALT activities.
In the present study, the administration of B. monnieri leaf extract caused a
significant decrease in the activities of serum marker enzymes (AST, ALT, ALP and
γ-GT) against ethanol-CCl4 induced toxicity, indicating its protective effect. The
protective effect of B. monnieri leaf extract was comparable to that of silymarin, a
standard hepatoprotectant.
LIPID PROFILE
The results of the present study revealed that, there was a significant increase in
the levels of serum cholesterol, triglycerides, free fatty acids and phospholipids in the
alcohol treated group and the levels were further increased in CCl4 and alcohol treated
group. This shows that these lipids are secreted from the liver at an increased rate. The
administration of B. monnieri leaf extract was effective in counteracting the oxidative
stress induced damage by decreasing the serum lipid levels of rats.
Our results are supported by a number of research findings, where plant extracts
have exhibited hypolipidemic effects. Aqueous extract of propolis, a resinous wax-like
beehive product, significantly reduced the levels of serum and tissue triglycerides, serum
cholesterol, total and esterified cholesterol in rat tissue (Bhadauria et al., 2008). An
aqueous extract of Ajuva iva lowered plasma cholesterol and triglyceride levels in
streptozotocin induced diabetic rats (El-Hilaly et al., 2006).
An ethanolic extract of Beta vulgaris roots exhibited significant dose-dependent
hepatoprotective activity against CCl4-induced hepatotoxicity in rats, by showing a
decline in the levels of serum markers, namely cholesterol and triglyceride (Agarwal
et al., 2006). The significant increase in the levels of cholesterol, triglycerides,
phospholipids and free fatty acids in the liver and kidney, caused by the administration of
streptozotocin in rats were brought down to normalcy on treatment with Aloe vera leaf gel
(Rajasekaran et al., 2006). The malathion-treated rats and the malathion plus vitamin-
40
treated group had significantly lower serum triglyceride levels when compared to the
control (Kalender et al., 2010).
ANTIOXIDANT STATUS IN THE LIVER AND KIDNEY OF RATS TREATED
WITH B. monnieri EXTRACT AFTER EXPOSURE TO CCl4
The antioxidant status in the experimental animals was assessed by analyzing the
enzymic and non-enzymic antioxidants in the liver and kidney of the rats exposed to
ethanol-CCl4 treatment.
ENZYMIC ANTIOXIDANTS
Metabolism of ethanol by CYP2E1 results in the production of ROS, leading to
oxidative stress (Chen et al., 2008; Haorah et al., 2008). Ethanol-induced oxidative stress
is the result of the combined impairment of the antioxidant status and production of ROS
(Reddy et al., 2009). Hence, it was felt imperative to study the effects of ethanol and CCl4
and the protective effect of the extract of B. monnieri leaves.
The enzymic antioxidants, namely SOD, CAT, POD, GR and GST, were analyzed
in the liver and the kidney of the experimental rats.
A significant reduction in the activities of all the enzymic antioxidants was
observed when treated with ethanol alone or in combination with CCl4. This effect was
counteracted by the administration of the methanolic extract of B. monnieri leaves and the
standard oxidant silymarin.
There are several reports published in the literature, wherein the antioxidant status
of organs is modulated by herbal extracts and compounds. These reports are in
accordance with our results and some of the salient ones are discussed below.
SOD, CAT and GPx constitute a mutually supportive team of enzymes, which
provide defense against the intermediates of dioxygen. CAT and GPx protect SOD
against inactivation by H2O2 and SOD protects CAT and GPx against superoxide anion
(Parthasarathy et al., 2006).
There was a marked decrease in the percentage inhibition of superoxide
dismutase, catalase and the level of GSH in the liver of CCl4 treated rats when compared
41
with the control group. However, the percentage inhibition of SOD, CAT and the level of
GSH were significantly increased on administration of the aqueous extract of Strychnos
henningsii in a dose-dependent manner (Oyedemi et al., 2010). The administration of the
aqueous extract of the bark of Terminalia arjuna significantly elevated the reduced SOD,
CAT and GST activities in the liver and kidney of CCl4 challenged mice (Manna et al.,
2006).
