chapter 5 discussion -...

Chapter 5Discussion

In this fashion, knowledge begets questions which beget newtechnology which provides answers; which in turn beget questions. Thisis the implacable carousel of research.

Richard Fortey (Earth: An Intimate History)

Bioprospection of Schleichera oleosa (Lour.) Oken for its Antiproliferative and Antioxidative PotentialV Discussion

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The process of carcinogenesis initiates from a set of mutations induced by carcinogens,

that affect regulation of proliferation and involves series of molecular events which trigger

progressive changes from preinvasive histological transformation to an invasive neoplastic

process (Mao et al., 1997; Hahn and Weinberg, 2002; Zandwijk, 2005). Chemopreventive

intervention involves a pharmacological approach that utilizes natural, synthetic or biologic

chemical agents with an objective to reverse, suppress or prevent carcinogenic progression. The

efficacy of a chemopreventive agent depends on its ability to inhibit the development of invasive

cancer, either by blocking the transformative, hyperproliferative and inflammatory processes that

initiate carcinogenesis or by arresting or reversing the progression of premalignant cells to

malignant by suppressing angiogenesis and metastasis. The appropriate use of chemopreventive

agent depends on the understanding of its mechanism of action at all levels i.e. at molecular,

cellular, tissue and organs levels, as well as in the animal as a whole (Sporn and Suh, 2000;

Dorai and Aggarwal, 2004; Mukhtar, 2012).

With increasing molecular mechanistic evidences coupled with considerations of quality,

safety and efficacy, phytochemicals from nondietary plants, such as herbs, have emerged as a

new and promising source of anticancer remedies or as adjuvant of chemotherapeutic drugs to

enhance their efficacy and to ameliorate their side effects (Li-Weber, 2009; Shu et al., 2010).

Bioactive phytocompounds are a potential alternative source to synthetic drugs which have been

plagued by unwanted side effects, toxicity and inefficiency, among other problems. Numerous

case-controlled epidemiological studies as well as experimental animal studies have also

demonstrated an inverse relationship between the intake of phytochemicals and cancer incidence

(Amin et al. 2009; Ma and Chapman 2009; Kumar et al., 2011). Phytochemicals use a plethora

of antisurvival mechanisms, boost the host's anti-inflammatory defense and sensitize malignant

cells to cytotoxic agents (D'Incalci et al., 2005). They have been reported to interfere with

specific stage of carcinogenic process, by modulating the proteins required in one or more signal

transduction pathways related to cellular proliferation, differentiation, apoptosis, inflammation,

angiogenesis and metastasis and thus inhibit, reverse or retard tumorigenesis (Surh, 2003; Sarkar

and Li, 2004; Ramos, 2008). Studies to date have demonstrated that phytochemicals can have

complementary and overlapping mechanisms of action, including scavenging of oxidative

agents, stimulation of the immune system, regulation of gene expression in cell proliferation and


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apoptosis, hormone metabolism, and antibacterial and antiviral effects (Waladkhani and

Clemens, 1998; Dasgupta and De, 2007). Current strategies for the evaluation of anticancer

phytochemicals are thus based on: (1) cell cycle and apoptosis regulation; (2) anti-oxidative

stress and anti-inflammatory activities; (3) drug resistance of cancer cells; and (4) specific

molecular targets targeting carcinogenesis and metastasis (Shu et al., 2010).

Source: Mehta et al. (2010)

Figure 5.1: The process of carcinogenesis involves initiation, promotion and progression.Progression is shown here to include the growth of malignant tumors, invasion and metastasis. Inthis figure for each of the stages, various major actions of phytochemicals involving signalingpathways are summarized (Mehta et al., 2010).

Increase in ROS formation, together with decrease in antioxidant defense, causes

oxidative stress that also plays a pivotal role in carcinogenesis. Although ROS have a short half

life, they can react with DNA, proteins and unsaturated fatty acids. This results in oxidative

damage to the cell that includes DNA strand breaks and formation of protein-protein and protein-

DNA crosslinks. Oxidation of lipids can result in lipid peroxides which persist much longer in


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the cell. They can initiate radical chain reactions and thus enhance the oxidative damage

(Steenvoorden and Henegouwen, 1997). Dietary chemopreventive phytochemicals have been

shown to protect the cells from the harmful effects of ROS by scavenging them, binding to metal

ions, quenching singlet and triplet oxygen or decomposing peroxides and thus inhibit their

reactions with biological entities viz. nucleic acids, proteins and lipids. These phytochemicals

also induce the expression of different antioxidant and detoxification enzymes like catalase, SOD

and glutathione peroxidase in the target tissues (Liu et al., 2003; Al-Dabbas et al., 2006; Surh et

al. 2008; Kilani-Jaziri et al., 2011). However, although collectively these phytochemicals are

good antioxidants, the roles and effect of individual compounds are often not well known and

elucidated. Moreover, a single plant could contain highly complex profiles of these compounds,

which sometimes are labile to heat, air and light, and they may exist at very low concentrations

in the plants. This makes the separation and detection of these protective phytochemicals a

challenging task (Tsao and Deng, 2004).

Keeping in mind the significant importance of phytochemicals as chemopreventive as

well as chemotherapeutic agents, coupled with the fact that the plant, Schleichera oleosa, has not

been explored for its bioactivities, the present study was planned to evaluate the protective

effects of different extracts and the active constituents from bark, leaves and roots of Schleichera

oleosa in different in vitro antioxidative and antiproliferative assays. The antioxidative effect

was evaluated in radical scavenging assays using DPPH free radical scavenging assay,

deoxyribose degradation assay, lipid peroxidation assay and plasmid nicking assay. The

reduction potential and chelating ability were also determined using reducing power and

chelating power assays. In vitro cytotoxicity against a panel of human cancer cell lines was

evaluated using Sulphorhodamine B (SRB) dye assay and MTT assay. Based on in vitro

cytotoxicity study profile, the methanolic extracts from bark and leaves of S. oleosa were subjected

to bioactivity guided fractionation to isolate the active constituents. This led to the isolation of 2

compounds, SOL-1 and SOL-2, from the methanolic extract of leaves (MEL) and 5 compounds,

SOB-1, SOB-2, SOB-3, SOB-4 and SOB-5, from the methanolic extract of bark (MEB). The

structures of isolated compounds were characterized and elucidated by NMR (1H, 13C, DEPT

135o), MS and FTIR studies and identified as chlorogenic acid (SOL-1), quercetin (SOL-2),

oleanolic acid (SOB-1), ursolic acid (SOB-2), lupeol (SOB-3), betulinic acid (SOB-4) and


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betulin (SOB-5). They were subjected to antiproliferative assays that involve various mechanistic

parameters typical of apoptosis viz. morphological analysis by microscopy, DNA fragmentation

analysis, flow cytometric analysis, colorimetric analysis for determination of caspases activity,

immunoblotting and determination of topoisomerase inhibitory activity.

The results obtained in different in vitro assays are discussed in the following sections in

the light of reports available in the literature:

5.1 In vitro Antioxidative Studies

In recent years, the studies on ‘‘oxidative stress’’ and its adverse effects on human health

have become a subject of considerable interest. Many recent studies have shown that exposure of

organisms to exogenous and endogenous factors generates a wide range of reactive oxygen

species (ROS), resulting in homeostatic imbalance that is implicated in several diseases, such as

cancer, arthritis, arteriosclerosis, heart disease, diabetes, inflammation, brain dysfunction and

acceleration of the ageing process (Halliwell, 1996; Gordon, 1996; Sies, 1997; Thomas and

Kalyanaraman, 1998; Halliwell and Gutteridge, 1999; Bonnefont et al., 2000; Feskanich et al.,

2000; Tripathi et al., 2007). It has been well documented in various previous studies that

antioxidant principles from herbal resources like terpenes, flavonoids, alkaloids etc. possess the

ability to protect the body from damage caused by free radical induced oxidative stress (Gerber

et al., 2002; Kris-Etherton et al., 2002; Serafini et al., 2002; Di Matteo and Esposito, 2003;

Ozsoy et al., 2008). In the present study different in vitro antioxidant radical systems are used to

elucidate the full profile of antioxidant potential of extracts and active constituents of S. oleosa.

The DPPH is a stable free radical by virtue of the delocalisation of the spare electron over

the molecule as a whole, so that the molecules do not dimerise and has been widely accepted as a

tool for estimating free radical scavenging activities of antioxidants. DPPH (purple in color) is

known as a good hydrogen abstractor yielding DPPH-H (yellow in color) as a byproduct, thus

measuring the hydrogen donating ability of the extracts (Sa´nchez-Moreno, 2002; Molyneux,

2004; Ionita, 2005). Out of different extracts of bark, it was found that methanol extract (MEB)

showed the maximum inhibition with lowest IC50 value of 70.86 µg/ml which was closely

followed by ethyl acetate extract (EEB) with IC50 value of 75.39 µg/ml. Hexane extract (HEB)


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showed the least activity among all the bark extracts. Among the leaves extracts, MEL was the

most potent with IC50 value of 57.99 µg/ml while among root extracts MER showed maximum

activity with IC50 value of 57.35 µg/ml. The hexane and chloroform extracts from different parts

of the plant showed negligible activity in this assay.

The potent activity of the extracts in DPPH radical scavenging assay may be attributed to

the presence of polyphenols as it was noted that the methanol extracts showed higher phenolic

and flavonoid content, as compared to other extracts. The studies conducted by Miliauskas et al.

