chapter 4 anti/ pro-oxidant and cytotoxicity...

Chapter 4 Anti/ Pro-oxidant and cytotoxicity studies

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4.1. INTRODUCTION

Reactive oxygen species (ROS) is a phrase used to describe a variety of molecules

and free radicals (chemical species with one unpaired electron) derived from molecular

oxygen (Halliwell, 1999). Molecular oxygen in the ground state is a bi-radical, containing

two unpaired electrons in the outer shell. Since the two single electrons have the same

spin, oxygen can only react with one electron at a time and therefore it is not very

reactive with the two electrons in a chemical bond. On the other hand, if one of the two

unpaired electrons is excited and changes its spin, the resulting species (known as singlet

oxygen) becomes a powerful oxidant (Turrens, 2003).

ROS include superoxide anion radical (O•2), singlet oxygen (

1O2), hydrogen

peroxide (H2O2), and the highly reactive hydroxyl radical (•OH). The deleterious effects

of oxygen are said to be the result from its metabolic reduction to these reactive and toxic

species (Buechter, 1988).

In body nitric oxide (NO) is produced during the metabolism of L-arginine. In

addition, O2¯•

may react with other radicals including nitric oxide (NO•) in a reaction

controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is also a

very powerful oxidant (Beckman and Koppenol, 1996; Radi et al. 2002). The oxidants

derived from NO• have been recently called reactive nitrogen species (RNS) (Turrens,

2003).

Reactive Oxygen Species normally exist in all aerobic cells in balance with

biochemical antioxidants. Oxidative stress occurs when this critical balance is disrupted

because of excess ROS, antioxidant’s depletion or both.


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Plants being the natural resources of antioxidants, endeavor was made to assess

whether plants under investigation possess any anti-oxidative properties or not in respect

to ROS and NO generation. This chapter deals with the anti/ pro-oxidative properties of

the extracts of the plants. The chapter also deals with the cytotoxicity of the same. In

order to test the safety on mammalian cells cytotoxicity of the extracts was evaluated.

4.2. REVIEW OF LITERATURE

4.2.1. Types and Sources of ROS

The term ROS is used for short-lived diffusible entities such as hydroxyl (•OH),

alkoxyl (RO•) or peroxyl (ROO

•) radicals and for some radical species of medium

lifetime such as superoxide (O•2) or nitroxyl radical (NO

•). It also includes the non-

radicals hydrogen peroxide (H2O2), organic hydroperoxides (ROOH) and hypochlorous

acid (HOCl) (Simon et al., 2000).


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Table 4.1. Types of ROS (Makhmoor, 2005)

ROS Types Symbol

Radicals

Superoxide O2-•

Hydroxyl OH•

Alkoxyl LO•/ RO

•

Peroxyl LOO•/ ROO

•

Nitric oxide NO•

Thiyl radical R-S•

Non- radicals

Hydrogen peroxide H2O2

Hypochlorous acid HOCl

Ozone O3

Singlet oxygen 1O2

Peroxynitrite ONOO-

Lipid peroxide LOOH

In living cells, the major source of endogenous ROS are hydrogen peroxide and

superoxide anion, which are generated as byproducts of cellular metabolism such as

mitochondrial respiration (Nohl et al. 2003). ROS are generated by mitochondria via the

release of electrons from the electron transport chain and the reduction of oxygen

molecules to superoxides (O2•). In vivo, O2

¯• is produced both enzymatically and

nonenzymatically.

Enzymatic sources include NADPH oxidases (Babior 2000; Vignais 2002; Babior

et al. 2002) and cytochrome P450-dependent oxygenases (Coon et al. 1992). The

proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another


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enzymatic source of both O2¯•

and H2O2 (and therefore constitutes a source of OH•) and

has been proposed to mediate deleterious processes in vivo (Yokoyama et al. 1990).

The non-enzymatic production of O2¯•

occurs when a single electron is directly

transferred to oxygen by reduced coenzymes or prosthetic groups (for example, flavins or

iron sulfur clusters) or by xenobiotics previously reduced by certain enzymes (for

example, the anticancer agent adriamycin or the herbicide paraquat) (Turrens, 2003).

Over the past 35 years several laboratories have identified a variety of mitochondrial

sources of O2¯•

including several respiratory complexes and individual enzymes.