The depletion in the activities of the antioxidant enzymes SOD, CAT and GPx
when treated with ethanol, was prevented by the methanolic extract of the root of Opuntia
ficus indica f. inermis in rat stomach tissues (Alimi et al., 2010). Administration of hydro
alcoholic extract of Nelumbo nucifera seeds to Wistar rats prior to CCl4 treatment caused
a significant dose-dependent increase in the level of SOD and CAT (Rai et al., 2006).
The co-administration of the ethanol extract of Aquilegia vulgaris or silymarin
resulted in a significant increase in the hepatic antioxidant enzyme activities, which was
significantly reduced after CCl4 administration in male Wistar rats (Jodynis-Liebert et al.,
2009). An ethanolic extract of the whole plant of Amaranthus spinosus was found to
increase the activities of SOD and CAT in CCl4 induced hepatotoxicity in rats (Zeashan
et al., 2008).
When the extract of Pleurotus ostreatus was used to treat rats with CCl4-induced
toxicity, it enhanced the mean activities of CAT, SOD, GPx and GST in kidneys, heart
and brain of rats (Jayakumar et al. 2008). Pretreatment of rats with Cystisus scoparius
plant extract caused a significant increase in the SOD, CAT, GPx, GST and GR activities
in the liver against CCl4 exposure (Raja et al., 2007).
Potato peel extract restored CCl4-induced altered antioxidant enzyme activities in
the liver to control levels (Singh et al., 2008). Aly et al. (2010) demonstrated that vitamin
C treatment to chlorpyrifos intoxicated mice decreased GST activity and normalized
CAT, SOD and G6PD activities in the liver. The co-administration of Kolaviron, a
biflavonoid complex from Garcinia kola seeds, during ethanol treatment ameliorated
hepatic SOD and GST activities (Adaramoye et al., 2009).
All these studies support our results, where the co-treatment of B. monnieri leaf
extract improved the antioxidant status and counteracted CCl4 and ethanol mediated
toxicity in vivo.
42
NON-ENZYMIC ANTIOXIDANTS
The levels of non-enzymic antioxidants such as vitamin C, vitamin A, vitamin E,
GSH and protein thiols were assessed in the liver and kidney of rats treated with CCl4 and
ethanol.
On treatment with alcohol and CCl4 the levels of vitamins C, A, GSH and protein
thiols were found to decrease. Their levels increased on co-treatment with the extract in
both the tissues (liver and kidney). In the case of vitamin E, CCl4 treatment caused a
significant increase in the levels when compared to the untreated controls in the liver. But
the values decreased to a significant extent in the kidney. Vitamin E is a membrane bound
antioxidant. Alcohol treatment might have destroyed the membranes leading to an
increase in the level of vitamin E in the alcohol treated group compared to control.
The methanolic extract of Solanum nigrum berries improved the GSH levels in the
gastric mucosa of rats treated with aspirin to induce ulceration (Jainu and Devi, 2006).
The oral administration of the ethanolic extract of Terminalia arjuna stem bark caused a
significant improvement in the decreased levels of GSH, vitamins C, A and E, total
sulfhydryl and non-protein sulfhydryl in the liver and kidney of alloxan-treated diabetic
rats (Ragavan and Kumari, 2006b). An ethanolic root extract of Tephrosia purpurea
enhanced the levels of vitamins C, E and GSH in DMBA induced hamster buccal pouch
carcinoma (Kavitha and Manoharan, 2006).
Polychlorinated biphenyls significantly diminished the levels of non-enzymatic
antioxidants, vitamins C and E, in a dose- and time-dependent manner in cultured rat
Leydig cells (Murugesan et al., 2008). A significant decrease in the non-enzymic
antioxidants (total sulphydryl groups, reduced glutathione, vitamin C and vitamin E) was
observed in the liver of Cd intoxicated rats, which was reverted by the administration of
diallyl tetrasulfide (Pari et al., 2007). Organotellurium compound induced the in vitro
oxidative stress in the cerebral cortex of rats and provoked a reduction of protein thiol
groups (Penz et al., 2009).
Thus, from the present study, it is clear that the levels of enzymic and non-
enzymic antioxidants, which were reduced on exposure to oxidative stress, were
counteracted on treatment with the leaf extract.