(2003), Sultana et al. (2007) and Yuan et al. (2009) established positive linear correlation

between DPPH radical scavenging activity and total phenol and flavonoid content. Similar

inference was reported by Chen et al. (2007), that heating of 50% ethanolic extract of Dioscorea

alata resulted in the loss of phenolic content and thus reduced its DPPH radical scavenging

activity. Furthermore, Liu et al., 2004 reported that the number and position of free phenolic

hydroxyl groups makes statistically significant contribution to the DPPH radical scavenging

activities of p-terphenyls obtained from edible mushrooms. The p-terphenyls with greater

number of OH groups exhibited higher radical scavenging activity in DPPH assay while in

compounds containing the same number of OH groups, with two pairs of OH groups in para-

positions with respect to each other showed stronger DPPH radical-scavenging activity than

compounds with four isolated OH groups. The order of activity of most active extracts from S.

oleosa bark, leaves and roots, in terms of IC50 values is summarized as: MER (57.35 µg/ml) >

MEL (57.99 µg/ml) > MEB (70.86 µg/ml)

Fe (III) reduction is often used as an indicator of electron-donating activity, which is an

important mechanism of phenolic antioxidant action and can be strongly correlated with other

antioxidant properties. Phytochemicals, which have reduction potential, react with potassium

ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+), which then reacts with ferric chloride

to form ferric ferrous complex that has an absorption maximum at 700 nm (Dorman et al., 2003;

Ozsoy et al., 2008; Singhal et al., 2011). The results of the assay in terms of reduction (%) were

calculated by comparing the absorbance of different extracts with that of the highest

concentration of gallic acid. Among the bark extracts, though the effect of EEB and MEB was

almost similar at 200 µg/ml concentration with reduction potential of 88.43% and 87.04%


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respectively but EEB had lower IC50 value (60.08 µg/ml) compared to MEB (93.70 µg/ml). In

case of leaves, MEL exhibited maximum reduction potential with IC50 value of 56.22 µg/ml,

which, among all extracts, is closest to gallic acid (40.07 µg/ml). The extracts from roots showed

lower reduction potential, compared to bark and leaves, wherein EER was found to be most

active with IC50 value of 85.94 µg/ml.

The results obtained in the present study are in accordance with the reports in literature

that establishes direct correlation between antioxidant properties and reducing power of plant

extracts (Amarowicz et al., 2004; Chung et al., 2005; Ferreira et al., 2007; Kanatt et al., 2007;

Goyal et al., 2010). The presence of reductones in the polyphenol rich extracts is associated with

their reducing properties and exerts antioxidant action by terminating the free radical chain

reaction by donating a hydrogen atom (Gordon, 1990; Osawa, 1994; Duh et al., 1999; Sahaa et

al., 2008). The extracts with high reducing power thus indicate their potential antioxidative

activity in other assay systems. The order of activity of most active extracts, in terms of IC50

values is summarized as: MEL (56.22 µg/ml) > EEB (60.08 µg/ml) > EER (85.94 µg/ml)

Hydroxyl radicals are extremely reactive oxygen species, capable of modifying almost

every molecule in the living cells by abstracting hydrogen atoms from biological molecules. This

radical has the capacity to cause strand damages in DNA leading to carcinogenesis, mutagenesis

and cytotoxicity (Arouma et al., 1987; Esmaeili and Sonboli, 2010). Hydroxyl radicals are

produced in vivo by Fenton-type reactions, in which transition metals like iron reduce hydrogen

peroxide and reducing agents such as ascorbic acid, which can accelerate their formation by

reducing Fe3+ ions to Fe2+. In deoxyribose degradation assay, hydroxyl radicals generated by

reaction of Fe3+-EDTA complex with H2O2 in the presence of ascorbic acid, were assessed by

monitoring the degraded fragments of deoxyribose, through malonaldehyde (MDA) formation

which upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added “radical

scavengers” compete with deoxyribose for the hydroxyl radicals produced and diminish

chromogen formation (Halliwell et al., 1987; Puppo, 1992; Li and Xie, 2000).

This assay was performed under two conditions i.e. in the presence or absence of EDTA

to access the role of any substance as hydroxyl radical scavenger or as metal chelator


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respectively and thus derive two separate inferences regarding their mechanism of action. In case

of presence of EDTA, condition referred to as ‘‘non-site-specific assay’’, EDTA forms a

complex with iron (III) and ˙OH are generated in solution. However, in the “site-specific assay”,

EDTA is not available, therefore, iron (III) can bind directly to the deoxyribose molecule and

produce hydroxyl radicals at this site itself. If any substance inhibits the deoxyribose degradation

more efficiently in absence of EDTA, then there is possibility that this drug is chelating the iron

ion and also trapping the hydroxyl radicals (Halliwell et al., 1987; Tiwari and Tripathi, 2007).

In the present investigation, the extracts from S. oleosa indicated the moderate ability to

bind with iron ions but a strong potential for direct scavenging action for the ˙OH thus

suggesting their greater role as radical scavengers rather than as metal chelators. Among the bark

extracts, methanol extract (MEB) was most effective in both non-site specific and site specific

assays with IC50 value of 41.56 µg/ml and 71.76 µg/ml respectively. In case of leaves, again the

methanol extract (MEL) was most potent with lowest IC50 values of 43.90 µg/ml and 64.02

µg/ml in non-site specific and site specific assays respectively. Ethyl acetate extract (EER) was

most effective, among different root extracts, in both non-site specific and site specific assays

with IC50 value of 48.70 µg/ml and 61.73 µg/ml respectively. Among different constituents

isolated from MEB, SOB-1, SOB-2 and SOB-3 exhibited strong activity in non-site specific

assay, with SOB-1 showing the lowest IC50 value of 35.22 µg/ml closely followed by SOB-2

with IC50 value of 43.42 µg/ml. In site specific assay, SOB-1, SOB-2 and SOB-3 showed

moderate activity with SOB-2 and SOB-1 showing comparable IC50 values of 79.15 µg/ml and

82.69 µg/ml respectively. SOB-4 and SOB-5 showed weak effect in both the assays. In case of

active constituents isolated from MEL, both SOL-1 and SOL-2 exhibited strong activity, both in

the presence and absence of EDTA. In non-site specific assay, SOL-1 and SOL-2 showed

comparable IC50 values of 24.10 µg/ml and 27.68 µg/ml respectively while in site specific assay,

SOL-1 and SOL-2 exhibited IC50 values of 28.77 µg/ml and 46.29 µg/ml respectively. The order

of activity of most active extracts from S. oleosa bark, leaves and roots, in terms of IC50 values in

both non-site specific and site specific assays is summarized as:

Non-site Specific Assay: MEB (41.56 µg/ml) > MEL (43.90 µg/ml) > EER (48.70 µg/ml)

Site Specific Assay: EER (61.73 µg/ml) > MEL (64.02 µg/ml) > MEB (71.76 µg/ml)


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Hydroxyl radical trapping potential of the extracts and their active constituents was

further confirmed by evaluating ˙OH induced damage to the bases or the deoxyribosyl backbone

of DNA, using plasmid nicking assay. Hydroxyl radicals, generated by Fenton’s reaction, would

act upon supercoiled plasmid DNA (Form I) and induce single strand or double strand breaks,

thus converting it into open circular (Form II) or relaxed (Form III) DNA. The results of the

plasmid nicking assay were in accordance with those of deoxyribose degradation assay and thus

confirmed the ˙OH scavenging potential of the extracts and active constituents isolated from S.

oleosa. All the extracts, except hexane and chloroform extracts from bark, leaves and roots of the

plant, were effective in protecting the supercoiled pBR322 from being degraded to Form II and

Form III DNA. Similarly among the active constituents isolated from MEB and MEL, all, except

SOB-4 and SOB-5, showed remarkable a DNA protective activity by scavenging hydroxyl

radicals.

The results of the study were in agreement with the previous studies that the enrichment

of phenolic compounds within plant extracts contribute significantly to ˙OH scavenging potential

of the extracts (Lopes at al., 1999; Russo et al., 2000; Zhao et al., 2005; Singh et al., 2007;

Özyürek et al., 2008). The strong protective effect of polyphenols isolated from leaves (SOL-1

and SOL-2) against ˙OH mediated damaging effect, both in the presence and absence of EDTA,

corroborate with the above findings. In case of terpenoids isolated from bark (SOB-1, SOB-2 and

SOB-3), the effect was more pronounced in presence of EDTA, as they are capable to scavenge

˙OH present in the free solution and thus protect the degradation of deoxyribose (detector

molecule) to thiobarbituric acid reactive material (Kammeyer, 1999; Graßmann, 2005; Kaur et

al., 2008a). Lee at al. (2002) reported that the ethanol extract of stem of Opuntia ficus-indica

(OFS) strongly inhibited hydroxyl radical-induced deoxyribose degradation in both site-specific

and non-site-specific assays as well as in plasmid nicking assay. These results proved the

excellent antioxidant activity of OFS extract in cell-free ROS-generating systems. Another study

by Cao et al. (2008) also demonstrated that that the ethanol extracts of Fagopyrum esculentum

and Fagopyrum Tartaricum can effectively inhibit non-site-specific and site-specific

deoxyribose degradation and also protects hydroxyl radical-mediated DNA strand breaks of

supercoiled pBR322 plasmid. Similar results were reported by Singh and coworkers (2009)

wherein the extracts of Acacia nilotica prevented strand break formation in supercoiled plasmid


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DNA and protein oxidation and thus exhibited strong and effective radical scavenging activity in

both deoxyribose degradation and plasmid nicking assays.