Superoxide formation occurs on the outer mitochondrial membrane, in the matrix and on

both sides of the inner mitochondrial membrane. Whilst the O2¯•

generated in the matrix

is eliminated in that compartment, part of the O2¯•

produced in the intermembrane space

may be carried to the cytoplasm via voltage-dependent anion channels (Han et al. 2003).

Fig. 4.1. Sites of superoxide formation in the respiratory chain


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4.2.2. Nitric oxide synthesis and sources

In body, NO is produced during the metabolism of L-arginine. The major sites of

arginine synthesis in vertebrates are liver and kidney. In liver, arginine is formed as well

as rapidly metabolized by urea cycle while in kidney; citrulline (extracted from the

plasma) is converted to arginine that is released into circulation (Morris, 1992). Many

other cell types also possess low levels of arginosuccinate synthetase (ASS) and

arginosuccinate lyase (ASL) that together synthesize arginine from citrulline and can,

therefore, play a role in NO synthesis (Beaudet et al., 1986). The terminal guanidino

nitrogen atom of L-arginine is the donor for NO (Palmer et al., 1988). The reaction

catalyzed by nitric oxide synthase (NOS) requires molecular oxygen (O2) and NADPH

(Gautam and Jain, 2007).Three major isoforms of this enzyme exist: a constitutively

expressed neuronal NOS (nNOS or NOS1), an endothelial NOS (eNOS or NOS3) and

inducible NOS (iNOS or NOS2). Inducible NOS is expressed in many cell types

including macrophages, muscle cells, hepatocytes, fibroblasts, astrocytes and endothelial

cells (Nathan and Shiloh, 2000).

Production of NO is induced in multiple cells types of the immune system,

including mast cells, dendritic cells, NK cells and phagocytic cells (neutrophils,


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eosinophils, macrophages, microglia cells, Kupffer cells) as well as other cells involved

in immune reactions, such as vascular smooth cells, keratinocytes, hepatocytes,

fibroblasts, cardiomyocytes, chondrocytes, mesangial cells, epithelial and endothelial

cells (Oswald et al., 1992; Bogdan, 2000).

4.2.3. Neutralization of ROS

Cellular redox balance is maintained by a powerful anti-oxidant system that

“neutralizes” ROS (Manda et al., 2009). Certain enzymes as well as non- enzymatic

cellular molecules are involved in the detoxification of ROS. Based on the nature of anti-

oxidants, the human anti-oxidant system can be categorized into two broader classes:

enzymatic and non- enzymatic (Jakus, 2000; Makhmoor, 2005).

Enzymatic Anti-oxidants: Enzymatic anti-oxidant system includes superoxide

dismutases (SOD), Catalase (CAT), the glutathione system (glutathione, glutathione

reductase, peroxidase (GPx) and transferase), the thioredoxin system (thioredoxins,

thioredoxin peroxidase and peroxiredoxins). In mitochondria, superoxide anion (O2−) can

be dismutated to hydrogen peroxide (H2O2) by two enzymes, namely copper-zinc

superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), that

are present in the mitochondrial matrix and in the intermembrane space, respectively

(Gibellini et al., 2010).

2 O2 + 2 H2O → O2 + H2O2 + 2 OH-

Once generated, H2O2 can be quenched by GPx in mitochondria, or by catalase in the

cytosol. Catalase, a major H2O2 detoxifying enzyme found in peroxisomes, is also present


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in heart mitochondria (Radi et al. 1991). However, this enzyme has not been found in

mitochondria from other tissues, including skeletal muscle (Phung et al., 1994).

2 H2O2 → 2 H2O + O2

Fig. 4.2. The generation of ROS by mitochondria. Electrons released from

mitochondria reduce oxygen molecules, thereby producing such ROS as superoxides

(O2.). Superoxide dismutase (SOD) catalyzes H2O2 formation from superoxides.

H2O2 might be deactivated by catalase (CAT). However, when H2O2 reacts with iron

or copper ions, hydroxyl radicals (OH•), the most reactive form of ROS, are

produced.

Hydrogen peroxide, the product of O2.-

dismutation and the main precursor of OH• in the

presence of reduced transition metals, is mostly decomposed by the enzyme glutathione

peroxidase (GPx). In the liver, mitochondria account for about one third of the total

glutathione peroxidase activity (Chance et al. 1979). A second GPx associated with the

mitochondrial membrane, known as phospholipid–hydroperoxide glutathione peroxidase,


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is specifically involved in reducing lipid peroxides associated with the membrane (Ursini

et al. 1999; Nomura et al. 2000). All GPxs require Glutathione (GSH) as a cofactor and

secondary enzymes, such as glutathione reductase and glucose-6-phosphate

dehydrogenase (G-6-PDH), to function. G-6-PDH generates NADPH to recycle the GSH

(Takebe et al., 2002; Ivanova and Ivanov, 2000).