43
The results obtained and the trends observed in both the in vitro (liver slices) and
in vivo (experimental animals) systems were quite similar except SOD and GST activities
in the liver slices. The activities of these enzymes did not show significant elevation in
liver slices, which may be due to the reason, that one hour exposure of the extract to the
oxidant-treated slices, was not sufficient to implicit a siginifcant response.
This observation validates the use of tissue slices as a viable alternative to the use
of live animals in research. The use of such a system would effectively bring down the
number of animals used in biomedical research, at the same time providing valuable clues
about the events occurring in vivo.
EXTENT OF LIPID PEROXIDATION
Lipid peroxidation in biological systems has been considered as one of the major
mechanisms of cell injury in aerobic organisms subjected to oxidative stress.
In the present study, ethanol caused an increase in the extent of LPO in the liver,
which was further increased on CCl4 challenge. The methanolic extract of B. monnieri
leaves brought down the level of LPO and the effect was found to be better compared to
standard antioxidant silymarin.
The treatment with an aqueous extract of Strychnos henningsii was able to lower
the rise in TBARS level dose-dependently in the liver of rats treated with CCl4 (Oyedemi
et al., 2010). The Kigelia africana, Calotropis procera, Hibiscus sabdariffa and
Alchornea cordifolia plant extracts statistically reduced the production of TBARS in a
concentration-dependent manner in the liver of rats with all the tested pro-oxidant-
induced oxidative stresses (Olalye and Rocha, 2007). Alimi et al. (2010) reported that the
methanolic extract of Opuntia ficus indica f. inermis inhibited the increase of MDA in rat
stomach tissues.
The aqueous extract of Mangifera indica L. fruit and gallic acid protected the
isolated rat hepatocytes against cumene hydroperoxide-induced LPO (Pourahmad et al.,
2010). The rats treated with the aqueous extract of Phyllanthus amarus showed a
significant decrease in plasma LPO (Karuna et al., 2009). Gymnema sylvestre extract
showed a significant hepatoprotective effect in irradiated mice by significantly lowering
44
the radiation induced LPO, measured as the levels of malondialdehyde (Bhatia et al.,
2008).
The administration of a hydro alcoholic extract of Nelumbo nucifera seeds to
Wistar rats caused a significant decrease in the level of thiobarbituric acid reactive
substances, when compared to CCl4 treated control in both liver and kidney (Rai et al.,
2006). The ethanolic extract of whole plant of Amaranthus spinosus showed potent
hepatoprotective activity against carbon tetrachloride induced hepatic damage in rats and
significantly altered malondialdehyde levels (Zeashan et al., 2008).
The oral administration of Arachniodes exilis at different doses resulted in
significant improvement of the levels of malondialdehyde in the liver of mice (Zhou et al,
2010). The treatment with repeated doses of either garlic or onion juices could restore the
concentration of thiobarbituric acid reactive substances to their normal levels in the
plasma, liver, brain, testes and kidney of the alloxan-induced diabetic rats (El-Demerdash
et al., 2005).
The treatment with the protein isolate of Phyllanthus niruri L. significantly altered
the increased lipid peroxidation in CCl4 induced liver changes to almost normal
(Bhattacharjee and Sil, 2007). The whole plant of Piper nigrum, Myristica fragrans
(Chatterjee et al., 2007) and holy basil (Juntachote et al., 2007) decreased the formation
of TBARS in the liver of experimental rats.
RADICAL SCAVENGING ACTIVITY
The overall antioxidant status of tissues treated with alcohol and CCl4 in the
presence and absence of the B. monnieri leaf extract was assessed by the ability of the
liver homogenate to scavenge the free radical DPPH.
The DPPH scavenging effect was not significantly high in alcohol and CCl4
treated groups as well as in the groups treated with the extract. Silymarin treated groups
alone showed slightly higher values compared to the controls.
An aqueous extract from Choerospondias axillaris fruit showed a high antioxidant
effect, especially scavenging of DPPH anions in d-galactose induced mouse aging model
(Wang et al., 2008c). The methanolic extracts of green tea and Ficus bengalensis
45
exhibited antioxidant property as reflected by their higher hydrogen donating ability
(Manian et al., 2008).
Telluroacetylenes showed effect of scavenging DPPH radicals in sodium
nitroprusside-induced oxidative damage in mouse brain (Souza et al., 2009). Menthe
spicata extract exhibited good antioxidant activity compared to the synthetic antioxidant,
butylated hydroxy toluene (Kannat et al., 2007).