Hydroxyl radicals are also capable of the quick initiation of lipid peroxidation (LPO)

process by abstracting hydrogen atoms from polyunsaturated fatty acids. Lipid peroxidation

mainly occurs in biomembranes where the content of unsaturated fatty acids is relatively high

and leads to the oxidative destruction of cellular membrane (Kappus, 1991; Esterbauer et al.

1992; Cheesemen and Slater 1993). A modified thiobarbituric acid-reactive species (TBARS)

assay was used to measure the lipid peroxide formed, using egg yolk homogenates as lipid rich

media. Malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated

fatty acids reacts with thiobarbituric acid (TBA) yielding a pinkish red chromogen with an

absorbance maximum at 532 nm (Janero, 1990; Halliwell and Chirico, 1993; Ruberto et al.,

2000; Tiwari and Tripathi, 2007). Among the bark extracts, methanol extract (MEB) was found

to be most potent with lowest IC50 value of 26.99 µg/ml while in case of leaves, ethyl acetate

extract (EEL) exhibited lowest IC50 value of 46.60 µg/ml and thus was most effective. Different

extracts from roots showed moderate effect in comparison to bark and leaves, where the

methanol extract (MER) was found to be most effective with IC50 value of 68.46 µg/ml. Among

the active constituents isolated from MEB, SOB-2 was most effective with lowest IC50 value of

42.13 µg/ml while SOB-1 and SOB-3 showed comparable activity with IC50 values of 46.70

µg/ml and 46.86 µg/ml, respectively. SOB-4 and SOB-5 were found to be weak peroxyl radical

scavengers. In case of active constituents isolated from MEL, SOL-2 exhibited strong activity

with IC50 value of 9.13 µg/ml, which was even lower than gallic acid (15.35 µg/ml) while SOL-1

showed IC50 value of 34.63 µg/ml. The extracts of S. oleosa and their active constituents are

effective in preventing lipid peroxidation by scavenging peroxyl radicals.

The results of the study are in concordance with the findings reported in the literature that

attribute the protective effect of the S. oleosa to high polyphenolic content of the extracts (Meyer

et al. 1998; Chen et al. 2001; Waddington et al. 2004; Lecumberri et al. 2007; Cooper et al.

2008). Polyphenols can inhibit lipid peroxidation either by acting as chain breaking hydrogen

donors or as metal chelators (Ahrene and O’Brien 1999). Although the extracts exhibited high

phenolic content but were found to be weak chelators therefore hydrogen donating property of


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the polyphenolic compounds may be responsible for the inhibition of free radical induced lipid

peroxidation (Yen et al. 1993). The polyphenolic compounds from leaves (SOL-1 and SOL-2) as

well as triterpenoids from bark (SOB-1, SOB-2 and SOB-3) also showed effective peroxyl

radical scavenging activity. Kaur et al. (2008b) reported that polyphenolic rich fractions of

Chukrasia tabularis extracts showed a remarkable protective effect against Fe3+ induced lipid

peroxidation by scavenging peroxyl radicals. Literature survey has also established the protective

effect of terpenoids in inhibiting lipid peroxidation (Jayaprakasam et al., 2007; Ramachandran

and Prasad, 2008; Thoppil and Bishayee, 2011; Kumari and Kakkar, 2012). The order of activity

of most active extracts from S. oleosa bark, leaves and roots, in terms of IC50 values is

summarized as: MEB (26.99 µg/ml) > EEL (46.60 µg/ml) > MER (68.46 µg/ml)

Iron and other transition metals (copper, chromium, cobalt, vanadium, cadmium, arsenic,

nickel) promote oxidation by acting as catalysts of free radical reactions. These redox-active

transition metals transfer single electrons during changes in oxidation states. Chelation of metals

by certain compounds decreases their prooxidant effect by reducing their redox potentials and

stabilizing the oxidized form of the metals (Reische et al., 2008; Končić et al., 2011). The

chelating power assay determines the ability of substance to chelate ferrous ions (Fe+2) by

measuring the decrease in the absorbance at 562 nm of the Fe (II)-ferrozine, a purple colored

complex (Carter, 1971; Dinis et al., 1994; Lim et al., 2007). In the present study, the results of

metal chelating ability suggested the inefficiency of extracts of S. oleosa to chelate metal ions.

Among all the extracts, ethyl acetate extract from roots (EER) was found to show comparatively

good activity with IC50 value of 142.72 µg/ml. EDTA a known chelator, showed IC50 value of

43.06 µg/ml while all other extracts exhibited negligible Fe+2 chelation activity with IC50>200

µg/ml.

The results of the metal chelation assay are supported by earlier report by Zhao and

coworkers (2006), in which it has been shown that the extract of Salvia miltiorrhiza possessed

strong reducing power and high scavenging activities against free radicals including superoxide

anion, hydroxyl and DPPH radicals but Fe+2 chelation activity was undetectable at the tested

concentrations (Zhao et al., 2006). Another study involving analysis of phytochemical and

antioxidant activities of ethanolic extracts from the twigs Cinnamomum osmophloeum also


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showed that the extracts showed poor metal chelating activity in spite of showing a remarkable

activity in DPPH, NBT, reducing power and lipid peroxidation assays (Chua et al., 2008). Moran

et al. (1997) studied the phenolic compounds from soyabean nodules and other legume tissues

for their ability to form complex with iron. It was found that though all phenolics tested were

able to show potent effect in radical scavenging assays but only those having catechol,

pyrogallol, or 3-hydroxy-4-carbonyl groupings were potent chelators and reductants of Fe3+ at

pH 5.5. According to Perron and Brumaghim (2009), the ability of the polyphenolic compounds

to chelate metal ions, depends on the pH as well as on the structure of the phenolic compound.

The results of the antioxidative assays, in the present study, corroborated with the reports

in literature. Ozsoy et al. (2008) evaluated the antioxidant activities of Smilax excelsa L. leaves

extracts using different antioxidant assays along with the levels of total phenolics, total

flavonoids and anthocyanins. They found that extracts of S. excelsa leaves contain high levels of

phenolic compounds and were capable of inhibiting lipid peroxidation, directly quenching free

radicals to terminate the radical chain reaction, acting as reducing agents and chelating transition

metals to suppress the initiation of radical formation. Li et al. (2008) evaluated antioxidant

capacities of 45 medicinal plants using ferric reducing antioxidant power (FRAP) and Trolox

equivalent antioxidant capacity (TEAC) assays and found strong correlation between the values

obtained in two assays (R2=0.9348). A high correlation between antioxidant capacities and their

total phenolic contents (R2=0.8672) indicated that phenolic compounds were a major contributor

of antioxidant activity of these plants. A similar correlation between the antioxidant activity and

total phenolic and flavonoid content has been reported in many other studies (Dorman and

Hiltunen, 2004; Kumaran and Karunakaran, 2007; Makris et al., 2007; Surveswaran et al., 2007;

Kalogeropoulos et al., 2009; Sacan and Yanardag, 2010; Michel et al., 2012).

The results further revealed that the extracts and their active constituents showed

different antioxidant activities in dose dependent manner. Yeşilyurt et al. (2008) reported that,

the acetone extract of the aerial parts of S. cedronella showed a high radical scavenging and

metal chelation ability in a dose dependent manner in DPPH and Fe2+–ferrozine test system

respectively but insignificant inhibition of lipid peroxidation in β-carotene–linoleic acid test

system. Further, phytochemical investigation led to the isolation of 3-methoxy-4-hydroxymethyl


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coumarin together with p-hydroxyphenylethyl docosanoate and two triterpenoids (oleanolic acid

and betulinic acid). The fact that the methanol and ethyl acetate extracts of S. oleosa bark, leaves

and roots were more potent than other extracts while hexane extract showed weak activity in all

antioxidant assays is supported by the study of Tachakittirungrod and coworkers (2007). The

results indicated that the methanol fraction of guava leaf extracts possessed the highest

antioxidant activity by free radical scavenging and reducing of oxidized intermediates, followed

by butanol and ethyl acetate fractions, while the hexane fraction showed the lowest antioxidant

activity. The phenolic content in guava leaf fraction was reported to play a significant role on the

antioxidant activity via reducing mechanisms. Another study by Razali et al. (2008) assessed the

total phenolic content and antioxidant activities of methanol, hexane and ethyl acetate extracts of

the shoots of Anacardium occidentale wherein their potency was found in the order of methanol

> ethyl acetate > hexane. The high levels of phenolic compounds in methanol extract were

responsible for its potent activity in scavenging the DPPH and ABTS radicals, superoxide anion

and nitric oxide as well as in reducing ferric ions whereas the hexane extract contained the

weakest antioxidants.

