1

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).

2

Several medicinal plants have been analyzed for their biological activity and

active constituents. One such medicinal plant is Bacopa monnieri, commonly known as

Brahmi. Research has focused primarily on Bacopa’s cognitive enhancing effects,

specifically memory, learning and concentrations and the results support the traditional

Ayurvedic claims. Not many studies have been done on the antioxidant and apoptosis-

modulating effects of B. monnieri. Hence, this study mainly focused on assessing the

antioxidant and apoptosis-modulating effects of B. monnieri.

The study was carried out in four phases, as explained in the methodology section.

The results obtained are discussed below.

PHASE I

Antioxidant status of different parts of B. monnieri

In Phase I of the study, different parts of the plant, namely leaves, stolon and

roots, were analysed for their antioxidant status. The results obtained showed that all the

three parts possessed considerable quantities of enzymic and non-enzymic antioxidants.

Among the three parts tested, the leaves possessed the maximum quantity of antioxidants

analyzed, followed by the stolon and the roots.

Several plants have been studied for their antioxidant status. The leaves of three

under-exploited medicinal plants were found to exhibit moderate activities of all the

enzymic antioxidants assessed (Nirmaladevi and Padma, 2008). Increased activities of the

enzymic antioxidants have been reported in the root and leaf samples of Phalaenopsis

(Ali et al., 2005).

Chamseddine et al. (2009) reported that the leaves of Solanum locopersicon

possessed higher superoxide dismutase activity. The leaves of Prunus cerasus L.

exhibited higher catalase activity (Chatizissavidis et al., 2008). Stajner and Popovic

(2009) reported that the highest carotenoid content was observed in the leaves of Allium

ursinum and the lowest in Allium scorodoprasum. The antioxidant potential of Clitoria

ternatea L. and Eclipta prostrata L. was analysed by Bhaskar et al. (2009) by assessing

enzymic and non-enzymic antioxidants.

Patel et al. (2010) have reported that when the stem and leaf parts of Calotropis

procera Linn., Hibiscus cannabinus L., Parthenium hysterophorus L., Gemelia arborea

3

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.

4

DPPH RADICAL SCAVENGING EFFECTS

The inhibition of free radical DPPH is one of the oldest and most frequently used

methods for total antioxidant potential/capacity of food and biological extracts. It is based

on the ability of an antioxidant to give hydrogen radical to synthetic long-lived nitrogen

radical compound DPPH.

In the present study, the different extracts of B. monnieri leaves were assessed for

their DPPH scavenging ability. The results of both qualitative dot blot screening and

quantitative spectrophotometric assays revealed that the methanolic extract exhibited the

highest activity.

The screening of herbal extracts and their components by the DPPH scavenging

assay has become a routine parameter for testing their antioxidant efficacy (Mothana

et al., 2008).

The aqueous, methanolic and ethanolic extracts of Melissa officinalis, Matricaria

recuttia and Cymbopogan citrates were found to possess DPPH scavenging activity

(Pereira et al., 2009). The alcohol–water extract of Ichnocarpus frutescens leaves

possessed 1,1-diphenyl-2-picrylhydrazyl radical and superoxide anion radical scavenging

activity (Kumarappan and Mandal., 2008).

The methanolic extracts of leaves and flowers of Lippia alba exhibited very

significant DPPH radical scavenging activity compared to the standard antioxidant

ascorbic acid (Ara and Nur, 2009). The methanolic extract of Manikara zapota showed

strong activity on scavenging DPPH radical, which implicates an essential defence

against the free radicals (Kaneria et al., 2009).

The hot water extract of Perilla frutescens stalk showed moderate DPPH radical

scavenging abilities than the leaf and seed extracts (Chou et al., 2009). The methanol

extract of Helichrysum plicatum subsp. Plicatum, a species of the Asteraceae family and

belonging to the subtribe Gnaphaliinae, has been reported to have antioxidant activity

using two in vitro methods, namely DPPH and β-carotene linoleic acid assays (Tepe

et al., 2005).

5

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

6

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

7

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.

8

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.

9

Anandjiwala (2007) reported that the methanolic extract of Bergia suffruticosa

showed inhibition of superoxide generation. Ogunlana et al. (2008) showed that the crude

extract of Newboudia leaves exhibited very high inhibition of superoxide generation.

The aqueous and ethanolic extracts of Iris germanica was found to exhibit strong

SO� scavenging activity (Nadaroglu et al., 2007). The methanol extracts of bark and

leaves of Cassia siamea and Cassia javanica plants showed effective superoxide anion

radical scavenging activity (Kaur and Arora, 2009).

Gymnema sylvestre extract showed antioxidant activity by inhibiting DPPH,

scavenging superoxide, as well as hydrogen peroxide (Rachh et al., 2009). Chloroform

and methanol extracts of the roots of Rhubarb (Rheum ribes L.) showed more potent

superoxide anion radical scavenging activity than BHT, and was comparable with well

known radical scavenger L-ascorbic acid (Ozturk et al., 2007).

Saito et al. (2008) reported that the extracts from Punica granatum (peel),

Syzygium aromaticum (bud), Mangifera indica (kernel) and Phyllanthus emblica (fruit)

scavenged superoxide anions, which was comparable to that of L-ascorbic acid. Kim

et al. (2009b) reported that α-chymotrypsin extract from the center of the root of Korean

Elk velvet antler showed high superoxide scavenging activity. The crude extract and

fractions of Vaccinium uliginosum L. containing polyphenol and pigment exhibited

superoxide scavenging activity (Kim et al., 2009c).

Li et al. (2009b) showed that Lysimachia foenum-graecum Hance exhibited SO●

radical-scavenging activities. Salvia brachyantha (Bordz) exhibited superoxide radical

scavenging activity at different magnitudes of potency (Esmaeili and Sonboli, 2010).

Inoue et al. (2005) reported that manuka honey had specific scavenging activity

for superoxide anion radicals. Caffeic acid, a major hydroxycinnamic acid present in

wine, was found to possess effective superoxide anion radical scavenging activity

(Gulcin, 2006). The superoxide anion radical scavenging activity was found to be

significantly higher in the raw and dry heated seed extracts than the hydrothermally

processed seed samples of cowpea (Vigna unguiculata) (Siddhuraju and Becker, 2007).

Yang et al. (2010a) reported that the methylated polysaccharides of longan fruit pericarp

resulted in a decrease in the superoxide anion radical scavenging activity with increasing

degree of methylation.

10

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.

11

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.

12

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

13

manner and their inhibitory effects were comparable to the reference compound, Trolox

(Hamad et al., 2010). The methanolic extract of the plant Hedyotis corymbosa

significantly reduced the accumulation of lipid peroxides in vitro in a dose-dependent

manner in rat liver homogenate (Sadasivan et al., 2006). The flowers of the plant

Dendrobium nobile inhibited the in vitro LPO significantly in both liver homogenate and

RBC ghosts (Devi et al., 2009).

Resveratrol strongly inhibited the LPO of linoleic acid emulsion (Gulcin, 2010).

Embelin (from Embelia ribes), a component of herbal drugs was found to inhibit LPO

and restore impaired Mn-superoxide dismutase in rat liver mitochondria (Joshi et al.,

2007). Quercitrin, a glycoside form of quercetin, was able to prevent the formation of

TBARS induced by pro-oxidant agents (Wagner et al., 2006).

These reports support our findings, wherein the leaf extracts were very effective in

inhibiting LPO in the different membrane systems. Since all the three systems comprise

of plasma membrane lipids, the observation that a better protection occurs in homogenate

than in plasma membrane and intact cell, make it clear that some endogenous component

in the cells is involved. It implies that an endogenous factor in the cells, interacts

synergistically with the leaf components to render better protection against lipid damage.

Thus, our results clearly demonstrate that the extracts of B. monnieri leaves are

very effective in protecting membrane lipids against oxidative damage.

EFFECTS OF B. monnieri LEAF EXTRACTS ON OXIDANT-INDUCED

DAMAGE TO DNA

The protective effect of B. monnieri leaf extracts (aqueous, methanol and

chloroform) to DNA was studied using DNA from different sources, i.e., commercially

available DNA and DNA from intact cells. In the commercial sources, DNA from

different sources namely λ DNA, pUC18 DNA, herring sperm DNA and calf thymus

DNA were used. The results showed that all the three extracts were able to revert the

damage caused by H2O2.

Many studies have reported the use of herbal extracts or products in rendering

protection to the damaged DNA.

14

Aphanes arvensis aqueous and methanolic extracts showed inhibitory effect on

DNA oxidation (Hamad et al., 2010). The aqueous extract of Cyanthillium cinereum

(Less.) H. Rob. evidently demonstrated the protective effect on pBR322 plasmid DNA

against oxidative breakdown (Guha et al., 2009).

The methanolic extract of Nelumbo nucifera flowers rendered protection to

pUC18 DNA exposed to H2O2 (Wong et al., 2005). Karawita et al. (2007) showed that

the enzymatic extracts from seven species of microalgae yielded promising DNA damage

inhibitory properties on mouse lymphoma L 5178 cells treated with H2O2.

Bitter cumin extract offered complete protection to DNA damage induced in calf

thymus DNA and reduced uncoiling or open circular form of pUC18 DNA (Ani et al.,

2006). Acorus calamus extract effectively reduced the disappearance of the covalently

closed circular form of plasmid DNA (pBR322) following exposure to γ-radiation

(Sandeep and Nair, 2010). Treatment with Azadirachta indica significantly mitigated

H2O2-induced oxidative damage to pBR322 DNA (Manikandan et al., 2009).

Kumar and Chattopadhyay (2007) showed that the n-butanol soluble fraction

derived from the methanol extract of Mentha spicata Linn. exhibited significant

protecting activity against DNA strand scission. Prakash et al. (2007) reported that some

of the food and medicinal plants were found to be effective in protecting plasmid DNA

nicking induced by hydroxyl radicals generated by Fenton's reaction.