From the results of the present study, it is clear that the treatment with B. monnieri
leaf extract improved the antioxidant status in the liver and kidney of rats and can render
protection against alcohol-CCl4 induced toxicity.
Therefore, the results of this study show that B. monnieri can be proposed to
protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective
effect might be correlated with its antioxidant and free radical scavenging effects.
PHASE IV
Various phytochemical components especially polyphenols (such as flavonoids,
phenolic acids, tannins, etc.) are known to be responsible for the free radical scavenging
and antioxidant activities of plants. Phenolic substances possess many biological effects.
These effects are mainly attributed to their antioxidant activities in scavenging free
radicals, inhibition of peroxidation and chelating transition metals (Nickavar et al., 2007).
The preliminary phytochemical analysis of the leaves of B. monnieri revealed the
presence of alkaloids, phenolics, flavonoids and saponins. To confirm the nature of the
active component present in B. monnieri leaves, spectral analysis (UV, HPLC, HPTLC,
IR and GC-MS) were carried out, which confirmed the presence of alkaloids, phenolics,
flavonoids and saponins.
Phytochemical screening of the leaves and roots of C.alata revealed the presence
of alkaloids, carbohydrates, tannins, saponins, phenols, flavonoids, anthraquinones and
cardiac glycosides (Makinde et al., 2007). Phytochemical screening of Euphorbia
heterophylla Linn, showed that the crude plant material contained some secondary
metabolites such as saponins, flavonoids and tannins (Falodun et al., 2008). Tridax
46
procumbens has antioxidant activity which was established to correspond to the amount
of total phenolic content of the plant samples (Habila et al., 2010).
Ramesh et al. (2009) reported that B. monnieri contains alkaloids (nicotine,
brahmine, herpestine), saponins (hersaponin, betulic acid, bacosides A, B, C and D) and
other chemicals like stigmastanol, β-sitosterol and stigmasterol. TLC analysis of the
Acacia nilotica (L) wild extract showed two spots at Rf values 0.48 and 0.64 (Singh and
Arora, 2009). Thin layer chromatographic analysis of ethanolic extract of B. monnieri
plant showed the presence of alkaloids and natural lipids (Sengupta et al., 2008). One of
the most common biological properties of alkaloids is their toxicity against cells of
foreign organisms. These activities have been widely studied for their potential use in the
elimination and reduction of human cancer cell lines. Alkaloids which are one of the
largest groups of phytochemicals in plants have amazing effects on humans and this has
led to the development of powerful pain killer medications (Igbinosa et al., 2009).
The UV spectrum of the flowers of Abelmoschus manihot showed peaks at 204,
210, 228, 262, and 277nm (Lai et al., 2007). Luteolin, a flavonoid widely occurring in
many medicinal plants, was quantified by Srinivasa et al. (2004) in three important herbal
drugs namely fruit of Cuminum cyminum, whole plant of B. monnieri and the flower of
Achillea millefolium by HPTLC.
The chromatographic analyses of Spirulina organic extracts with TLC and HPLC
showed that carotenoids, chlorophyll-derived and phenol compounds were the main
constituents (El-Baky et al., 2008). A simple and sensitive reversed phase high
performance liquid chromatographic (HPLC) procedure identified the presence of Bacopa
saponins present in the extracts of the medicinal plant, B. monnieri (Ganzera et al., 2004;
Deepak et al., 2005; Murthy et al., 2006). Zehl et al. (2007) analysed the presence of two
dammarane-type triterpenoid saponins from B. monnieri by the application of a variety of
MS techniques.
These results are in accordance with the results of the present study, where the
presence of alkaloids, phenolics, flavonoids and saponins has been confirmed.
The outcome of the study, thus, provides a significant contribution in establishing
the antioxidant properties of B. monnieri leaves, which includes not only radical
scavenging properties, but also biomolecular protection. This effect is also observed
47
in vivo, wherein no toxic effects were observed. The bioactivity of antioxidant and
anticancer property of the plant is possibly due to the presence of phytochemicals.
Thus, the present study emphasizes the medicinal value of B. monnieri leaves,
against oxidant-induced damage in vitro and in vivo.
The results obtained in the present study and the conclusions drawn from them are
presented in the next chapter.