In the present investigation, different active constituents isolated from methanolic

extracts of the bark and leaves of S. oleosa also showed effective antioxidant activity except

SOB-4 and SOB-5. The isolated compounds identified as phenols and terpenoids, exhibited

potent radical scavenging, reducing power and metal chelation abilities. Likewise, Haraguchi et

al. (1997) demonstrated effective radical scavenging activity of sesquiterpenoids (7-hydroxy-3,4-

dihydrocadalin, 7-hydroxycadalin) and flavonoids (quercetin, kaempferol and their glycosides)

isolated from Heterotheca inuloides in DPPH radical scavenging, superoxide radical scavenging

and lipid peroxidation assays. Various other studies have also established the role of different

classes of active compounds, including terpenoids, flavonoids and phenolic acids, as potent

antioxidants (Pietri et al., 1997; Boveris and Puntarulo, 1998; Lu and Foo, 2001; Choi et al.,

2002; Grassmann et al., 2002; Prakash et al., 2007; Dioufa et al., 2009; Yuan et al., 2012)

Though chemical assays do not reflect the cellular physiological conditions but the

potency of the plant extracts in cell free experimental system ascertains their role as potential

source of natural antioxidants. Although, the antioxidant activity found in in vitro experiments is


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only indicative of the potential health benefit, these results remain important as the first step in

screening the antioxidant activity of S. oleosa bark, leaves and roots.

5.2 In vitro Antiproliferative Studies

Natural products have been isolated as biologically active compounds with great

therapeutic potential as they are involved in various metabolic processes including free radical-

scavenging, regulation of gene expression in cell proliferation, cell-cycle arrest and apoptosis

induction, modulation of enzyme activities in detoxification, and stimulation of the immune

system (Pessoa et al., 2006; Ramos, 2008; Nzaramba et al., 2009). Compelling data from in vitro

and in vivo laboratory studies, epidemiological investigations and human clinical trials indicate

that these phytochemicals have important effects on cancer chemoprevention and therapy (Aziz

et al., 2003; Galati and O'Brien, 2004; Russo, 2007). The need for biologically active compounds

with low profiles of adverse reactions compared to synthetic chemotherapeutic drugs has

triggered an extensive investigation of herbal phytochemicals and their mechanisms of action

(Issa et al., 2006). The present study was thus aimed at determining the in vitro cytotoxicity

profile of different extracts and their active constituents of S. oleosa against various cancer cell

lines and elucidating mechanism of action of isolated compounds.

MTT assay is a tetrazolium-based colorimetric assay where MTT is reduced by

dehydrogenases and reducing agents present in metabolically active cells to yield water insoluble

violet-blue formazan crystals which are dissolved in organic solvents and measured

spectrophotometrically (van Meerloo et al., 2011; Stockert et al., 2012). The assay was

performed on HL-60 (human pro-myelocytic leukemia) cell line. The results of the assay showed

that among the bark extracts methanol extract (MEB) was most effective with lowest IC50 value

of 24.37 µg/ml. Among the leaves extracts again the methanolic extract (MEL) showed

maximum effect with IC50 value of 32.84 µg/ml while in case of roots maximum effect was seen

in ethyl acetate extract (EER) with IC50 value of 66.62 µg/ml. Among the active constituents

isolated from MEB and MEL, SOL-2 was found to be most effective with lowest IC50 value of

8.91 µg/ml. All other compounds also showed good effect with IC50 value ranging between

16.41 µg/ml to 28.47 µg/ml.


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SRB assay relies on the uptake of the negatively charged pink aminoxanthine dye,

sulphorhodamine B (SRB) by basic amino acids in the living cells, which after fixing and lysing

give a more intense colour and thus greater absorbance (Skehan et al., 1990). 12 cancer cell lines

from 9 different tissues were used in the study to assess the selectivity of extracts. The

effectiveness of the extracts was determined on the basis of their inhibitory activity against

maximum number of cell lines as well as their IC50 values. The methanolic extracts from

different parts of S. oleosa viz. MEB, MEL and MER were found to be most effective among

bark, leaves and roots respectively. The cytotoxicity profile of most active extracts from S.

oleosa bark, leaves and roots was determined in terms of IC50 values, which shows that MEB

was most potent, followed by MEL and MER respectively. MEB and MEL being most potent

were subjected to bioactivity guided fractionation, leading to isolation of 5 compounds from

MEB and 2 compounds from MEL. The compounds were also subjected to in vitro cytotoxicity

evaluation against 16 cancer cell lines wherein they exhibited moderate to strong inhibitory

activity against different cell lines depending on their selectivity.

Further to assess the selective toxicity of the most active extracts, MEB and MEL as well

as their bioactive constituents, towards cancer cells, they were also assessed for in vitro

cytotoxicity towards hGF normal human gengevial fibroblast cells, in MTT assay, at the at the

highest concentration tested towards cancer cells i.e. 100 µg/ml for extracts and 100 µM for

compounds. The extracts and the compounds were not found to produce any significant

cytotoxicity at the above mentioned concentration which suggested their disparity of action

towards normal versus cancerous cells, that may be attributed to differences in tissue-specific

levels and expression patterns of cytochrome P450 (CYP) isoforms mediating cell-specific

cytotoxicity. CYPs' is a multigene family consisting of constitutive and inducible enzymes which

can either activate or detoxify anticancer agents and thus, may influence the response of tumors

to anti-cancer drugs (Crewe et al., 1997; Sridar et al., 2002; Nigam et al., 2008). The results are

strengthened by the study of Bhushan et al. (2009) that evaluated the pentacyclic triterpenediol

(TPD) from Boswellia serrata for their apoptosis inducing potential in HeLa and SiHa cells. It

was found that whereas TPD induced effective cytotoxicity through activation of various

apoptotic signalling cascades, it was found to be ineffective in inducing toxicity in normal hGF

and monkey kidney CV-1 cells, at the same concentration.


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In vitro cytotoxicity profiling of the extracts as well as compounds revealed their

potential anticancer capacity. Fricker and Buckley (1996) obtained similar cytotoxicity profiles

in MTT and SRB assay for four anti-cancer drugs across the panel of seven human cancer cell

lines and recommended the suitability of both assays as cytotoxicity endpoints. SRB assay being

simple, easily reproducible and having stable end-point that does not require a time-sensitive

measurement as in formazan based assays, was preferred for testing the extracts and their

bioactive constituents across different cell lines (Keepers et al., 1991; Houghton et al., 2007).

The differential cytotoxic effect of extracts as well as the isolated compounds against different

cell lines may be attributed to the variations in their biochemical characteristics as well as the

molecular structure of the receptors (Creasey et al., 1987; Martin-Kleiner et al., 2007; Thipnate

et al., 2011).

The biological activities of medicinal plants are attributed to their bioactive chemicals

mostly being secondary metabolites. Various studies have reported the use of cytotoxicity

evaluation as a measure to determine potency of plant extracts for their bioactivity guided

fractionation to isolate bioactive constituents (Kaur et al., 2005; Hymavathi et al., 2009; Pan et

al., 2009; Hyun et al., 2010; Pan et al., 2010; Ludwiczuk et al., 2011; Forgo et al., 2012).

Therefore, the cytotoxicity values in SRB assay were taken as an index for the bioactivity guided

fractionation of active extracts/fractions. The in vitro cytotoxicity assays can only provide a

snapshot of cell behavior in the course of drug treatment, which may underestimate the number

of dead cells due to their possible rapid degradation into cellular debris (Brunelle and Zhang,

2010). Therefore to ascertain the mechanism of action of cell death as well as the probable

pathway of action of active constituents isolated from MEB and MEL, the compounds were

subjected to bioassays that involve various mechanistic parameters typical of apoptosis. HL-60

(human pro-myelocytic leukemia) cell line was selected for mechanistic evaluation, since, being

suspension cell line, its easier to passage and does not require enzymatic or mechanical

dissociation, that can adversely affect the morphology of cells in culture. MTT assay was

performed on HL-60 cells to calculate IC50 values of the isolated compounds to determine their

test concentrations for mechanistic studies i.e. 10 µg/ml and 30 µg/ml.


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Defects in apoptosis pathways are implicated in the development of cancer and are

believed to play a major role in the survival and proliferation of neoplastic cells. Therefore,

bioactivity of potential anticancer agents can be measured, in theory, by assessing any critical

event within the cell death pathway. An increasing number of anticancer drugs are being

developed to target specific aberrant signaling components of cell death or survival pathways

(Fesik, 2005; Qiao and Wong, 2009; Wong, 2009; Brunelle and Zhang, 2010). The anticancer

potential of chemotherapeutic agents lies in their ability to kill cancer cells by inducing apoptosis

and not necrosis, which can promote an inflammatory response and other negative effects on

normal cells (Coussens and Werb, 2002; Grivennikov et al., 2010). A considerable amount of

research has been devoted to the development of anticancer therapeutics from plants, in view of

their low profiles of adverse reactions and also elucidating the molecular mechanisms by which

herbal products inhibit cancer (Issa et al., 2006). As discussed above, the present study

established the strong cytotoxic potential of the bioactive constituents isolated from MEB and

MEL against a panel of human cancer cell lines including HL-60. Further, these isolated

compounds were subjected to various assays to ascertain the molecular mechanism of cell death.