The addition of Bambusar caulis in Liquamen, provided a protective barrier

against hydroxyl radical-induced damage to pBR322 supercoiled DNA in a dose-

dependent manner (Je et al., 2009). The supplementation of water cress in the diet

reduced lymphocyte DNA damage in healthy adults (Gill et al., 2007). Hydroxylated-4-

thiaflavans, a group of antioxidants, showed protection against oxidative DNA damage in

herring sperm DNA induced by cumene hydroperoxide (Lodovici et al., 2006).

In the present study, the extracts of B. monnieri leaves were found to offer very

significant protection against oxidative damage in purified DNA samples under

physiological conditions. Following this, the extent of DNA damage in the presence and

the absence of the leaf extracts was followed in intact cells using the comet assay.

15

Among the methods to measure oxidative damage to DNA, the comet assay has

been shown to be the most accurate method for measuring DNA oxidation (Stoyanova

et al., 2010).

In the present study, untransformed cells (peripheral blood lymphocytes) were

used to assess the oxidant-induced damage in intact cells using the comet assay. The leaf

extracts were very effective in bringing down the extent of oxidative DNA damage. The

damage caused by the oxidant was completely nullified on exposure to the extracts.

The results obtained with the purified DNA preparations showed that the damage

could not be completely reverted by the leaf extracts. The fact that the same dose of leaf

extracts could completely revert the damage to basal levels in the intact cells suggests the

possible involvement of some endogenous cellular component that works in conjunction

with the leaf components to protect DNA. A similar indication has also been obtained

from our results with the inhibition of LPO in different membrane preparations.

Jang et al. (2010) have shown that the solvents (acetone, ethanol and methanol)

extracts of calyx and seed of Diospyros kaki exhibited the greatest protective effect on

H2O2 induced DNA damage in human leukocytes. The protective effect of Cnidium

officinale extract against H2O2-induced oxidative damage in the human skin fibroblasts

was revealed by Jeong et al. (2009). Acorus calamus extract effectively protected DNA

from radiation induced strand breaks and enhanced the DNA repair process in murine

cells and human peripheral blood leukocytes (Sandeep and Nair, 2010).

Diphlorethohydroxycarmalol isolated from Ishige okamurae exerts profound

antioxidant effects against H2O2-mediated cell damage and significantly enhanced cell

viability against H2O2-induced oxidative damage (Heo and Jeon, 2009). Garlic extracts

efficiently enhanced the ability of normal human leukocytes to resist hydrogen peroxide

and 4-hydroxynonenal induced oxidative damage (Park et al., 2009).

Ecbalium elaterium fruit juice significantly reduced the frequency of comet

bearing cells in human lymphocytes (Rencuzogullari et al., 2006). A red mixed berry

juice and a corresponding polyphenol-depleted juice, serving as control, revealed a

decrease of oxidative DNA damage (Weisel et al., 2006). Fabiani et al. (2008) showed

that phenolic compounds, when used both as purified compounds and in complex crude

extracts in olive oil, prevented hydrogen peroxide-induced DNA damage.

16

Miranda et al. (2008) reported that ingestion of mate tea (Ilex paraguariensis)

increased the resistance of DNA to H2O2-induced DNA strand breaks and improved the

DNA repair after H2O2 challenge in liver cells, irrespective of the dose ingested. Miorelli

et al. (2008) revealed that ebselen at a concentration of 5-10 µM diminished the extent of

the DNA damage induced by hydrogen peroxide.

The cytoprotective effects of the Kojizyme extract against H2O2-induced DNA

damage increased significantly with increasing extract concentrations in comet assay tests

(Heo and Jeon, 2008). Athukorala et al. (2005) also reported that antioxidant compounds

from seaweed extracts exhibited superior ROS scavenging activity and cytoprotective

effects against H2O2-induced DNA damage.

The results of the present study are in agreement with the above reports. Our

results of the assays probing the extent of protection rendered by B. monnieri leaf extracts

to lipids and DNA strongly suggest the synergistic involvement of some, as yet

unidentified, endogenous factor in the target cells, that acts in conjunction with the plant

components to greatly reduce the extent of oxidative damage. Thus, our study shows that

B. monnieri leaves are an excellent source of naturally occurring antioxidant compounds

with potent lipid and DNA damage inhibition potential.

EFFECTS OF B. monnieri LEAF EXTRACTS ON OXIDATIVE STRESS-

INDUCED APOPTOTIC CHANGES

Damage to cells and tissues involve the generation of ROS and RNS, followed by

alterations in lipids, DNA and proteins, which eventually lead to cellular dysfunction and

cell death. The consequent role of free radicals in the mechanism of cell death, especially

in the induction of apoptotic death has been receiving growing attention in the field of

cancer therapy (Mishra, 2004).

With this backdrop, in the present study, the effect of B. monnieri leaf extract on

the oxidative stress-induced apoptosis was analysed in untransformed (S. cerevisiae and

chick embryo fibroblasts) and transformed (Hep2 laryngeal carcinoma) cells. The

oxidants used were H2O2 (in S. cerevisiae cells) and etoposide, a standard

chemotherapeutic drug (in chick embryo fibroblasts and Hep2 cells).

17

EFFECT OF B. monnieri LEAF EXTRACT ON H2O2-INDUCED DEATH IN

S. cerevisiae CELLS

The results obtained clearly indicated that the exposure to H2O2 resulted in a steep

rise in the number of cells undergoing apoptosis. B. monnieri leaf extract, by itself, did

not cause an increase in the extent of apoptosis. When co-administered along with H2O2,

the plant extract resulted in a markedly decreased number of apoptotic cells. Thus, it is

evident that the leaf extract protect the yeast cells from oxidative stress-induced death.

EFFECT OF B. monnieri LEAF EXTRACT ON ETOPOSIDE-INDUCED DEATH

IN UNTRANSFORMED AND TRANSFORMED CELLS

The results of the present study revealed that on exposure to B. monnieri leaf

extract, a differential response was evoked in the untransformed and transformed cells.

On exposure to etoposide, the number of cells undergoing apoptosis was significantly

increased in both the cell types. The leaf extract, by itself, did not induce apoptosis in

normal cells, but apoptosis was induced in cancer cells. This indicated the anticancer

activity of the B. monnieri leaf extract.

The administration of the leaf extract effectively counteracted the apoptosis-

inducing effect in primary cells, whereas, the apoptosis-inducing effect of etoposide was

further augmented in Hep2 cells in the presence of the extract.

This observation has a very significant bearing. It is deducible from our results

that the B. monnieri leaf extract protects the normal cells from oxidative death, while

rendering the cancer cells more susceptible to the cytotoxic action of the

chemotherapeutic drug etoposide. Thus, based on our results, it can be suggested that

B. monnieri leaves can be administered as a supportive therapy during cancer

chemotherapy, to minimize the toxic side effects to non-cancerous cells and to maximize

the anticancer drug action.

The above results were confirmed by a battery of tests, namely MTT assay, SRB

assay, staining procedures (with Giemsa, PI, EtBr and DAPI) and DNA fragmentation.

All these are highly reliable assays as is evident from the rich literature support, which is

discussed below.

18

MTT VIABILITY ASSAY

The viability of both untransformed and transformed cells was significantly

decreased on exposure to the oxidant H2O2/etoposide. B. monnieri leaf extract increased

the viability of the untransformed cells, whereas the viability was further increased in

cancer cells.

MTT is considered to be a reliable assay to determine the extent of cell viability,

as observable from the vast number of studies reported in the literature.

The ethanolic extract of Pleurotus ostreatus inhibited the cell proliferation in a

dose-dependent manner in HL-60 leukemia cells (Venkatakrishnan et al., 2010). The

methanolic extract of Piper sarmentosum possessed anticarcinogenic properties in HepG2

cells (Ariffin et al., 2009). An aqueous extract of several Asteraceae species exhibited its

antiproliferative activity against HeLa (cervix epithelial adenocarcinoma), A431 (skin

epidermoid carcinoma) and MCF-7 (breast epithelial adenocarcinoma) cells, in the MTT

assay (Rthy et al., 2007).

The ethanolic extract of Celastrus orbiculatus exhibited a cytotoxic effect on

human melanoma A375-S2 and human cervical carcinoma HeLa cell lines (Xu et al.,

2008). Je et al. (2009) reported that Bambusae caulis in Liquamen significantly protected

against hydrogen peroxide-induced apoptosis in PC-12 cells. MTT assay in U87 and

U251n glioma cells treated with various concentrations of gallic acid showed decreased

cell viability (Lu et al., 2010a).

Honokiol, an active component isolated and purified from the Magnolia induced

cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes (Chen

et al., 2010b). Phloroglucinol derivative (2,4-bis(2-fluorophenylacetyl) phloroglucinol)

induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but

not in primary chondrocytes (Liu et al., 2010b).

Treatment with celecoxib resulted in a concentration-dependent decrease of

cellular viability in U87-MG, U251 and A172 (Gaiser et al., 2008). Withaferin A, a major

constituent of the dietary component Withania somnifera, induced Par-4-dependent

apoptosis in androgen-refractory prostate cancer cells (Srinivasan et al., 2007).

19

The cell viability estimated by the MTT assay showed that Unearia rhynchophylla

induced cytotoxicity in HT-29 (human colon carcinoma) cells (Jo et al., 2008). Similarly,

Zhang and Poporich (2008) estimated, using the MTT assay, the inhibition of cell

proliferation in Hep G2 (liver carcinoma) cells by soya saponins, which was found to be

dose-dependent. Koon et al. (2006) investigated the effect of curcumin on the viability of

nasopharyngeal carcinoma cells using MTT.