GPx

2GSH + H2O2 GSSG + 2 H2O

Non-Enzymatic Anti-oxidants: Non-enzymatic anti-oxidants may be further

classified into two groups: endogenous and exogenous anti-oxidants. The major

extracellular endogenous anti-oxidants found in human plasma are transition metal

binding proteins. These include ceruloplasmin are the copper ions sequesters.

Hepatoglobin binds with haemoglobin while ferritin and transferring bind with free iron

(Ivanova and Ivanov, 2000; Halliwell and Gutteridge, 1990). Lipoic and uric acids,

bilirubin, ubiquinone and glutathione are non-protein endogenous anti-oxidants which

inhibit the oxidation processes by scavenging free radicals (Shahidi, 1997).

Many effective exogenous anti-oxidants are generally of dietary origin. The best

known are vitamins such as ascorbic acid, vitamin E, carotenoids, quinines and

polyphenols. These molecules can inhibit oxidative reactions by scavenging free radicals,

while certain compounds may chelate redox active metals or inhibit particular oxidative

enzymes. Vitamin E is a lipid soluble, chain breaking anti-oxidant which reacts with lipid

peroxyl radicals to yield a relatively stable lipidhydroperoxide and thus protects against


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membrane lipid peroxidation (Maguire et al., 1989). On the other hand, vitamin C has

multiple anti-oxidant properties, including the ability to regenerate α-tocopherol by

reducing α-tocopheroyl radicals at the membrane surfaces of membranes. It also

scavenges others free radicals and certain non-radicals such as hypochlorous acid (HOCl)

(Packer and Cadenas, 2002; Makhmoor, 2005).

4.2.4. ROS, NO and disease states

ROS normally exist in all aerobic cells in balance with biochemical antioxidants.

Oxidative stress occurs when this critical balance is disrupted because of excess ROS,

antioxidants depletion, or both (Waris and Ahsan, 2006). There is growing awareness that

oxidative stress plays a role in various clinical conditions such as malignant diseases,

diabetes, atherosclerosis, chronic inflammation, viral infection, and ischemia-reperfusion

injury (Behrend et al., 2003; Apel and Hirt, 2004; Bergamini et al., 2004; Reddy and

Clark, 2004; Shah and Channon, 2004; Willner, 2004).

ROS levels are increased in cells exposed to various stress agents, including

anticancer drugs (Jabs, 1999). Excessive anti-oxidants scavenge these beneficial ROS and

can thereby interfere with the protective functions of phagocytes (Cedro et al., 1994).

Chronic hepatitis B (HBV) and hepatitis C virus (HCV) infections are associated with an

increased production of ROS within the liver that is responsible for the oxidation of

intracellular macromolecules (Waris and Ahsan, 2006). Chronic HBV infection results in

an increased total intra hepatic iron and/or increase in the pro-oxidant low molecular

weight iron compartment of the liver. Previously, a strong correlation between the

presence of HBV surface antigen and iron deposition in the Kupffer cells and spleens of


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infected individuals has been reported (Senba et al., 1985). The exogenous oxygen

radical load is contributed by a variety of environmental agents (inhaled smoke and

polluted air) and dietary antioxidants (Frei et al., 1991; Nagashima et al., 1995; Churg,

2003). Mutagens, tumor promoters and a variety of carcinogens including benzene,

aflatoxin and benzo(a)pyrene may exert their partly by generating ROS during their

metabolism (Mauthe et al., 1995; Pagano, 2002; Schins, 2002).

ROS are essential mediators of apoptosis which eliminates cancer and other cells

that threaten our health (Kerr et al., 1994; Blackstone and Green, 1999; Slater et al.,

1995; Johnson et al., 1996; Kroemer et al., 1997; Hickman, 1992). Excessive anti-

oxidants interfere with this highly important protective mechanism (Verhaegen et al.,

1995; McGovan et al., 1996; Labriola and Linvingston, 1999; Salganik et al., 2001).

H2O2 can kill both the promastigote and amastigote forms of Leishmania

donovani parasite (Das et al., 2001). Most importantly, reactive oxygen species,

including H2O2, generated by anti-parasitic agents or macrophages can kill the

intracellular parasites (Nabi, 1984; Mauel et al., 1984) and are therefore important

regulators of protozoal infection (Solbach and Laskay, 2000; Schirmer et al., 1987).