Morphological changes in the cells are regarded as the gold standard for apoptosis

detection in vitro. Morphologically, apoptotic cells are characterized by membrane blebbing, cell

shrinking, nuclear condensation, chromatin aggregation, degradation of chromosomal DNA and

formation of apoptotic bodies (Pulido and Parrish, 2003; Cummings et al., 2004 ). In the present

study, HL-60 cells treated with compounds isolated from MEB and MEL exhibited typical

morphological changes characteristic of apoptosis in light microscopy, fluorescence microscopy

using DAPI stain and scanning electron microscopy (SEM) studies. To further confirm the

apoptotic events induced by the bioactive constituents isolated from MEB and MEL, flow

cytometry studies, involving various parameters of apoptotic pathway, were done. The majority

of structural and biochemical events occurring during cell death at cellular level can be analysed

by flow cytometry. Along with detecting the changes in cell volume and granularity, it also helps

to quantify apoptosis (Schmid et al., 1992; Lecoeur et al., 2002).

Since cancer cells are characterized by uncontrolled cell division due to anomalies in cell

cycle check points, regulation of cell cycle is a key to restrict unregulated cell proliferation.


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Literature survey has shown that most of the antineoplastic agents act by damaging genomic

DNA, mediated by endonuclease cleavage, thus triggering cell cycle arrest and finally apoptosis

(Hartwell and Kastan, 1994; Shapiro and Harper, 1999; Liu et al., 2005; Mertens-Talcott and

Percival, 2005; Sun and Liu, 2006). Flow cytometric analysis can be employed for precise

evaluation of cellular DNA content and subsequent identification of hypodiploid cells, which

generally appear in the sub-G0/G1 peak region in the histogram. This analysis is based on the

ability to stain the cellular DNA with a fluorochrome in a stoichiometric manner and the

proportion of hypodiploid cells in total cell population represents the intensity of apoptosis-

inducing activity of the tested sample. Therefore cell cycle analysis was performed by staining

the cellular DNA in different phases of cell cycle using propidium iodide (PI), a fluorescent dye

that intercalates with cellular DNA (Li et al., 2005c; Riccardi and Nicoletti, 2006; Agrawal et al.,

2011). As evident from the DNA content histogram analysis, all the compounds isolated from

MEB and MEL showed concentration-dependent increase in the apoptotic sub G0 fraction,

therefore indicating that the compounds induced cell cycle arrest at the G0/G1 phase. Among all

the compounds, SOB-4 was found to be most effective with 73.63% of hypodiploid DNA at 30

µM concentration.

Cells undergoing apoptosis loose the phospholipid asymmetry of their plasma membrane

due to translocation of phosphatidylserine (PS), a membrane phospholipid, from the inner layer

of the membrane to the outer layer and thereby exposing PS to the external environment.

Annexin V–FITC assay is used to quantitatively determine the percentage of cells within a

population that are actively undergoing programmed cell death at the early phases of apoptosis

even before the loss of cell membrane integrity or appearance of other morphological changes

associated with apoptosis (Shounan et al., 1998; Grosse et al., 2009). The assay was performed

in conjugation with a dye exclusion test using propidium iodide (PI), to establish integrity of the

cell membrane and thus discriminate between intact cells (FITC−/PI−), apoptotic cells

(FITC+/PI−) and necrotic cells (FITC+/PI+) (Vermes et al., 1995). Among all the compounds

isolated from MEB and Mel, SOB-5 and SOB-4 were observed to show considerable effect with

62.69% and 61.27% cells in early apoptosis at concentration of 30 µM. All other compounds

also showed concentration dependent increase in apoptotic cell population.


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After determining the early apoptotic events induced by the isolated compounds in

Annexin V/PI staining assay, they were further investigated for caspases dependent degradation

of nuclear DNA into nucleosomal units. In the later stage of apoptosis, the fragmentation of the

genomic DNA is the biochemical hallmark, an irreversible event that commits the cell to die

(Schwartzman et al., 1993; Vermes et al., 2000; Zhang et al., 2009). DNA fragmentation in an

inter-nucleosomal pattern by activated endogenous endonucleases was assayed by two methods:

for nucleosomal ladders by agarose gel electrophoresis and for quantitative studies by TUNEL

assay. It was observed that the active extracts MEB and MEL along with their active constituents

exhibited the characteristic nuclear ladder pattern in agarose gel electrophoresis, thus confirming

the activation of endonucleases leading to endonucleocytic cleavage of genomic DNA. Further

analysis by TUNEL assay, that allows monitoring the percentage of DNA-fragmented cells,

showed that among all the compounds, maximum fragmentation of 88.7% was seen with SOB-5

while others exhibited fragmentation in range of 58.4% - 80.5%.

The role of mitochondria, as a key executioner of intrinsic pathway of apoptosis is well

established. The intrinsic pathway for programmed cell death is activated by non-receptor–

mediated intracellular signals like oncogenes, hypoxia, radiation, ROS overproduction or direct

DNA damage. These stimuli induce changes in the inner mitochondrial membrane that result in

the loss of mitochondrial transmembrane potential causing the release of apoptogenic factors

from the intermembrane space into the cytoplasm (Yang et al., 1997; Elmore, 2007; Ashkenazi

et al, 2008). ROS are known triggers of the intrinsic apoptotic cascade via interactions with

proteins of the mitochondrial permeability transition complex, thus resulting in cytochrome c

release from the intermembrane space. Significant mitochondrial loss of cytochrome c will lead

to further ROS increase due to a disrupted electron transport chain (Chen and Lesnefsky, 2006;

Tsujimoto and Shimizu, 2007; Circu and Aw, 2010). The ROS generation ability of different

compounds isolated from MEB and MEL was assessed using a peroxide sensitive fluorescent

probe, DCFH-DA that showed SOB-4 and SOB-5 to be effective generators of ROS

intermediates leading to activation of redox sensitive transcription factors. Further all the other

compounds were observed to be weak elicitors of intracellular ROS. The integrity of

mitochondrial membranes in HL-60 cells following treatment with different isolated compounds

was examined by measuring their ability to retain Rh-123, a fluorescent dye that gets integrated


140

in the membranes of intact mitochondria (Kim at al., 2007). The results revealed that though only

SOB-4 and SOB-5 were able to generate ROS stimuli, yet all the isolated compounds exhibited

remarkable mitochondrial membrane depolarization in HL-60 cells, with SOB-4 showing the

maximum effect with 74.4% treated cells exhibiting loss of mitochondrial membrane potential

(MMP). Further, on analysis of cytochrome c in the cytoplasm of HL-60 cells, treated with

different compounds, it was observed that all the compounds promoted release of cytochrome c,

with SOB-5 showing maximum cytosolic cytochrome c fraction of 75.5% in Hl-60 cells at 30

µM concentration. The release of cytochrome c has been reported to be regulated by Bax (Bcl2

Associated X-protein) which translocates from cytosol to mitochondria in response to apoptotic

signals. Bax is implicated in the formation of pores in the outer mitochondrial membrane after

translocation from cytosol allowing subsequent release of cytochrome c and apoptosis activating

factors (Wei et al., 2001; Kumar et al., 2008). Expression analysis of HL-60 cells treated with

different concentrations of MEB and MEL, the active extracts from which compounds were

isolated, showed that there was decline in the cytosolic level of Bax with corresponding increase

in the mitochondria in both MEB and MEL treated cells.

Cytochrome c release results in formation of the apoptosome, a catalytic multiprotein

platform that activates caspase-9 which further leads to activation of downstream caspase

cascade. Thus, the ability of isolated constituents from MEB and MEL to effect the activation of

different caspases was assessed by measuring the caspase activity in the HL-60 cells. It was

observed that there was almost 3-fold to 6-fold increase in the activity caspase-3 and caspase-6,

that act as executioner caspases and cleave various substrates in the cell, leading to cell

morphological changes. SOB-4 and SOB-5 showed almost comparable effects and exhibited

significant increase in the activation of these effector caspases. Since caspase-3 activity is known

to be up-regulated through signaling cascades emanating from both, caspase-9 in mitochondria-

initiated intrinsic pathway and from caspase-8 in the death receptor-triggered extrinsic pathway,

therefore HL-60 cells were examined for any corresponding increase in the activity of either

caspase-8 or caspase-9 following treatment with different compounds (Kumar et al., 2008). HL-

60 cells exposed to isolated compounds from MEB and MEL displayed almost 1.5-fold to 2.5-

fold increase in caspase-8 and caspase-9 activity. It was observed that, although the isolated

compounds exhibited activation of effector caspases (caspase-3 and caspase-6) over a wide range


141

(3-fold to 6-fold), but the activation of initiator caspases (caspase-8 and caspase-9) depicted

narrow range of activity (1.5-fold to 2.5-fold), with all the isolated constituents showing almost

similar and comparable activities in HL-60 treated cells. Therefore, signaling pathways activated

by the compounds isolated from MEB and MEL appears to involve both extrinsic and intrinsic

cascades as evidenced by activation of both caspase-8 and caspase-9, respectively. The results of

other assays discussed above (MMP, cytochrome-c and Bax analysis) add to the considerable

evidence in favor of the involvement of intrinsic mitochondrial mediated pathway. Furthermore

as the activation of caspase-8 was observed, it was thought that the receptor-mediated pathway

may have been triggered due to increased expression of upstream apical death receptors. The

expression of TNF-R1 was observed to increase in HL-60 cells treated with MEB and MEL, the

most active extracts from which bioactive constituents were isolated. The activation of TNF-R1

might engage Fas associated death domain (FADD) and death inducing signaling complex

(DISC) allowing the release of active form of caspase-8 (Kumar et al., 2008). The activation of

an effector caspase, caspase-3, further results in proteolytic cleavage of downstream substrate

Poly (ADP-ribose) polymerase1 (PARP1), a DNA repair enzyme and finally culminating into

apoptosis (Yu et al, 2006). PARP cleavage analysis by FACS (Fluorescence-activated cell

sorting) revealed that all the compounds isolated from MEB and MEL exhibited effective PARP

cleavage activity, with SOB-4 showing maximum cleavage of 76.7% at 30 µM concentration.