Resveratrol rendered protection against arsenic trioxide induced cardiotoxicity

in vitro and in vivo. Pretreatment with resveratrol dose-dependently attenuated the As2O3-

induced reduction in cell viability (Zhao et al., 2008a). Wali et al. (2009) analysed the

cell viability in neoplastic mouse +SA mammary epithelial cells by the MTT assay, and

reported that the cell viability was significantly decreased by α-tocotrienol in a dose- and

time-dependent manner.

Peng et al. (2007) determined the cytotoxic activity of the root of Spiranthes

australis on A549 (lung carcinoma), BEL-7402 (hepatoma cell line), SGC-7901 (gastric

cancer), MCF-7 (breast cancer), HT-29 (colon cancer), K562 (leukemia) and A498 (renal

cancer) cell lines by MTT assay, where the cell viability was reduced by the plant extract.

Human hepatoma SMMC-7721 cells treated with the Rosa roxburghii extract showed a

decrease in the cell viability (Yu et al., 2007). The extract of Linum persicum and

Euphorbia cheiradenia induced the reduction of K562 cell (leukemia cell lines) viability

through apoptosis, which was quantified by the MTT assay (Amirghofran et al., 2006).

From the results of the MTT assay in the present study, it can be inferred that

B. monnieri leaf extract was potent in inducing apoptosis in cancerous cells and

augmenting it in the presence of an oxidant. The extract effectively counteracted the toxic

effect of the oxidant in non-cancerous cells.

SRB ASSAY

The results of the SRB assay also followed the same trend as the MTT assay in the

present study. Several studies have used the SRB assay to ascertain the cytotoxic effect of

phytocomponents against cancer cells.

The phenolic ethanolic extract of Clitocybe alexandri inhibited the growth of four

different tumor cell lines (lung, breast, colon and gastric cancer) (Vaz et al., 2010).

20

Primary rat hepatic stellate cells and the human hepatic stellate cell line LX-2 treated with

Withagulatin A showed a dose-dependent decrease in cell viability as determined by SRB

assay (Liu et al., 2010a). The extracts of Phyllanthus emblica and Terminalia bellerica

are reported to have high cytotoxic effects against the human hepatocellular carcinoma

(HepG2) and lung carcinoma (A549) cells (Pinmai et al., 2008).

The ethylacetate extract of Ageratum conyzoides exhibited high cytotoxic activity

on A-549 and P-388 cancer cells (Adebayo et al., 2010). Cistus incanus L. and

Cistus monspeliensis L. extracts treatment on prostate cell lines resulted in a significant

decrease in cell viability (Vitali et al., 2010). The potential antiproliferative activity of the

Ailanthus excelsa extract and isolated flavonoids against five human cancer cell lines

(ACHN, COR-L23, A375, C32, and A549) was investigated in vitro by the SRB assay in

comparison with one normal cell line, 142BR. The extract exhibited the highest inhibitory

activity against C32 cells (Said et al., 2010).

An anthocyanin extract from blueberry and an anthocyanin-pyruvic acid adduct

extract significantly reduced cell proliferation in two breast cancer cell lines (MDA-MB-

231 and MCF7) as assessed by the SRB assay (Faria et al., 2010). A new diterpene

alkaloid named delphatisine isolated from the aerial parts of Delphinium chrysotrichum,

showed significant cytotoxic activity against the A549 cell line (He et al., 2010).

Andrographolide, a diterpenoid lactone isolated from a traditional herb

Andrographis paniculata, has been reported to inhibit prostate cancer (PC-3) cells in a

dose-dependent manner, as determined by sulphorhodamine B assay (Zhao et al., 2008b).

The cytotoxic effect of 7-O-methylaromadendrin and tectorigenin against MCF-7 (breast

cancer) and C32 amelanotic melanoma cell line was determined by the SRB assay (Fang

et al., 2008).

The cytotoxicities of the 14 alkaloids isolated from Corydalis ternata were

evaluated on several human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15)

using the SRB assay (Kim et al., 2010b). Abu-Dahab and Afifi (2007) determined the

antiproliferative activity of several medicinal plants against a breast adenocarcinoma cell

line by SRB analysis and observed that the plant extracts exhibited high cytotoxicity

against MCF-7 (breast cancer) cells. Patel et al. (2009) reported that the methanolic

21

extract of Solanum nigrum fruits, when screened for its cytotoxicity against HeLa and

Vero cell lines at different concentrations, exhibited significant toxicity on HeLa cell line.

Fibrarecisin, a novel triterpenoid, isolated from the chloroform extract of the stem

bark of Fibraurea recisa Pierre showed cytotoxicity against A549 (lung cancer) cells (Fu

et al., 2007). Mucronulatol from Caribbean propolis was found to possess cytotoxic

effect against cancer cells (Carballo et al., 2008). The cytotoxic effect of etoposide,

vinblastine sulfate and dacarbazime against B16–F10 and HMEC-1 cells was determined

by SRB assay (Dandamudi and Campbell, 2007).

From the observations made from the cell viability assays in the present study, it

can be inferred that oxidative stress caused significant death in both untransformed and

transformed cells. B. monnieri leaves showed their protective effect against oxidative

stress in untransformed cells, while enhancing the cytotoxic effect of etoposide in cancer

cells.

MORPHOLOGICAL CHANGES ASSOCIATED WITH APOPTOSIS

The morphological changes associated with apoptosis are membrane blebbing,

cell shrinkage, membrane-bound apoptotic bodies and chromatin condensation. In the

present study, a marked increase in the number of apoptosing cells in the cancer (Hep2)

cells exposed to methanolic extract of B. monnieri leaves was observed. The oxidative

stress-induced cellular death in normal cells was effectively counteracted by the presence

of B. monnieri leaf extract.

Staining with giemsa has been reported by many researchers characterizing the

effect of herbal components on apoptosis in different cells.

Apoptotic cell death was observed in WM793B cell line as evident from the

morphological changes (Lesiak et al., 2010). Morphological changes of L02 cells

observed by Giemsa staining showed that Polygonum multiflorum could induce L02 cell

apoptosis (Zhang et al., 2010). Studies have demonstrated that wogonin, a monoflavonoid

extracted from the root of Scutellaria baicalensis, could effectively inhibit the

proliferation of several cancer cell lines as determined by Giemsa staining (Zhang et al.,

2008b).

22

Clitocine, a natural biologically active substance isolated from the mushroom

Leucopaxillus giganteus, induced cell death as characterized with the changes in cell

morphology (Ren et al., 2008). The julibroside J8 isolated from the Albizia julibrisin,

inhibited the growth in the BGC-823, BEL-7402 and HeLa cell lines (Zheng et al., 2006).

Zhou et al. (2007) reported that oridonin, a diterpenoid extracted from medicinal herbs,

had potent antitumor activity with low adverse effects on U937 cells.

Artesunate induced apoptosis of tumor cells in vitro, as observed from the

morphological changes of Raji and Jurkat cells under light microscopy after Wright-

Giemsa dyeing (Zeng et al., 2009). Apoptosis in ECV-304 cell line supplemented with

sodium morrhuate and liposodium morrhuate was also demonstrated by Giemsa staining

(Tu et al., 2006).

Gao et al. (2007) investigated the effect of lidamycin in human hepatoma BEL-

7402 cells, showing cell multinucleation by Giemsa staining. Histone deacetylase

inhibitor showed visible morphological changes of U266 cells when subjected to Wright-

Giemsa staining (Ma et al., 2009). Morphological changes were observed in human

leukemia U937 and KG1 cells treated with guanosine 5'-triphosphate (Yazdanparast

et al., 2006).

Rosiglitazone treated cells displayed morphological characteristics of cell

differentiation, and more evident signs of differentiation were observed when combined

with all trans-retinoic acid (Huang et al., 2009a). Trans-resveratrol, a compound found in

grapes, berries, peanuts and red wine, exerted anticancer roles in T47D cells and caused

apoptosis, as demonstrated by Giemsa staining (Alkhalaf, 2007a).

The effects of arsenic trioxide and/or transforming growth factor-beta1 (TGF-β1)

of NB4 cells showed apoptotic morphological changes when stained with Giemsa (Liang

et al., 2009). Song and Zhang (2006) showed that parthenolide inhibited the proliferation

of human hepatocellular carcinoma cell line BEL-7402. The apoptotic effect of Berberine

on K562 cells showed inhibition of proliferation of K562 cells in a dose- and time-

dependent manner (Jin et al., 2009).

The results of the present study revealed that the methanolic extract of B. monnieri

leaves can exert a differential response against the oxidative stress-induced apoptosis in

different types of cells.

23

NUCLEAR CHANGES ASSOCIATED WITH APOPTOSIS

Apoptotic nuclei undergo typical changes including chromatin condensation,

peripheral marginalization, nuclear shrinkage and subsequent fragmentation (Cohen et al.,

2003).

The characteristic nuclear changes associated with apoptosis were quantified by

EtBr, PI and DAPI staining. The results of the study revealed that the number of cells

undergoing apoptosis increased in both untransformed and transformed cells subjected to

oxidative stress. The administration of B. monnieri leaf extract exerted no cytotoxic effect

in normal cells, but significant cytotoxicity was observed with cancer cells. These results

show that the leaf extract exhibited anticancer potential.

PROPIDIUM IODIDE STAINING

In this study, staining with propidium iodide showed that the methanolic extract of

B. monnieri leaves reduced apoptosis in untransformed cells, and not in cancer cells,

indicating the anti-apoptotic and anticancer property of the plant.

Propidium iodide intercalates into double-stranded nucleic acids. It is excluded by

viable cells but can penetrate cell membranes of dying or dead cells. Propidium iodide

staining is a routine parameter in most studies centering on apoptosis.

