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).

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 Bacopas 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 apoptosismodulating 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

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.

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 alcoholwater 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 -carotene linoleic acid assays (Tepe et al., 2005).

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

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

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.

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.

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 -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.

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 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 -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 dosedependent 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-4thiaflavans, 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.

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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.

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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 M 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 STRESSINDUCED 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).

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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 apoptosisinducing 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.

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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).

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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 As2O3induced 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 -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).

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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-MB231 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

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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 B16F10 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).

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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 WrightGiemsa 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 BEL7402 cells, showing cell multinucleation by Giemsa staining. Histone deacetylase inhibitor showed visible morphological changes of U266 cells when subjected to WrightGiemsa 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-1) 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 timedependent 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.

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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.

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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.

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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 SGC7901 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

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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

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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,

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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 chlorideinduced 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

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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).

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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).

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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.

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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)

32

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.

33

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.

34

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 -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,

35

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 antibacterial, including antioxidant, antiviral and

hepatoprotectant,

anti-inflammatory,

antiallergic,

antineoplastic effects (Upadhyay et al., 2007).

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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 -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 -glutamyl transpeptidase (-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).

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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[]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-1,6diene-3,5-dione] to CCl4-induced rats for a period of three months significantly decreased the levels of serum marker enzymes AST, ALP and -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 -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).

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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-dglucosamine 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 -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-

39

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

40

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.

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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 nonenzymic antioxidants, which were reduced on exposure to oxidative stress, were counteracted on treatment with the leaf extract.

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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-oxidantinduced 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

43

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

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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

45

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, -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

46

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.

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