DNA topoisomerases play an essential role in the maintenance of genetic material

integrity by regulating DNA topology during transcription, replication and recombination

processes. Topoisomerase inhibitors stabilize topoisomerase–DNA cleavable complexes and are

converted to permanent DNA strand breaks in the cell when replication or transcription

complexes collide with the covalently attached enzyme (Liu and D’Arpa, 1992; Binaschi et al.,

1994; Boege, 1996; Li and Liu, 2001; Godard et al., 2002; Wilstermann and Osheroff, 2003;

McClendona and Osheroff, 2007). These permanent DNA strand breaks ultimately initiate the

cytotoxic effects of topoisomerase poisons, thus establishing them as potential chemotherapeutic

agents (Kaufmann, 1998). Topoisomerase inhibitors are among the most efficient inducers of

apoptosis. The main pathways leading from topoisomerase-mediated DNA damage to cell death

involves activation of caspases in the cytoplasm by proapoptotic molecules released from

mitochondria, though in some cells, apoptotic response has been seen to involve activation of Fas


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death receptors by topoisomerase inhibitors (Fortune and Osheroff, 2000; Sordet et al., 2003).

The results of topoisomerase I inhibition assay showed that MEL as well as its isolated

constituents were ineffective topoisomerase I inhibitors. MEB along with SOB-2 and SOB-4,

isolated from MEB were found to be effective topoisomerase I inhibitors while SOB-5 showed

moderate topoisomerase I inhibitory activity. In case of topoisomerase II inhibition assay both

MEB and MEL were found to inhibit plasmid relaxation, mediated through topoisomerase II

enzyme. Further analysis of bioactive constituents isolated from MEB and MEL in the above

assay revealed that whereas SOB-1, SOB-2, SOB-4 and SOL-2 were found to be strong

inhibitors of topoisomerase II activity, SOB-3 and SOB-5 exhibited weak effect in inhibiting

topoisomerase II mediated relaxation of DNA.

A compelling body of evidence in literature supports the results of various

antiproliferative assays performed in the current study. Bhushan et al. (2006) observed

remarkable cytotoxicity by AP9-cd, a standardized lignan composition from Cedrus deodara

consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, on human leukemia

Molt-4 and HL-60 cells. Apoptosis was confirmed by observation of apoptotic bodies and DNA

ladder formation. Flowcytometric analysis revealed increased sub-G0 cell fraction along with

time-related increase in apoptosis and post-apoptotic necrosis in annexinV-FITC/PI-stained cells.

Further analysis showed increase in mitochondrial depolarization and activation of caspases-3,-8

and -9. The continuing study by Saxena et al. (2010) showed that the apoptotic potential of AP9-

cd was significantly enhanced in HL-60 cells in the presence of three natural antioxidants:

curcumin, silymarin and acteoside. It was confirmed by using various models like MTT assay,

DNA fragmentation, nuclei condensation, sub-G0 DNA population, Annexin-V-FITC binding,

ROS depletion and immunoblotting in HL-60 cells. Alosi and coworkers (2010) assessed the

antiproliferative activity of pterostilbene, an analogue of resveratrol found in blueberries,

towards breast cancer cell lines, MCF-7 and MDA-231 in MTT assay that depicted apoptosis in

DNA fragmentation and TUNEL assay. The study attributed in vitro cancer cell growth

inhibition to intrinsic apoptotic pathway as exhibited by mitochondrial depolarization, altered

cell cycle and increased caspase-3/7 activity. A study by Won et al (2006) demonstrated that the

cytotoxicity of HaBC18, an active fraction from butanol extract of Albizzia julibrissin, toward

Jurkat T cells is attributable to apoptosis mediated by mitochondria-dependent death-signaling


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pathway regulated by Bcl-xL, as established by various biochemical events such as

mitochondrial cytochrome c release, activation of caspase-9 and -3, degradation of PARP and

DNA fragmentation.

A recent study by Plastina et al. (2012) investigated the effects of Ziziphus jujube extracts

(ZEs) on two breast cancer cell lines, MCF-7 and SKBR3, where ZEs were reported to induce

cell death by apoptosis in both malignant breast cells as demonstrated in DNA fragmentation and

terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays. Luo et al.

(2010) observed strong cytotoxic effect of cajanol, an isoflavanone from roots of Cajanus cajan,

towards MCF-7 human breast cancer cells in a time and dose-dependent manner. Further

analysis of mechanism of action of cajanol revealed that it induced apoptosis via mitochondria-

dependent pathway where ROS-mediated mitochondrial cytochrome c release resulted in the

activation of caspase-9 and caspase-3 cascade and active-caspase-3 was involved in PARP

cleavage. Inhibition of Bcl-2 expression and induction of Bax expression, observed in western

blot analysis, was associated with the disintegration of the outer mitochondrial membrane,

causing cytochrome c release. Bakar et al. (2010) reported that the extract of Mangifera pajang

kernel induced cytotoxicity in MCF-7 and MDA-MB-231 cells with IC50 values of 23 and

30.5 μg/ml, respectively. Apoptotic mode of cell death was confirmed by the induction of cell

cycle arrest at the sub-G1 phase of the cell cycle by the kernel extract along with Annexin V-

FITC/PI staining and caspases-3 and -9 induction. A study by Sreejith and coworkers (2012)

demonstrated that the ethanol extract of Glycosmis pentaphylla and its active fractions induced

apoptosis in Hep3 B cell line, as confirmed in DNA fragmentation, Hoechst staining,

morphological studies, RT-PCR, PARP cleavage studies. Chemoprofiling data revealed the

presence of flavonoid in the active fraction that increased the expression ratio of Bax/Bcl2 genes

in a time and dose dependent manner.

The collective results of the antiproliferative mechanistic studies demonstrated that MEB

and MEL as well as their isolated bioactive constituents might regulate cell cycle progression

and induce HL-60 cell apoptosis through both extrinsic and intrinsic pathways. Kumar et al.

(2008) evaluated the apoptosis inducing ability of an essential oil from a lemon grass variety of

Cymbopogon flexuosus (CFO) and its major chemical constituent sesquiterpene isointermedeol


144

(ISO) in human leukaemia HL-60 cells. It was found that both CFO and ISO induced

concentration dependent strong and early apoptosis as measured by various end-points, e.g.

annexinV binding, DNA laddering, apoptotic bodies formation and an increase in hypo diploid

sub-G0 DNA content during the early 6 h period of study which may be attributed to early surge

in ROS formation with concurrent loss of mitochondrial membrane potential. Further CFO and

ISO were observed to activate apical death receptors TNFR-1, DR4 and caspase-8 activity along

with increased expression of mitochondrial cytochrome c protein with its concomitant release to

cytosol leading to caspase-9 activation. A study by Kang et al. (2010) reported that the apoptotic

effect of oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, correlates with

high expression and activation of Bax, FADD, caspase-8 as well as caspase-3 and decreased

protein levels of Bcl-2 and SIRT-1, suggesting that both the extrinsic and intrinsic apoptosis

pathways are involved in the apoptotic processes. Yang et al. (2011) revealed that celastrol, a

major biologically active component of Tripterygium regelii, induced sub-G1 DNA

accumulation, formation of apoptotic bodies, nuclear condensation and a DNA ladder in MCF-7

cells along with the decreased expression of anti-apoptotic Bcl-2 protein and increased

expression of pro-apoptotic Bax protein, release of cytochrome c and finally PARP cleavage.

Celastrol was also found to trigger the activation of caspase-7, -8, and -9 as well as caspase-8-

mediated bid cleavage thereby suggesting the involvement of both the mitochondrial-dependent

and independent pathways in induction of apoptosis. Pal and coworkers (2011) investigated the

cytoprotective role of arjunolic acid (AA), a triterpenoid saponin, that increased the levels of

caspase-9, -8, -3, Fas and Bid, increased reactive oxygen species (ROS) production, decreased

mitochondrial membrane potential (MMP), enhanced cytochrome c release in the cytosol,

disturbed the Bcl-2 family protein balance, cleaved PARP protein and ultimately led to apoptotic

cell death through both intrinsic and extrinsic apoptotic pathways. Lin et al. (2012) demonstrated

leaf extract H.sabdariffa L. (HLE) to be rich in polyphenols, including catechin and ellagic acid

(EA) and also examined the anticancer properties of HLE and EA that indicated chromatin

condensation, apoptotic morphology by inverted microscopy studies as well as quantitative

evaluation of DNA strand breaks using TUNEL assay and detection of large number of

hypodiploid cells in sub-G1 region. Further analysis to assess the pathway revealed decreased

protein expression of Bcl-2 and Mcl-1, increased translocation of Bax to the mitochondria,


145

release of cytochrome c from mitochondria to cytosol and increased expression of FasL, TNFα

and TNFR1 thus indicating that HLE induces cell death in LNCaP cells via both intrinsic

(Bax/cytochrome c-mediated caspases-9) and extrinsic (Fas-mediated caspases-8/t-Bid)

apoptotic pathways.