An ethanolic extract of Serenoae repentis fructus induced apoptosis in MDA

MB231 prostate, breast carcinoma, renal Caki-1, urinary bladder J82, colon HCT 116 and

lung A549 cancer cells, which was assessed by PI staining (Hostanska et al., 2007). The

methanolic and methane dichloride extract of the aerial parts of Larrea divariculata

exerted cytotoxic activity on MCF-7 cell line (Bongiovanni et al., 2008).

Platycodon grandiflorum extract induced cell apoptosis in SKOV3 ovarian cancer

cells as observed by PI staining (Hu et al., 2010). Panax quinquefolins leaf extract was

tested for cytotoxic activity in THP-1 leukemia cells using PI and showed significant

increase in apoptosis (Kitts et al., 2007). Huang et al. (2009b) showed that Antrodia

camphorate induced apoptosis in a concentration-dependent manner on human oral

cancer cells.

24

Some Chinese herbs, screened for antiproliferative properties in human HaCaT

keratinocytes model, induced cell arrest and death as assayed by PI (Tse et al., 2007).

Fenugreek seed polyphenolic extract treatment showed an increased hepatocyte viability

and reduced apoptotic nuclei in ethanol-induced hepatic injury in rats (Kaviarasan and

Anuradha, 2007). The apoptosis-inducing effects of Iris tectorum Maxim on MCF-7 and

C32 cell lines was shown by Fang et al. (2008).

An aqueous extract of Rhodiola imbricata rhizome significantly decreased the

proliferation of k-562 cells, as determined by PI staining (Mishra et al., 2007). In HepG2

(hepatocellular carcinoma), PI staining displayed the apoptotic cells when treated with

Trichosanthes kirilowii tuber extract (Shin et al., 2008). Ma et al. (2010) indicated that

Pinus massoniana bark extract, a mixture of flavonoids, selectively induced apoptosis in

HepG2 cells through caspase-dependent pathways.

Propidium iodide staining revealed that bovine aortic endothelial cells exposed to

methylglyoxal after DL-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione

biosynthesis, displayed features characteristic of apoptosis (Takahashi et al., 2010).

Gambogic acid showed significant effects in inducing apoptosis as assessed by PI staining

(Wang et al., 2010a). 4',7-diacetoxyapigenin increased apoptotic population as observed

by PI staining (Xu et al., 2010).

Brucea javanica oil induced apoptosis of T24 cells, which was analyzed by PI

staining (Lou et al., 2010b). Imatinib treatment resulted in an increased number of

apoptotic cells in a time- and dose-dependent manner on rat C6 glioma cell (Yang et al.,

2010b).

Liu et al. (2010c) reported that aeoniflorin reduced the rate of neuronal apoptosis

in corticosterone-induced primary cultures of rat cortical neurons as measured by PI

staining. Cai et al. (2008) examined the cytotoxic effect of emodin against human

pancreatic adenocarcinoma cell lines Mia Paca-2, BxPC-3, Panc-1, and L3 6pl. WST-1.

In the light of these reports, the results of PI staining in the present study are

highly validated.

25

ETHIDIUM BROMIDE STAINING

EtBr, which is an intercalating agent, can be used to visualize the changes that

occur in the nucleus during apoptosis. Apart from EtBr alone, the treatment with a

combination of acridine orange and ethidium bromide has been used as a reliable index of

cellular degeneration (Campos-Dapaz et al., 2008).

The ethanolic extract of Curcuma aromatica induced apoptosis and inhibited

angiogenesis in Ehrlich Ascites Tumor cells as assessed by EtBr staining (Thippeswamy

and Salimath, 2006). Acridine orange / ethidium bromide (AO/EB) staining indicated that

the cytotoxicity induced by 2',4'-dihydroxychalcone, one of the main components in

Herba oxytropis was mediated by apoptosis in human gastric cancer MGC-803 cells (Lou

et al., 2009). Analysis of the acridine orange / ethidium bromide staining revealed that the

crude extract of Solanum hydratum reduced cell proliferation and induced apoptosis in a

time- and dose-dependent manner in human colon cancer Colo205 cells (Hsu et al.,

2008).

Sanguinarine, a benzophenanthridine alkaloid derived from the root of

Sanguinaria canadensis, induced apoptosis in human cancer cells, which was assessed by

AO/EB staining (Hau et al., 2008). Sanguinarine, a benzophenanthridine alkaloid derived

from the root of Sanguinaria canadensis, induced apoptosis in human cancer cells, which

was assessed by AO/EB staining (Han et al., 2008). Hahnvajanawong et al. (2010)

reported the growth suppression by caged xanthones from Garcinia hanburyi in

cholangiocarcinoma KKU-100 and KKU-M156 cells.

The percentage of norepinephrine-induced apoptosis in the primary cultured rat

cardiac myocytes was decreased, as observed by EtBr staining assay, after pretreatment

with crocetin (Shen et al., 2009). The cytotoxicity of Pristimerin isolated from Maytenus

ilicifolia was evaluated in human tumor cell lines and in human peripheral blood

mononuclear cells. The cells stained with AO/EB, showed the occurrence of necrosis and

apoptosis in a concentration-dependent manner in HL-60 cell lines (Costa et al., 2008).

Cells treated with different concentrations of beta-sitosterol showed

morphological changes by acridine orange / ethidium bromide double staining in SGC-

7901 human stomach cancer cells (Zhao et al., 2009a). Fluorescence microscopy using

AO/EB staining indicated that kauren-19-oic acid induced apoptosis in HL-60 cell

26

cultures (Cavalcanti et al., 2009). The apoptotic morphology induced by cycloheximide in

human leukocytes was detected by AO/EB staining (Baskic et al., 2006).

In the present study, EtBr staining also confirmed that the methanolic extract of

B. monnieri leaves significantly protected the yeast and primary (untransformed) cells

from oxidant-induced damage. In the case of Hep2 (transformed) cells, the extract caused

an increase in the number of cells undergoing apoptosis.

DAPI STAINING

The nuclear changes that are characteristic of apoptosis can be monitored by

fluorescence microscope using DAPI staining. Epigallocatechin gallate, the major

component of polyphenols in green tea protected H9c2 cardiomyoblasts against hydrogen

dioxides-induced apoptosis as analysed by DAPI staining (Sheng et al., 2010).

The cell death in human hepatocellular carcinoma Hep 3B cells treated with the

ethanolic extracts of Euchresta formosana radix was identified as apoptosis using DAPI

staining (Hsu et al., 2007). Human breast cancer cells treated with the extracts of

Astrodaucus persicus also showed potential decrease in the cell proliferation by staining

with DAPI (Abdolmohammadi et al., 2008).

Phyllanthus niruri treatment, counteracted the changes and maintained normalcy

in hepatocytes against tertiary butyl hydroperoxide-induced apoptosis, as evidenced by

DAPI staining (Sarkar and Sil, 2010). Gallic acid induced apoptosis via caspase-3 and

mitochondrion-dependent pathways in vitro and suppressed lung xenograft tumor growth

in vivo in NCI-H460 cells as examined by DAPI staining (Ji et al., 2009).

Studies with kaempferol, a natural flavonoid, have reported that kaempferol had

anti-proliferation activities and induced apoptosis in human osteosarcoma cells (Huang

et al., 2010b). The effect of curcumin on apoptosis in Sk-hep-1 cells was investigated by

DAPI staining and the results indicated that curcumin was able to inhibit proliferation and

induce apoptosis in Sk-hep-1 cells (Wang et al., 2010b).

Aiyar et al. (2010) reported that 2,4,3',5'-tetramethoxystilbene, a chemically

modified herbal derivative of resveratrol, induced cell death by targeting Bax, as

evidenced by DAPI staining. Epigallocatechin-3-gallate effectively inhibited cellular

27

proliferation and induced apoptosis of the synovial sarcoma cells, as indicated by DAPI

staining (Sun et al., 2010).

Rajabalan (2008) observed that anticancer drugs caused leukemic lines to undergo

apoptosis, using DAPI staining. DAPI staining confirmed apigenin-induced DNA

condensation and damage in A549 cells (Lu et al., 2010b).

The results of DAPI staining in our studies indicate that the methanolic extract of

B. monnieri leaves brought the proportion of apoptosing cells down in untransformed

cells, while causing an increase in the number of apoptosing cells in transformed cells.

DNA FRAGMENTATION

Apoptosis is the predominant form of programmed cell death and occurs under a

variety of physiological and pathological conditions. One of the most prominent

biological features of apoptosis is nucleosomal DNA fragmentation. DNA fragments are

considered to be the hallmark of apoptosis and have been followed by many researchers

(Cheah et al., 2007). It is acknowledged to be a late event in apoptosis.

In the present study, in the yeast cells, eventhough apoptosis was observable by

both the assays of viability, as well as all the staining procedures, the characteristic DNA

fragmentation could not be observed.

It has been reported that the DNA laddering during apoptosis has been observed in

most, but not all, apoptotic systems (Cornillon et al., 1994). Several researchers have

reported that DNA fragmentation did not occur in S. cerevisiae cells undergoing apoptotic

cell death (Madeo et al., 2002; Mazzoni et al., 2003; Lundin et al., 2005).

DNA fragmentation is a late event in apoptosis. Large fragments of 50-300kb are

observed in the early stages and only later do they get further fragmented to 200bp

fragments or its multiples (Schliephacke et al., 2004). Jeon et al. (2002) have shown that

the process of apoptosis in yeast cells can be triggered by oxidative stress much earlier

than DNA fragmentation, which took over four hours to manifest. Similar observations

have been reported by Collins et al. (1997).

Methyl methane sulfonate did not cause DNA fragmentation upto 90 minutes of

exposure in rad52 mutant cells. In our study, an exposure interval of one hour was used,

28

which is the probable reason for the lack of observation of DNA fragmentation. This

observation of lack of DNA fragmentation in yeasts can also be explained by the fact that

S. cerevisiae chromatin structure has short or no linker DNA between nucleosomes

(Lowary and Widom, 1989; Madeo et al., 1999).