Numerous reports have shown that Leishmania parasites are susceptible to ROS-

mediated killing (Zarley et al., 1991; Wilson et al., 1994; Pearson et al., 1983 Channon

and Blackwell, 1985; Murray, 1982) and RNS-mediated killing (Roach et al., 1991;

Bhattacharyya et al., 2002; Green et al., 1990; Mauel et al., 1991; Liew et al., 1990c;

Evans et al., 1993; Vouldoukis et al., 1995; Murray and Nathan, 1999). It has been

shown that RNS alone is both necessary and sufficient to control Leishmania donovani


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infection in mice (Murray and Nathan, 1999) and more recently that both ROS and RNS

produced by macrophages act together early to control infection by L. chagasi (Gantt et

al., 2001) and L. donovani (Bhattacharyya et al., 2002; Murray and Nathan, 1999).

4.2.5. Anti-oxidant/ Pro-oxidant activity of plant extracts

Plants contain active components, namely phenolics and polyphenolics, that are

known to act as anti-oxidants (Cai et al., 2003). Every anti-oxidant is in fact a redox

agent and might become a pro-oxidant to accelerate lipid peroxidation and induce DNA

damage under special conditions and concentrations (Lastra and Villegas, 2007).

Studies have revealed pro-oxidant effects of anti-oxidant vitamins and several

classes of plant-derived polyphenols such as flavonoids (Rahman et al., 1990), tannins

(Singh et al., 2001) and curcumin (Ahsan and Hadi, 1998). As reported earlier, resveratol

(Lastra and Villegas, 2007) and phloroglucinols from Garcinia subelliptica (Wu et al.,

2008) can exhibit pro-oxidant properties, leading to oxidative breakage of cellular DNA

in the presence of transition metal ions such as copper. The pro-oxidant and anti-oxidant

effect of plant extracts are due to the balance of two activities: free radical-scavenging

activity and reducing power on iron ions, which may drive the Fenton reaction via

reduction of iron ions. In a Fenton reaction, Fe2+

reacts with H2O2, resulting in the

production of hydroxyl radicals, which are considered to be the most harmful radicals to

biomolecules. Fe2+

is initially oxidized to Fe3+

in the Fenton reaction. By the action of

many reductants, such as ascorbic acid, the oxidized forms of iron ion can be reduced

later to the reduced form (Fe2+

), which can enhance the generation of hydroxyl radicals.


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A predominant reducing power (on iron ions) over the free radicalscavenging activity in a

mixture of compounds results in the pro-oxidant effect (Tian and Hu, 2005).

Desmarchelier et al., (1997) studied anti oxidant and pro-oxidant activities of

lyophilized aqueous extracts of 19 Argentine plants using the hydroperoxide-initiated

chemiluminescence assay in liver homogenates and noted that two extracts, from

Baccharis grisebachii and Terminalia triflora exhibited pro-oxidant activity in the 10–

1000 µg range. Yen et al., (1997) investigated anti-oxidant and pro-oxidant effects of

various tea extracts and observed dual effects of tea extracts in the model system that was

dependent on the ability of both reducing iron and scavenging oxy-radicals. Perez- Garcia

et al., (2001) evaluated aqueous, methanol and dichloromethane extracts from Artemisia

copa, Baccharis grisebachii, Baccharis incarum, Baccharis latifolia, Mutisia kurtzii and

Pluchea sagittalis, plants used in the Traditional Medicine of South America, for activity

on the respiratory burst and the inducible heat shock protein of 72 kD (hsp72) synthesis

and found that the aqueous extract from Mutisia kurtzii caused a clear increase of the

hsp72 production and showed pro-oxidant activity. Ling et al., (2010) analyzed the anti-

oxidant activity of several Malaysian plant extracts with their pro-oxidant capacity.

Khawaga and Abou- Seif, (2010) assessed the anti-oxidant activities of the aqueous

extracts of dry green pods of Phaseolus vulgaris, leaves of Olea europaea, unripe fruits

of Bitter melon and leaves of Morus nigra and also observed pro-oxidant activities of the

aqueous extracts of the above plants towards protein and estimation of some markers of

the protein oxidation.