The active constituents isolated from MEB and MEL, were characterized as triterpenoids

and polyphenolic compounds, respectively and they showed remarkable cytotoxicity through

apoptotic pathway of cell death. Recent study by Kim et al. (2012) explored the underlying

mechanisms of the anticancer activity of the ethanolic extract of mango peel (EEMP) and found

that it induced apoptosis in HeLa cells as evidenced by the increased cell population in the sub-

G1 phase and the appearance of fragmented nuclei. They attributed the apoptosis-mediated cell

death in human cervical carcinoma cells to the presence of plant phenolics and the lipophilic

principles of EEMP, as confirmed by HPLC–PDA–ESI–MS and GC-MS analysis. Treatment of

the cells with EEMP was also found to downregulate anti-apoptotic Bcl-2 expression, resulting

in the proteolytic activation of caspase-3, -7, -8, and -9 and the degradation of poly (ADP-ribose)

polymerase (PARP) protein. Another recent study by Ham and coworkers (2012) isolated 4

serratane-type triterpernoids, tohogenol, serratenediol (SE) and its two derivatives: 21-epi-

serratenediol and 21-epi-serratenediol-3-acetate from an alcoholic extract of Lycopodium

serratum (LSE) and investigated the ability of LSE and SE to induce apoptosis in cultured

human promyelocytic leukemia HL-60 cells. Both LSE and SE exhibited formation of apoptotic

bodies and fragmented DNA, as well as the accumulation of DNA in the sub-G1 phase of the cell

cycle. Further analysis of the mechanism of these events indicated that SE treated HL-60 cells

had an increased ratio of Bax/Bcl-xL, released the cytochrome c, activated caspase-9, -3, and

cleaved poly-ADP-ribose polymerase (PARP), thus suggesting that SE-induced apoptosis likely

occurs through the caspase-dependent pathway. Zunino et al (2009) isolated polyphenolic

compounds viz. quercetin, kaempferol, and ellagic acid from Fragaria xananassa (strawberries)

and revealed significant apoptosis was induced by purified strawberry fractions as well as

isolated compounds, in pre-B acute lymphoblastic leukemia (ALL) cell line as measured by loss

of nuclear DNA, loss of mitochondrial membrane potential, and activation of caspase-3. The role

of terpenoids and polyphenolic compounds as potent cytotoxic agents as well as inducers of

apoptotic mode of cell death has been established by various studies (Akihisa et al., 2003;


146

Mertens-Talcott and Percival, 2005; Ríos and Recio, 2006; Manu and Kuttan, 2008; Alesiani et

al., 2010; Lage et al., 2010; Mahaira et al., 2011; Santos et al., 2011; Rezaei et al., 2012; Wu et

al., 2012; Yang et al., 2012).

The findings of the current study established that the isolated polyphenols (SOL-1 and

SOL-2) as well as three terpenoids (SOB-1, SOB-2 and SOB-3) exhibited strong antioxidant

properties along with activating the mitochondrial mediated pathway of cell death. Since ROS

stimuli is well known to trigger intrinsic pathway, as they act as secondary messengers, therefore

ROS generation was measured by DCFH-DA that measures ROS largely localized in the

cytoplasm where DCF is mainly trapped. It was observed that the ROS generation by the

compounds exhibiting potent antioxidant activity was comparatively lesser than the compounds

with low antioxidant activity. The lower ROS generation, after 6 hr of treatment with

compounds, may be attributed to the time dependent decline in the generation of ROS. Kumar

and coworkers (2008) observed that ROS stress generated in mitochondria during the first hour

of treatment with CFO, an essential oil from Cymbopogon flexuosus declined from 60% to 15%

after 6 h of treatment. Similar trend was reported for its major chemical constituent sesquiterpene

isointermedeol (ISO) that showed decline from 29% after 1 hr of treatment to 0.05% after 6 hr

treatment. They attributed the decline to increased mitochondrial expression of cytochrome c,

which by virtue of its strong antioxidant activity possibly caused quenching of ROS to rescue

cells from DNA damage. Moreover, being highly reactive and short lived, exact ROS levels are

difficult to detect directly in complex biological systems. Thus time dependent, real time analysis

of drug treatment on cancer cells is required in order to characterize the flow of ROS generation

in cells and to ascertain their maximum peak.

Furthermore, the ROS generation by the antioxidant compounds may be attributed to

their pro-oxidant behavior that is triggered depending on their concentration as well as the

surroundings, like the presence of metal ions. Plant polyphenols, recognized as naturally

occurring antioxidants, also act as prooxidants catalyzing DNA degradation in the presence of

transition metal ions such as copper (Ahmad et al., 1992; Inoue et al., 1994; Hadi et al., 2000).

Copper is one of the most redox reactive metal ion present in chromatin and closely associated

with DNA bases. Antioxidants like polyphenols are able to bind to both DNA and Cu(II) forming


147

a ternary complex that may result in redox reaction leading to the reduction of Cu(II) to Cu(I),

whose reoxidation generates a variety of reactive oxygen species (ROS). Since cellular copper

levels are considerably elevated in various cancers, therefore cancer cells may be more prone to

electron transfer between copper ions and antioxidants to generate ROS (Margalioth et al., 1987;

Ebadi and Swanson, 1988; Yoshida et al., 1993; Ebara et al., 2000; Zheng et al., 2006; Hadi et

al., 2007). Quercetin, an antioxidant polyphenol, has been reported to generate ROS, thus acting

as pro-oxidant and causing radical-induced apoptosis in various cancer cell lines (Zhang and

Zhang, 2009; Gibellini et al., 2010; Lee et al., 2010). The pro-oxidant action of flavonoids viz.

quercetin, curcumin and a stilbene resveratrol has been shown to involve mobilization of

endogenous copper ions leading to ROS generation (Rahman et al., 1990; Ahsan and Hadi; 1998;

Ahmad et al., 2000). Chlorogenic acid, another polyphenolic antioxidant, has been reported to

induce apoptosis of Bcr-Abl(+) chronic myeloid leukemia (CML) cell lines and clinical leukemia

samples by both, upregulating death receptor DR5 and triggering loss of mitochondrial

membrane potential accompanied by release of cytochrome c from the mitochondria to the

cytosol. The results of the study established the role of ROS for chlorogenic acid mediated

preferential killing of Bcr-Abl(+) cells (Rakshit et al., 2010). Chlorogenic and caffeic acids have

also been reported to switch from anti- to pro-oxidant activity, depending on the presence of free

transition metal ions, or on their redox status (Morishita and Ohnishi, 2001). Thus ROS exert

multifaceted role at cellular level, by acting both as cytotoxic agents and signal transducers

involved in physiological cell proliferation and death. Therefore, anti-neoplastic therapies

targeting ROS or the antioxidant system actually play on the edge, to kill tumors and to preserve

the normal cells homeostasis (Manda et al., 2009).

In the present study it was observed that the extracts, MEB and MEL as well as their

isolated bioactive constituents selectively inhibited topoisomerase I and topoisomerase II enzyme

activity, thus suggesting their involvement in the apoptosis-inducing mechanism. As already

discussed, topoisomerase inhibitors generate DNA damage by stabilizing the covalent

topoisomerase-DNA cleavage complex. This topoisomerase inhibitor-mediated DNA damage

triggers the phosphorylation of the transcription factor, p53 that in turn is upregulated by protein

kinases viz. DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM).

Phosphorylation of p53 has been reported to down-regulate several anti-apoptotic genes and

activate pro-apoptotic protein Bax, which translocates from cytosol to mitochondria and in turn


148

triggers mitochondrial permeability transition (MPT) that results in cytochrome c release and

activation of caspases cascade (Bennett et al., 1998; Liao et al., 1999; Schuler et al., 2000; Matas

et al., 2001; Baptiste et al., 2002; Sordet et al., 2003; Valkov and Sullivan, 2003; D’Archivio et

al., 2008). The results of the present study thus indicate the probable involvement of p53

mediated intrinsic apoptotic pathway, triggered by topoisomerase inhibitors and strengthened by

the reports in literature. Etoposide, an anticancer agent with topo II inhibitory activity, has been

reported to induce apoptosis in L929 fibroblasts through p53 phosphorylation mediated

upregulation of the pro-apoptotic protein, Bax, (Karpinich et al., 2002). A study by Li and

coworkers (2005a) demonstrated that ellagic acid, a phenolic compound with reported

topoisomerase I and II inhibition, induced G0/G1-phase cell cycle arrest through increased p53

and p21 level and decreased CDK2 gene expression and induce apoptosis through activation of

caspase-3 activity in human bladder cancer T24 cells. Another study by Priyadarsini et al. (2010)

examined the molecular mechanism of quercetin-induced cell death in human cervical cancer

(HeLa) cells which demonstrated that it suppressed the viability of HeLa cells in a dose-

dependent manner by inducing cell cycle arrest and mitochondrial apoptosis through a p53-

dependent mechanism. The apoptotic cell death triggered by quercetin, a topoisomerase II

inhibitor, involved characteristic changes in nuclear morphology, phosphatidylserine

externalization, mitochondrial membrane depolarization, modulation of cell cycle regulatory

proteins and NF-κB family members, upregulation of proapoptotic Bcl-2 family proteins,

cytochrome c, Apaf-1 and caspases and downregulation of antiapoptotic Bcl-2 proteins and

surviving.