While DNA fragmentation was not observable in the S. cerevisiae cells,

significant DNA damage was observed in the primary cells and Hep2 cancer cells, which

was altered by the methanolic extract of B. monnieri, which results supported the trend

observed by the earlier assays. This observation further reiterated the fact that yeast cells

are unique in their DNA fragmentation pattern. On the other hand, it is also possible that

the DNA damage is observable in the primary and cancer cells, as they were exposed to

the oxidant and the leaf extract for 24 hours as against one hour for the yeast cells.

A methanolic extract of Oroxylum indicum induced DNA fragmentation in HL60

(promyelocytic leukemia) cells (Roy et al., 2007). A549 human non-small cell lung

cancer cells exposed to ethanol extract of Dunaliella salina showed significant DNA

fragmentation (Shen et al., 2008).

Inagaki et al. (2007) demonstrated that a compound purified from the ethyl acetate

extract of black soybean vinegar induced DNA fragmentation and the development of

apoptotic bodies in U937 cancer cells. Duchesnea indica phenolic fraction significantly

inhibited SKOV-3 cell proliferation and markedly induced apoptosis by characteristic

nuclear DNA fragmentation (Peng et al., 2008).

Clitocine, a natural biologically active substance isolated from the mushroom

Leucopaxillus giganteus, induced DNA fragmentation (Ren et al., 2008). Agarwala et al.

(2010) demonstrated the cytoprotective potential of mangiferin, against mercury chloride-

induced toxicity in HepG2 cell line using DNA fragmentation as an index. A progressive

increase in fragmented DNA was also observed in esophageal cancer cells (TE-2) treated

with the natural antioxidant gallic acid, which was isolated from the fruits of a medicinal

Indonesian plant (Faried et al., 2007).

DNA fragmentation was observed in human breast cancer cells treated with

cajanol, a novel anticancer agent from pigeonpea [Cajanus cajan (L.) Millsp.] roots (Luo

et al., 2010). The fragmented DNA ladder in d-gal-treated mice was inhibited by

troxerutin, a naturally occurring bioflavonoid (Liu et al., 2010d). Ethanolic extracts of

29

Sophora moorcroftiana seeds significantly inhibited SGC-7901 (gastric cancer) cell

proliferation and induced apoptosis by characteristic nuclear DNA fragmentation (Ma

et al., 2006).

When MDA-MB-231 breast cancer cells were treated with resveratrol, DNA

fragmentation was found to occur in a dose-dependent manner (Alkhalaf, 2007b). The

exposure of doxorubicin caused DNA fragmentation in cardiomyocytes (Du and Lou,

2008). DNA fragmentation was also observed in melanoma cells (B16F1) treated with

laurinterol (Kim et al., 2008b). A progressive increase in fragmented DNA was observed

in gastric cancer cells (NCL-N87) and bile duct cancer cells (YGIC-6B) treated with

paclitaxel combined with photodynamic therapy (Park et al., 2008).

Thus, from the results obtained, it can be inferred that B. monnieri leaves, alone or

in combination with chemotherapy drugs, could inhibit the proliferation of cancer cell

lines in vitro, through the mechanism of apoptosis, which suggest that B. monnieri is

likely to be a potential drug in the treatment of cancer.

PHASE III

EFFECT OF B. monnieri LEAF EXTRACT ON THE ANTIOXIDANT STATUS

OF GOAT LIVER SLICES SUBJECTED TO OXIDATIVE STRESS

The results obtained so far revealed that the leaves of B. monnieri were very

effective in protecting cellular biomolecules from oxidative damage. The extract was also

found to alleviate the oxidative stress imposed on untransformed cells, while causing an

increase in the extent of death in the transformed cells. As the next step, the influence of

the leaf extract was tested on the antioxidant status of cells maintained in their tissue

architecture. For this, thin slices of liver tissue exposed to the oxidant in the presence and

the absence of the extract were used.

Liver slices are an intermediate between liver cells and isolated organs. A major

advantage of hepatic slices compared to isolated hepatocytes is the lack of disruption of

cell-to-cell contacts as occurring during the hepatocyte isolation procedure. With liver

slices, the normal tissue architecture, cell heterogeneity and cell-cell interactions are

maintained; the native cell types and integrity of the organ remain intact (Scott, 2005).

30

In the present study, the protective effects of the B. monnieri leaf extract in vitro

against hydrogen peroxide-induced oxidative stress were evaluated. The enzymic and

non-enzymic antioxidants were analyzed in the slices, and the results are discussed below.

ENZYMIC ANTIOXIDANTS

The enzymic antioxidants analysed were SOD, CAT, POD and GST.

SUPEROXIDE DISMUTASE

SOD is considered as the first line of defense against the deleterious effects of oxy

radicals in the cell by catalyzing the dismutation of superoxide radicals to H2O2 (Chandra

et al., 2007).

The activities of SOD in the slices subjected to oxidative stress decreased

significantly. The exposure to the extract did not improve the values. The extract, by

itself, did not decrease the activity of SOD, when compared to the control group.

Several studies in the literature have reported a modulation in SOD activity after

exposure to herbal extracts and components.

Intragastric administration of a polyphenolic extract from Pinus koraiensis seed

increased the levels of SOD in vivo (Su et al., 2009). The activity of SOD increased

significantly in the liver of mice in Kigelia africana, Calotropis procera, Hibiscus

sabdariffa and Alchornea cordifolia extract treated groups and counteracted the

paracetamol-induced oxidative stress (Olalye and Rocha, 2008).

Flavonoids extracts from Inula britannica inhibited oxidative stress in rat aorta

after balloon injury and the activity of SOD was increased significantly in both serum and

vascular tissues (Zhang et al., 2009). An increased level of SOD was reported in a

glycoportein isolated from Rhus verniciflua in the liver of hyperlipidemic mice (Oh et al.,

2006).

The green pods of Acacia nilotica caused a significant increase in the levels of

SOD in the liver, lungs, kidneys and blood in CCl4 treated rats (Singh et al., 2009b).

Preincubation of primary cultured rat cardiac myocytes with crocetin remarkably

prevented the decrease in SOD activity (Shen et al., 2009).

31

In the present study however, B. monnieri leaf extract failed to improve the

activities of superoxide dismutase. The reason for this non-response is presently unclear.

CATALASE

By catalyzing the conversion of H2O2 to O2 and H2O, catalase has been shown to

shift the redox balance towards antioxidant and leading to increased antioxidant capacity,

which may alleviate the detrimental effect of H2O2 (Ren et al., 2004).

In the present study, the activity of catalase was found to decrease on exposure of

the slices to the oxidant hydrogen peroxide. This may be due to the utilization of the

enzyme in the detoxification of hydrogen peroxide. However, on administration of the

extract, the activity of the enzyme was found to increase significantly.

Catalase decomposes hydrogen peroxide and helps to protect from highly reactive

hydroxyl radicals. Wang et al. (2008a) showed that, in Douchi extracts treated,

cholesterol-fed rats, CAT activity in the liver, increased significantly compared to the

negative control group. Aphanamixis polystachya bark extract supplementation resulted in

dose-dependent and significant improvement in hepatic catalase level in rats (Krishnaraju

et al., 2009).

Sumnath and Rana (2006) reported that the pretreatment with hydroalcoholic

extracts of Tarxacum officinale roots improved the levels of catalase and peroxidase in

the liver of rats intoxicated with CCl4. The ethanol:water (7:3) extract of Coriandrum

sativum increased the activities of SOD, CAT and GPx in the liver of CCl4 treated rats

(Sreelatha et al., 2009).

Our results clearly show that B. monnieri leaf extract increased the activities of

both CAT and SOD, which will bring about effective and complete scavenging of

superoxide-peroxide oxidants.

PEROXIDASE

The treatment with the oxidant decreased the activity of peroxidase in the liver

slices. The activity was reversed by the administration of the leaf extract.

32

The antioxidant enzymes CAT and GPx have been reported to protect SOD

against inactivation by H2O2 (Parthasarathy et al., 2006). The oral administration of

methanolic extract of Phyllanthus significantly elevated the activity of hepatic glutathione

peroxidase during CCl4 stress in rats (Lee et al., 2006). Glutathione peroxidase activity in

the kidney of cholesterol-fed rats increased significantly compared with the negative

control group when treated with Douchi extract (Wang et al., 2008a).

Li et al. (2010b) reported that saponins from Panax japonicus protected against

alcohol-induced hepatic injury in mice by up-regulating the expression of GPx, and SOD.

The statistically significant losses in the activities of SOD, CAT and Px in

acetaminophen-induced liver damage in mice was counteracted by all the tested plants

(Kigelia africana, Calotropis procera, Hibiscus sabdariffa and Alchornea cordifolia)

except Calotropis procera as reported by Olalye and Rocha (2008).

The oral administration of Coscinium fnestratum stem extract in graded doses

caused a significant increase in peroxidase activity in the liver and kidney of

streptozotocin-nicotinamide induced type II diabetic rats (Punitha et al., 2005). The

treatment with the alcoholic extract of Momordica charantia increased the activity of

peroxidase in the liver and brain tissues of ammonium chloride treated rats (Thenmozhi

and Subramanian, 2010).

From the available literature, it is obvious that the treatment with the extract

significantly increased the activities of SOD, CAT and POD, which form the first line of

defense against ROS.

GLUTATHIONE S-TRANSFERASE

Glutathione S-transferases are a group of enzymes that catalyse the conjugation of

GSH to a variety of electrophiles, thereby decreasing its reactivity with cellular

components (Bickers and Athar, 2006).

The activities of GST were slightly decreased on exposure to H2O2 but the leaf

extract did not alter the activity of GST in the oxidant exposed slices.

The ethanolic extract of Hibiscus sabdariffa increased the activity of hepatic GST

in sodium arsenate-induced oxidative stress in rats (Usoh et al., 2005). Lima et al. (2005)

33

reported a significant increase of liver GST activity in rats and mice of sage drinking

groups.

Lee et al. (2007a) showed that pre-treatment of rats with tea seed oil (Camellia

oleifera Abel.) could increase the activities of glutathione peroxidase, glutathione

reductase and glutathione S-transferase in liver when compared with CCl4-treated group.

In our study, however, the activities of GST in the various treatment groups did not vary

significantly in the goat liver slices.

Our results show that on exposure to the oxidant H2O2, the activities of the major

enzymic antioxidants were significantly affected, which was improved by the

administration of B. monnieri leaf extract.

NON-ENZYMIC ANTIOXIDANTS

ROS are continuously produced in the cells, which are regulated by a variety of

cellular defenses consisting of enzymic and non-enzymic antioxidants. The non-enzymic

antioxidants analysed were vitamins C, E, A and reduced glutathione. The results

obtained are discussed below.

VITAMIN C

Vitamin C, a major ubiquitous non-enzymic antioxidant, has a crucial role in

scavenging several reactive oxygen species. At physiological concentrations, vitamin C is

a potent free radical scavenger in the plasma, protecting cells against oxidative damage

caused by ROS (Pavana et al., 2007).

In the present study, the levels of vitamin C decreased in the goat liver slices

exposed to the oxidant H2O2, which was effectively improved by the leaf extract.

Sumathi and Padma (2008) reported that Withania somnifera extracts effectively

counteracted the damage produced by H2O2 and replenished vitamin C levels in hydrogen

peroxide induced goat liver slices. Jia et al. (2009) reported a significant increase in the

plasma and liver antioxidant status in streptozotocin-induced diabetic rats when treated

with the polysaccharides from Ganoderma lucidum root extract.

34

Asparagus racemosus root powder administration significantly elevated the

decreased activities of hepatic SOD, CAT and vitamin C levels in hypercholesterolemic

rats (Visavadiya and Narasimhacharya, 2009). Exposure to the oxidant CCl4 significantly

depleted vitamin C, which was counteracted effectively by the oral administration of

curcumin (Kamalakkannan et al., 2005).

In the present study also, the extract of B. monnieri leaves effectively counteracted

the damage produced by H2O2 to liver slices and replenished the vitamin C levels.

VITAMIN E

Vitamin E is one of the major chain breaking lipophilic antioxidant, within the cell

membrane, where it protects membrane fatty acids from peroxidation. Vitamin E has been

effective in blocking peroxyl mediated chain reaction and in combination with ascorbate,

it scavenges SO� in the lipid membrane (Manikandan and Devi, 2005).

A significant reduction in the levels of vitamin E was observed in the slices

exposed to the oxidant, which were improved on exposing the oxidant exposed slices with

the B. monnieri leaf extract.

The levels of vitamin E in the liver and kidney of streptozotocin-induced type II

diabetic rats improved by the administration of alcoholic extract of Coscinium

fenestratum (Punitha et al., 2005). The co-administration of vitamin E with the extract of

Withania somnifera, Ocimum sanctum and Zingiber officinalis elevated the antioxidant

status in the cardiac, skeletal, hepatic tissues, cerebrum and cerebellum of oxidative stress

imposed rats (Misra et al., 2005).

The effect of atrazine in the liver of atrazine-induced male Wistar rats was

ameliorated by the administration of vitamin E (Singh et al., 2010). Thus, it is clear from

the present study that the increase in the vitamin E levels by the leaf extract of

B. monnieri proves its antioxidant potential.

VITAMIN A

The exposure of the slices to the oxidant decreased the levels of vitamin A. On

administration of B. monnieri leaf extract the levels increased significantly when

compared to the control.

35

Ragavan and Kumari (2006b) reported that the oral administration of Trigonella

arjuna reversed the levels of vitamin A to normal in the liver and kidney of diabetic rats.

Vitamin A administration has been reported to prevent hepatic injury caused by CCl4

treatment in rats (Noyan et al., 2006).

The chemopreventive potential of vitamin A and β-carotene during early

hepatocarcinogenensis induced by 5-azacytidine in Wistar rats has been demonstrated

(Sampaio et al., 2007). Consumption of water cress (cruciferous vegetable) can decrease

the damage to DNA and cause a modification in the antioxidant status by increasing

carotenoid concentration in healthy adults (Gill et al., 2007).

Thus, it is perceivable from these studies, that an increase in the levels of vitamin

A by the leaf extract proves the ability of the extract to increase the antioxidant status in

the tissues.

REDUCED GLUTATHIONE

GSH is an important intracellular defense against damage by ROS. The reduced

form of glutathione is necessary to maintain the normal reduced state of cells so as to

alleviate all the injurious effects of oxidative stress (Ahmad et al., 2009).

In the present investigation, a marked depletion in the levels of GSH was observed

when the slices were exposed to the oxidant. The levels improved when the slices were

co-treated with the extract.

Administration of chloroform and methanol extracts of Ichnocarpus frutescens

significantly increased the level of glutathione in the liver of paracetamol-treated rats in a

dose-dependent manner (Dash et al., 2007). Aqueous extract of Phyllanthus amarus

treated rats showed a significant decrease in plasma LPO and a significant increase in

plasma vitamin C, uric acid and GSH levels, and GPx, CAT and SOD activities (Karuna

et al., 2009).

Treatment with Ocimum basilicum and Trigonella foenum-graecum showed

significant increase in reduced glutathione levels in the H2O2 and CCl4 intoxicated goat

liver (Meera et al., 2009). In the present investigation, treatment with the extract

significantly increased the activities of GSH-dependent enzymes like GST and POD,

36

which further strengthens the fact that the extract improves the antioxidant status of the

liver slices exposed to oxidative stress.

Thus, the results obtained revealed that the methanolic extract of B. monnieri

leaves influences favourably the antioxidant status of oxidatively stressed goat liver

slices.

EFFECT OF B. monnieri LEAF EXTRACT ON THE ANTIOXIDANT STATUS

OF EXPERIMENTAL ANIMALS SUBJECTED TO OXIDATIVE STRESS

The in vivo assessment of drug metabolism in experimental animals provides the

most physiologically relevant test system and gives information that relates to the

complexity of sequential and parallel routes of metabolic clearance, not only by multiple

enzymes, but also by multiple organs. The results obtained in the in vitro studies showed

that B. monnieri leaves possessed antioxidant and hepatoprotective activity. Many factors

such as signaling pathways involving hormones, regulatory substances and other

molecules, physiological nature and multiple organ systems play an important role in

influencing the in vivo environment. To confirm the results obtained in vitro, in vivo

studies were carried out using male Wistar rats. Oxidative stress was induced in the

animals by the administration of CCl4 following the induction of CYP2E1 with alcohol

pre-treatment.

Ethanol-induced oxidative stress plays a major role in the mechanisms by which

ethanol produces liver injury (Cederbaum et al., 2009). CCl4, a widely used

hepatotoxicant, is biotransformed by cytochrome P450 systems to produce the

trichloromethyl and trichloromethyl peroxy radicals that causes lipid peroxidation and

thereby, produce liver damage (Sisodia and Bhatnagar, 2010).

Silymarin, an antioxidant flavonoid complex, has long been used in the treatment

of liver diseases. It has been demonstrated that silymarin acts as an antioxidant, reducing

free radical-mediated damage in tissues and inhibiting lipid peroxidation. It has been

reported as having multiple pharmacological activities including antioxidant,

hepatoprotectant, anti-inflammatory, antibacterial, antiallergic, antiviral and

antineoplastic effects (Upadhyay et al., 2007).

37

In the present investigation, male Wistar rats were exposed to oxidative stress and

the effect of the leaf extract on the stress was studied. Liver and kidney were used to

assess the antioxidant potential of B. monnieri. Liver is one of the tissues showing a high

rate of free radical generation with high metabolic capacity and detoxifying capacity. The

other organ, kidney is an excretory organ and is relevant for measuring biochemical

changes and oxidative damage. Thus, in the present study, the oxidative status of both the

organs was studied in rats exposed to CCl4. Ethanol pretreatment sensitizes the liver to

toxic challenge by CCl4 so as to potentiate the liver damage.

The extent of liver damage in different treatment groups were evaluated by the

activities of the liver marker enzymes and the lipid profile in the serum. The results

obtained are discussed below.

SERUM MARKER ENZYMES

Cellular damage exhibits good correlation with enzyme leakage (Sherawat and

Sultana, 2006). Serum AST, ALT, ALP and γ-GT are the most sensitive markers

employed in the diagnosis of hepatic damage. The increase in the activities of these

enzymes in serum and subsequent fall in the tissue might be due to the leakage of these

cytosolic enzymes into the circulatory system, resulting from hepatocellular damage

during ethanol administration. This is indicative of the onset of hepatocellular damage

due to liver dysfunction and disturbance of the biosynthesis of these enzymes, with

alteration in the permeability of liver membrane (Pradeep et al., 2010).

The serum markers analyzed were aspartate transaminase (AST), alanine

transaminase (ALT), alkaline phosphatase (ALP) and γ-glutamyl transpeptidase (γ-GT).

A significant increase in the activities of the serum markers were observed in the groups

treated with ethanol both with and without CCl4.

Opoku et al. (2007) showed that the administration of Rhoicissus tridentata

extracts after CCl4 intoxication in rats resulted in significantly reduced concentrations of

ALT and ASP. CCl4-induced hepatotoxicity in rats, as judged by the raised serum

enzymes, was prevented by pretreatment with the aqueous and methanolic extracts of

Phyllanthus niruri, demonstrating their hepatoprotective action (Harish and

Shivanandappa, 2006).