Li et al. (2005d) demonstrated that a potent triterpene 3α-hydroxy-15α-acetoxy-lanosta-

7,9(11),24-trien-26-oic acid, ganoderic acid X (GAX), isolated from Ganoderma amboinense,

showed characteristic apoptotic events like degradation of chromosomal DNA, decreased level

of Bcl-xL, disruption of mitochondrial membrane, cytosolic release of cytochrome c and

activation of caspase-3 in human hepatoma HuH-7 cells. Along with the growth of cancer cell

lines, GAX was also shown to inhibit the activities of topoisomerases I and IIα in vitro. Kawiak

and coworkers (2007) investigated the role of ROS and topo II inhibition in the induction of

apoptosis mediated by plumbagin, a naphthoquinone and found that plumbagin induced DNA

cleavage in HL-60 cells, having high topo II activity, whereas in a cell line with reduced topo II

activity, HL-60/MX2, the level of DNA damage was significantly decreased, while ROS were


149

generated at a similar rate in both cell lines. This suggested the mechanism of indirect damage to

DNA by plumbagin-induced ROS, mediated through topo II. Verma et al. (2008) reported the

anticancer potential of P. longifolia leaves extract (A001) and its chloroform fraction (F002) on

various human cancer cell lines along with inhibition of DNA topoisomerase I enzyme activity

by F002. Furthermore, F002 was also found to induce apoptosis in human leukemia HL-60 cells,

through the mitochondrial-dependent pathway, as observed by apoptotic bodies formation, DNA

ladder, enhanced annexin-V-FITC binding of the cells, increased sub-G0 DNA fraction, loss of

mitochondrial membrane potential (ΔΨmt), release of cytochrome c, activation of caspase-9,

caspase-3, and cleavage of poly-ADP ribose polymerase (PARP). Various other studies have also

documented the involvement of DNA topoisomerases in the triggering of apoptosis by natural

products (Negri et al., 1995; Berger et al., 2001; Søe et al., 2004, de Mejía et al., 2006; Cai et

al., 2008; Díaz-Carballo et al., 2008; Neukam et al., 2008; Azarova et al., 2010; Moukharskaya

and Verschraegen, 2012).

The study establishes the protective effects of different extracts from bark, leaves and

roots of Schleichera oleosa with respect to their antioxidative and antiproliferative potential. The

most active extracts, MEB and MEL, as well as the isolated bioactive constituents were observed

to possess significant radical scavenging and apoptosis-induced cytotoxicity activities, in

different in vitro assay systems. The antioxidant and cytotoxic potencies of different extracts of

S. oleosa plant parts as well as the isolated compounds from MEB and MEL, at the highest

concentration tested, in different in vitro assay systems is summarized in Table 5.1-5.8.

Apoptotic cell death induced by the isolated compounds, was found to utilize a wide range of

molecular targets, thereby suggesting the involvement of parallel death pathways that converged

in induction of caspases-3. Due to the involvement of multiple signaling pathways by different

isolated compounds, combinational studies are required to assess their synergistic, additive or

antagonistic behavior. Therefore, more detailed investigations involving target based and time

dependent mechanistic studies, utilizing their potential molecular targets as well as in vivo

animal models are warranted to determine clinical potential of the isolated principles from S.

oleosa for the treatment of leukemia.

Table 5.1: Antioxidant potency of extracts of bark of Schleichera oleosa at the highest concentration tested in differentin vitro antioxidant assays+

+ Categorization of antioxidant activities: >75%: A (Very strong); 50%-75%: B (Strong); 25%-50%: C (Moderate); <25%: D (Weak)++ Categorization of plasmid protection activity: >60%: A (Very strong); 40%-60%: B (Strong); 20%-40%: C (Moderate); <20%: D (Weak)* % age of supercolied DNA at the highest concentration tested

Extracts DPPH(200 µg/ml)

ReducingPower

(200 µg/ml)

ChelatingPower

(200 µg/ml)

Deoxyribose DegradationLipid

Peroxidation(100 µg/ml)

PlasmidNicking++

(1000 µg/ml)*

Non-siteSpecific

(100 µg/ml)

Site Specific(100 µg/ml)

Hexane(HEB)

D D D D D D D

Chloroform(CEB)

C D D C D C C

Ethyl acetate(EEB)

A A D A B A A

Methanol(MEB)

A A D A B A A

Water(WEB)

B B D A C A A

Table 5.2: Antioxidant potency of extracts of leaves of Schleichera oleosa at the highest concentration tested in differentin vitro antioxidant assays+



ReducingPower

(200 µg/ml)

ChelatingPower

(200 µg/ml)



PlasmidNicking++

(1000 µg/ml)*

Non-siteSpecific

(100 µg/ml)


Hexane(HEL)

D D D D D D D

Chloroform(CEL)

C C D C D C C

Ethyl acetate(EEL)

A A C A B A A

Methanol(MEL)

A A C A A A A

Water(WEL)

B B D A B A A

Table 5.3: Antioxidant potency of extracts of roots of Schleichera oleosa at the highest concentration tested in differentin vitro antioxidant assays+



ReducingPower

(200 µg/ml)

ChelatingPower

(200 µg/ml)



PlasmidNicking++

(1000 µg/ml)*

Non-siteSpecific

(100 µg/ml)


Hexane(HER)

D D D D D D C

Chloroform(CER)

D D D C D D C

Ethyl acetate(EER)

A A B A B A A

Methanol(MER)

A B C B B A A

Water(WER)

B B D B B A A

Table 5.4: Antioxidant potency of compounds isolated from methanolic extracts of bark and leaves of Schleichera oleosaat the highest concentration tested in different in vitro antioxidant assays+


Extracts Compounds

Deoxyribose DegradationLipid Peroxidation

(100 µg/ml)Plasmid Nicking++

(1000 µg/ml)*Non-siteSpecific

(100 µg/ml)


Methanol extract ofbark (MEB)

SOB-1 A B A A

SOB-2 A B A A

SOB-3 B B B A

SOB-4 C C B C

SOB-5 C C B B

Methanol extract ofleaves (MEL)

SOL-1 A A A A

SOL-2 A B A A

Table 5.5: Cytotoxic potency of extracts of bark of Schleichera oleosa at the highest concentration tested in differentin vitro cytotoxicity assays+ *

Extracts

SRB MTTTissue

Colon Ovary Prostate Lung Cervix Leukemia Liver Breast Brain LeukemiaCell Lines

Colo-205 HCT-15 OVCAR-5 PC-3 DU-145 A-549 Hela MOLT-4 THP-1 Hep-G2 MCF-7 IMR-32 HL-60

Hexane(HEB) D D D D D D D D D D D D D

Chloroform(CEB) D D D D D D D D D D D D D

Ethyl Acetate(EEB) B C D C D D C D C C B D C

Methanol(MEB) A B C B C C B A B B B D B

Water(WEB) D D D B C D D C D B D D B

+ Categorization of antioxidant activities: >90%: A (Very strong); 70%-90%: B (Strong); 50%-70%: C (Moderate); <50%: D (Weak)* Highest concentrationtested: 100 µg/ml

Table 5.6: Cytotoxic potency of extracts of leaves of Schleichera oleosa at the highest concentration tested in differentin vitro cytotoxicity assays+ *

Extracts

SRB MTTTissue



Hexane(HEL) D D D D D D D D D D D D D

Chloroform(CEL) D D D D D D D D D D D D D

Ethyl Acetate(EEL) D D B B D D D C D C C D C

Methanol(MEL) C B C B C C B B B C A D B

Water(WEL) C C D D B D C D C B D D B


Table 5.7: Cytotoxic potency of extracts of roots of Schleichera oleosa at the highest concentration tested in differentin vitro cytotoxicity assays+ *

Extracts

SRB MTTTissue



Hexane(HER) D D D D D D D D D D D D D

Chloroform(CER) D D D D D D D D D D D D D

Ethyl Acetate(EER) D D D C D C C C D D D D C

Methanol(MER) C B D B C D C C C D C D C

Water(WER) C D D C D D D C D D C D C


Table 5.8: Cytotoxic potency of compounds isolated from methanolic extracts of bark and leaves of Schleichera oleosaat the highest concentration tested in different cytotoxicity assays+ *

Compounds

SRB MTTTissue

Colon Ovary Prostate Lung Cervix Leukemia Liver Breast Brain Leukemia

Cell LinesColo-205

HCT-15

OVCAR-5

IGR-OV-1

PC-3 DU-145

A-549 Hop-62

Hela SiHa MOLT-4

THP-1

Hep-G2

MCF-7

SK-N-SH IMR-32

HL-60

Methanol extractof bark(MEB)

SOB-1 C C A B B B A C B B B B B B D C C

SOB-2 D C B A B B D C C C B A C B C D A

SOB-3 B C D A B B B C B A B B D B D D B

SOB-4 C B B C B B C D B B A B C A B C B

SOB-5 B B C B A B B B B B B A D B C C B

Methanol extractof Leaves

(MEL)

SOL-1 C C C B B C B C C B B B B B D C B

SOL-2 B B C C B A C C B B A A B B C C A+ Categorization of antioxidant activities: >90%: A (Very strong); 70%-90%: B (Strong); 50%-70%: C (Moderate); <50%: D (Weak)* Highest concentrationtested: 100 µM

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