38

The partially purified petroleum ether extractable fraction of the whole plant

Aerva lanata restored the elevated activities of liver marker enzymes against liver damage

induced by carbon tetrachloride (CCl4) in Sprague Dawley rats (Nevin and Vijayammal,

2005). Oral pretreatment with the chloroform extracts of Terminalia catappa significantly

reduced the increased serum AST and ALT activities in CCl4 treated rats (Tang et al.,

2006).

Oral administration of aqueous suspension of dried latex of Calotropis procera

produced a dose-dependent reduction in the serum levels of liver enzymes AST and ALT

against CCl4 induced hepatotoxicity in rats (Padhy et al., 2007). The treatment with the

alkaloid fraction of Hygrophila auriculata leaves showed a significant decrease in AST,

ALT and ALP activities in serum when compared with CCl4 treated rats (Raj et al.,

2010).

Pretreatment with Wild Panax ginseng C.A. Meyer for 4 weeks completely

abrogated increases in the ALT, AST and LPO levels in the rats, when challenged with

benzo[α]pyrene (Gum et al., 2007). Shim et al. (2010) have reported that ginsan, a

polysaccharide extracted from Panax ginseng, on CCl4-induced liver injury in rats,

markedly suppressed the serum ALT and AST levels.

Pre-treatment and post-treatment with dehydrocavidine, a main active ingredient

of Corydalis saxicola Bunting, significantly prevented increases in serum enzymatic

activities of ALT, AST and ALP in the liver of CCl4 intoxicated rats (Wang et al., 2008b).

Oral administration of curcumin and BDMC-A [bis-1,7-(2-hydroxyphenyl)-hepta-1,6-

diene-3,5-dione] to CCl4-induced rats for a period of three months significantly decreased

the levels of serum marker enzymes AST, ALP and γ-GT (Kamalakkannan et al., 2005).

Resveratrol treatment was able to mitigate hepatic damage induced by acute

intoxication of CCl4 and showed pronounced curative effect and deviated serum

enzymatic variables in rats (Fan et al., 2009). Kim et al. (2005) have reported that the

pretreatment of momordin Ic and oleanolic acid obtained from Kochiae fructus fruit

significantly lowered the increased activities of AST, ALT and γ-GT in CCl4 treated rats.

Rutin prevents changes in the activities of ALT and AST in the serum, liver and heart,

indicating the protective effect of rutin against the hepatic and cardiac toxicity caused by

streptozotocin (Fernandes et al., 2010).

39

The hepatoprotective effects of kahweol and cafestol, coffee-specific diterpenes,

on CCl4-induced liver damage revealed an increase in the serum levels of hepatic enzyme

markers ALT and AST in a dose-dependent manner (Lee et al., 2007b). Yan et al. (2006)

showed that administration of chitosan oligosaccharide, d-glucosamine and N-acetyl-d-

glucosamine on CCl4-induced hepatotoxicity in mice, induced marked change in serum

AST and ALT activities.

In the present study, the administration of B. monnieri leaf extract caused a

significant decrease in the activities of serum marker enzymes (AST, ALT, ALP and

γ-GT) against ethanol-CCl4 induced toxicity, indicating its protective effect. The

protective effect of B. monnieri leaf extract was comparable to that of silymarin, a

standard hepatoprotectant.

LIPID PROFILE

The results of the present study revealed that, there was a significant increase in

the levels of serum cholesterol, triglycerides, free fatty acids and phospholipids in the

alcohol treated group and the levels were further increased in CCl4 and alcohol treated

group. This shows that these lipids are secreted from the liver at an increased rate. The

administration of B. monnieri leaf extract was effective in counteracting the oxidative

stress induced damage by decreasing the serum lipid levels of rats.

Our results are supported by a number of research findings, where plant extracts

have exhibited hypolipidemic effects. Aqueous extract of propolis, a resinous wax-like

beehive product, significantly reduced the levels of serum and tissue triglycerides, serum

cholesterol, total and esterified cholesterol in rat tissue (Bhadauria et al., 2008). An

aqueous extract of Ajuva iva lowered plasma cholesterol and triglyceride levels in

streptozotocin induced diabetic rats (El-Hilaly et al., 2006).

An ethanolic extract of Beta vulgaris roots exhibited significant dose-dependent

hepatoprotective activity against CCl4-induced hepatotoxicity in rats, by showing a

decline in the levels of serum markers, namely cholesterol and triglyceride (Agarwal

et al., 2006). The significant increase in the levels of cholesterol, triglycerides,

phospholipids and free fatty acids in the liver and kidney, caused by the administration of

streptozotocin in rats were brought down to normalcy on treatment with Aloe vera leaf gel

(Rajasekaran et al., 2006). The malathion-treated rats and the malathion plus vitamin-

40

treated group had significantly lower serum triglyceride levels when compared to the

control (Kalender et al., 2010).

ANTIOXIDANT STATUS IN THE LIVER AND KIDNEY OF RATS TREATED

WITH B. monnieri EXTRACT AFTER EXPOSURE TO CCl4

The antioxidant status in the experimental animals was assessed by analyzing the

enzymic and non-enzymic antioxidants in the liver and kidney of the rats exposed to

ethanol-CCl4 treatment.

ENZYMIC ANTIOXIDANTS

Metabolism of ethanol by CYP2E1 results in the production of ROS, leading to

oxidative stress (Chen et al., 2008; Haorah et al., 2008). Ethanol-induced oxidative stress

is the result of the combined impairment of the antioxidant status and production of ROS

(Reddy et al., 2009). Hence, it was felt imperative to study the effects of ethanol and CCl4

and the protective effect of the extract of B. monnieri leaves.

The enzymic antioxidants, namely SOD, CAT, POD, GR and GST, were analyzed

in the liver and the kidney of the experimental rats.

A significant reduction in the activities of all the enzymic antioxidants was

observed when treated with ethanol alone or in combination with CCl4. This effect was

counteracted by the administration of the methanolic extract of B. monnieri leaves and the

standard oxidant silymarin.

There are several reports published in the literature, wherein the antioxidant status

of organs is modulated by herbal extracts and compounds. These reports are in

accordance with our results and some of the salient ones are discussed below.

SOD, CAT and GPx constitute a mutually supportive team of enzymes, which

provide defense against the intermediates of dioxygen. CAT and GPx protect SOD

against inactivation by H2O2 and SOD protects CAT and GPx against superoxide anion

(Parthasarathy et al., 2006).

There was a marked decrease in the percentage inhibition of superoxide

dismutase, catalase and the level of GSH in the liver of CCl4 treated rats when compared

41

with the control group. However, the percentage inhibition of SOD, CAT and the level of

GSH were significantly increased on administration of the aqueous extract of Strychnos

henningsii in a dose-dependent manner (Oyedemi et al., 2010). The administration of the

aqueous extract of the bark of Terminalia arjuna significantly elevated the reduced SOD,

CAT and GST activities in the liver and kidney of CCl4 challenged mice (Manna et al.,

2006).

The depletion in the activities of the antioxidant enzymes SOD, CAT and GPx

when treated with ethanol, was prevented by the methanolic extract of the root of Opuntia

ficus indica f. inermis in rat stomach tissues (Alimi et al., 2010). Administration of hydro

alcoholic extract of Nelumbo nucifera seeds to Wistar rats prior to CCl4 treatment caused

a significant dose-dependent increase in the level of SOD and CAT (Rai et al., 2006).

The co-administration of the ethanol extract of Aquilegia vulgaris or silymarin

resulted in a significant increase in the hepatic antioxidant enzyme activities, which was

significantly reduced after CCl4 administration in male Wistar rats (Jodynis-Liebert et al.,

2009). An ethanolic extract of the whole plant of Amaranthus spinosus was found to

increase the activities of SOD and CAT in CCl4 induced hepatotoxicity in rats (Zeashan

et al., 2008).

When the extract of Pleurotus ostreatus was used to treat rats with CCl4-induced

toxicity, it enhanced the mean activities of CAT, SOD, GPx and GST in kidneys, heart

and brain of rats (Jayakumar et al. 2008). Pretreatment of rats with Cystisus scoparius

plant extract caused a significant increase in the SOD, CAT, GPx, GST and GR activities

in the liver against CCl4 exposure (Raja et al., 2007).

Potato peel extract restored CCl4-induced altered antioxidant enzyme activities in

the liver to control levels (Singh et al., 2008). Aly et al. (2010) demonstrated that vitamin

C treatment to chlorpyrifos intoxicated mice decreased GST activity and normalized

CAT, SOD and G6PD activities in the liver. The co-administration of Kolaviron, a

biflavonoid complex from Garcinia kola seeds, during ethanol treatment ameliorated

hepatic SOD and GST activities (Adaramoye et al., 2009).

All these studies support our results, where the co-treatment of B. monnieri leaf

extract improved the antioxidant status and counteracted CCl4 and ethanol mediated

toxicity in vivo.

42

NON-ENZYMIC ANTIOXIDANTS

The levels of non-enzymic antioxidants such as vitamin C, vitamin A, vitamin E,

GSH and protein thiols were assessed in the liver and kidney of rats treated with CCl4 and

ethanol.

On treatment with alcohol and CCl4 the levels of vitamins C, A, GSH and protein

thiols were found to decrease. Their levels increased on co-treatment with the extract in

both the tissues (liver and kidney). In the case of vitamin E, CCl4 treatment caused a

significant increase in the levels when compared to the untreated controls in the liver. But

the values decreased to a significant extent in the kidney. Vitamin E is a membrane bound

antioxidant. Alcohol treatment might have destroyed the membranes leading to an

increase in the level of vitamin E in the alcohol treated group compared to control.

The methanolic extract of Solanum nigrum berries improved the GSH levels in the

gastric mucosa of rats treated with aspirin to induce ulceration (Jainu and Devi, 2006).

The oral administration of the ethanolic extract of Terminalia arjuna stem bark caused a

significant improvement in the decreased levels of GSH, vitamins C, A and E, total

sulfhydryl and non-protein sulfhydryl in the liver and kidney of alloxan-treated diabetic

rats (Ragavan and Kumari, 2006b). An ethanolic root extract of Tephrosia purpurea

enhanced the levels of vitamins C, E and GSH in DMBA induced hamster buccal pouch

carcinoma (Kavitha and Manoharan, 2006).

Polychlorinated biphenyls significantly diminished the levels of non-enzymatic

antioxidants, vitamins C and E, in a dose- and time-dependent manner in cultured rat

Leydig cells (Murugesan et al., 2008). A significant decrease in the non-enzymic

antioxidants (total sulphydryl groups, reduced glutathione, vitamin C and vitamin E) was

observed in the liver of Cd intoxicated rats, which was reverted by the administration of

diallyl tetrasulfide (Pari et al., 2007). Organotellurium compound induced the in vitro

oxidative stress in the cerebral cortex of rats and provoked a reduction of protein thiol

groups (Penz et al., 2009).

Thus, from the present study, it is clear that the levels of enzymic and non-

enzymic antioxidants, which were reduced on exposure to oxidative stress, were

counteracted on treatment with the leaf extract.

43

The results obtained and the trends observed in both the in vitro (liver slices) and

in vivo (experimental animals) systems were quite similar except SOD and GST activities

in the liver slices. The activities of these enzymes did not show significant elevation in

liver slices, which may be due to the reason, that one hour exposure of the extract to the

oxidant-treated slices, was not sufficient to implicit a siginifcant response.

This observation validates the use of tissue slices as a viable alternative to the use

of live animals in research. The use of such a system would effectively bring down the

number of animals used in biomedical research, at the same time providing valuable clues

about the events occurring in vivo.

EXTENT OF LIPID PEROXIDATION

Lipid peroxidation in biological systems has been considered as one of the major

mechanisms of cell injury in aerobic organisms subjected to oxidative stress.

In the present study, ethanol caused an increase in the extent of LPO in the liver,

which was further increased on CCl4 challenge. The methanolic extract of B. monnieri

leaves brought down the level of LPO and the effect was found to be better compared to

standard antioxidant silymarin.

The treatment with an aqueous extract of Strychnos henningsii was able to lower

the rise in TBARS level dose-dependently in the liver of rats treated with CCl4 (Oyedemi

et al., 2010). The Kigelia africana, Calotropis procera, Hibiscus sabdariffa and

Alchornea cordifolia plant extracts statistically reduced the production of TBARS in a

concentration-dependent manner in the liver of rats with all the tested pro-oxidant-

induced oxidative stresses (Olalye and Rocha, 2007). Alimi et al. (2010) reported that the

methanolic extract of Opuntia ficus indica f. inermis inhibited the increase of MDA in rat

stomach tissues.

The aqueous extract of Mangifera indica L. fruit and gallic acid protected the

isolated rat hepatocytes against cumene hydroperoxide-induced LPO (Pourahmad et al.,

2010). The rats treated with the aqueous extract of Phyllanthus amarus showed a

significant decrease in plasma LPO (Karuna et al., 2009). Gymnema sylvestre extract

showed a significant hepatoprotective effect in irradiated mice by significantly lowering

44

the radiation induced LPO, measured as the levels of malondialdehyde (Bhatia et al.,

2008).

The administration of a hydro alcoholic extract of Nelumbo nucifera seeds to

Wistar rats caused a significant decrease in the level of thiobarbituric acid reactive

substances, when compared to CCl4 treated control in both liver and kidney (Rai et al.,

2006). The ethanolic extract of whole plant of Amaranthus spinosus showed potent

hepatoprotective activity against carbon tetrachloride induced hepatic damage in rats and

significantly altered malondialdehyde levels (Zeashan et al., 2008).

The oral administration of Arachniodes exilis at different doses resulted in

significant improvement of the levels of malondialdehyde in the liver of mice (Zhou et al,

2010). The treatment with repeated doses of either garlic or onion juices could restore the

concentration of thiobarbituric acid reactive substances to their normal levels in the

plasma, liver, brain, testes and kidney of the alloxan-induced diabetic rats (El-Demerdash

et al., 2005).

The treatment with the protein isolate of Phyllanthus niruri L. significantly altered

the increased lipid peroxidation in CCl4 induced liver changes to almost normal

(Bhattacharjee and Sil, 2007). The whole plant of Piper nigrum, Myristica fragrans

(Chatterjee et al., 2007) and holy basil (Juntachote et al., 2007) decreased the formation

of TBARS in the liver of experimental rats.

RADICAL SCAVENGING ACTIVITY

The overall antioxidant status of tissues treated with alcohol and CCl4 in the

presence and absence of the B. monnieri leaf extract was assessed by the ability of the

liver homogenate to scavenge the free radical DPPH.

The DPPH scavenging effect was not significantly high in alcohol and CCl4

treated groups as well as in the groups treated with the extract. Silymarin treated groups

alone showed slightly higher values compared to the controls.

An aqueous extract from Choerospondias axillaris fruit showed a high antioxidant

effect, especially scavenging of DPPH anions in d-galactose induced mouse aging model

(Wang et al., 2008c). The methanolic extracts of green tea and Ficus bengalensis

45

exhibited antioxidant property as reflected by their higher hydrogen donating ability

(Manian et al., 2008).

Telluroacetylenes showed effect of scavenging DPPH radicals in sodium

nitroprusside-induced oxidative damage in mouse brain (Souza et al., 2009). Menthe

spicata extract exhibited good antioxidant activity compared to the synthetic antioxidant,

butylated hydroxy toluene (Kannat et al., 2007).

From the results of the present study, it is clear that the treatment with B. monnieri

leaf extract improved the antioxidant status in the liver and kidney of rats and can render

protection against alcohol-CCl4 induced toxicity.

Therefore, the results of this study show that B. monnieri can be proposed to

protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective

effect might be correlated with its antioxidant and free radical scavenging effects.

PHASE IV

Various phytochemical components especially polyphenols (such as flavonoids,

phenolic acids, tannins, etc.) are known to be responsible for the free radical scavenging

and antioxidant activities of plants. Phenolic substances possess many biological effects.

These effects are mainly attributed to their antioxidant activities in scavenging free

radicals, inhibition of peroxidation and chelating transition metals (Nickavar et al., 2007).

The preliminary phytochemical analysis of the leaves of B. monnieri revealed the

presence of alkaloids, phenolics, flavonoids and saponins. To confirm the nature of the

active component present in B. monnieri leaves, spectral analysis (UV, HPLC, HPTLC,

IR and GC-MS) were carried out, which confirmed the presence of alkaloids, phenolics,

flavonoids and saponins.

Phytochemical screening of the leaves and roots of C.alata revealed the presence

of alkaloids, carbohydrates, tannins, saponins, phenols, flavonoids, anthraquinones and

cardiac glycosides (Makinde et al., 2007). Phytochemical screening of Euphorbia

heterophylla Linn, showed that the crude plant material contained some secondary

metabolites such as saponins, flavonoids and tannins (Falodun et al., 2008). Tridax

46

procumbens has antioxidant activity which was established to correspond to the amount

of total phenolic content of the plant samples (Habila et al., 2010).

Ramesh et al. (2009) reported that B. monnieri contains alkaloids (nicotine,

brahmine, herpestine), saponins (hersaponin, betulic acid, bacosides A, B, C and D) and

other chemicals like stigmastanol, β-sitosterol and stigmasterol. TLC analysis of the

Acacia nilotica (L) wild extract showed two spots at Rf values 0.48 and 0.64 (Singh and

Arora, 2009). Thin layer chromatographic analysis of ethanolic extract of B. monnieri

plant showed the presence of alkaloids and natural lipids (Sengupta et al., 2008). One of

the most common biological properties of alkaloids is their toxicity against cells of

foreign organisms. These activities have been widely studied for their potential use in the

elimination and reduction of human cancer cell lines. Alkaloids which are one of the

largest groups of phytochemicals in plants have amazing effects on humans and this has

led to the development of powerful pain killer medications (Igbinosa et al., 2009).

The UV spectrum of the flowers of Abelmoschus manihot showed peaks at 204,

210, 228, 262, and 277nm (Lai et al., 2007). Luteolin, a flavonoid widely occurring in

many medicinal plants, was quantified by Srinivasa et al. (2004) in three important herbal

drugs namely fruit of Cuminum cyminum, whole plant of B. monnieri and the flower of

Achillea millefolium by HPTLC.

The chromatographic analyses of Spirulina organic extracts with TLC and HPLC

showed that carotenoids, chlorophyll-derived and phenol compounds were the main

constituents (El-Baky et al., 2008). A simple and sensitive reversed phase high

performance liquid chromatographic (HPLC) procedure identified the presence of Bacopa

saponins present in the extracts of the medicinal plant, B. monnieri (Ganzera et al., 2004;

Deepak et al., 2005; Murthy et al., 2006). Zehl et al. (2007) analysed the presence of two

dammarane-type triterpenoid saponins from B. monnieri by the application of a variety of

MS techniques.

These results are in accordance with the results of the present study, where the

presence of alkaloids, phenolics, flavonoids and saponins has been confirmed.

The outcome of the study, thus, provides a significant contribution in establishing

the antioxidant properties of B. monnieri leaves, which includes not only radical

scavenging properties, but also biomolecular protection. This effect is also observed

47

in vivo, wherein no toxic effects were observed. The bioactivity of antioxidant and

anticancer property of the plant is possibly due to the presence of phytochemicals.

Thus, the present study emphasizes the medicinal value of B. monnieri leaves,

against oxidant-induced damage in vitro and in vivo.

The results obtained in the present study and the conclusions drawn from them are

presented in the next chapter.