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4.2.6. Cytotoxicity and Cytoxicity assays

Cytotoxicity assays are widely used by the pharmaceutical industry to screen for

cytotoxicity in compound libraries. The development of in vitro cytotoxicity assays has

been driven by the need to rapidly evaluate the potential toxicity of large numbers of

compounds, to limit animal experimentation whenever possible, and to carry out tests

with small quantities of compound. Assessing cell membrane integrity is one of the most

common ways to measure cell viability and cytotoxic effects. Compounds that have

cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan

blue or propidium iodide are normally excluded from the inside of healthy cells;

however, if the cell membrane has been compromised, they freely cross the membrane

and stain intracellular components (Riss and Moravec, 2004). Alternatively, membrane

integrity can be assessed by monitoring the passage of substances that are normally

sequestered inside cells to the outside. One commonly measured molecule is lactate

dehydrogenase (LDH) (Decker and Lohmann-Matthes, 1988). Protease biomarkers have

been identified that allow researchers to measure relative numbers of live and dead cells

within the same cell population. The live-cell protease is only active in cells that have a

healthy cell membrane, and loses activity once the cell is compromised and the protease

is exposed to the external environment. The dead-cell protease cannot cross the cell

membrane, and can only be measured in culture media after cells have lost their

membrane integrity (Niles et al., 2007).

Cytotoxicity can also be monitored using the 3- (4, 5-dimethyl thiazol-2-yl)-2, 5-

diphenyl tetrazolium bromide (MTT) or 3-(4,5 deimethylthiazol-2-yl)-5-(3-


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carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H- tetrazolium (MTS) assay. This assay

measures the reducing potential of the cell using a colorimetric reaction. Viable cells will

reduce the MTS reagent to a colored formazan product. A similar redox-based assay has

also been developed using the fluorescent dye, resazurin (Riss and Moravec, 2004). In

addition to using dyes to indicate the redox potential of cells in order to monitor their

viability, researchers have developed assays that use ATP content as a marker of viability

(Riss and Moravec, 2004). Such ATP-based assays include bioluminescent assays in

which ATP is the limiting reagent for the luciferase reaction (Fan and Wood, 2007).

4.3. MATERIALS AND METHODS

4.3.1. Reagents

Starch, Penicillin, Streptomycin, N (1-napthyl) ethylene diamine dihydrochloride

(NED), Sulphanilimide, Roswell Parker Memorial Institute (RPMI) 1640 was obtained

from Sigma (St Louis). 2′, 7′-Dichloro dihydrofluorescein diacetate (DCFDA) was

obtained from Molecular Probes (Eugene, OR). Fetal bovine serum (FBS) was obtained

from Invitrogen Corporation (Carlsbad, CA). 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT), Trypan Blue Dimethyl sulfoxide (DMSO) were purchased

from Sigma (St Louis).


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4.3.2. Buffers and Solutions

1. Phosphate buffered saline: 20 mM Phosphate buffer, 150 mM NaCl, pH

7.2

2. Phosphate buffer: 136 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4,

1.46mM KH2PO4, pH 7.2

3. HANKS buffer: 135 mM NaCl, 5 mM KCl, 5 mM D- glucose, 3mM

Na2HPO4, 4 mM KH2PO4, 10 mM HEPES, 4 mM

NaHCO3, pH 7.2

4. Griess reagent (Green et al., 1982)

Name of the

Reagent

Ingredients Concentration

Colour Reagent – I Sulfanilamide (SRL, India) 1% (w/v) (dissolved in 5%

aqueous H3PO4)

Colour Reagent – II 1-N-(napthyl)-ethylene

diamine dihydrochloride

0.1% (w/v) in double

distilled water

Equal volumes of Reagent-I and Reagent-II are mixed to prepare the Griess reagent.


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4.3.3. Animals and cell culture

BALB/c mice reared in Indian Institute of Chemical Biology (IICB) was used for

experimental purposes with prior approval of the animal ethics committee of the institute.

RPMI-1640 buffered with 20 mM HEPES and 0.2 g NaHCO3, supplemented with

10% (v/v) heat inactivated FBS, 500 M ME, 100U/ml penicillin, 100g/ml

streptomycin was used for cell culture.

4.3.4. Isolation of macrophages

Macrophages (MΦs) were isolated from mice 36-48 h after injection (i.p.) with

2% (w/v) hydrolyzed starch by peritoneal lavage with ice-cold Phosphate buffered saline

(PBS) (Mookerjee Basu et al., 2006). Cells were washed and cultured for 18-24 hr (for

adherence) in Roswell Parker Memorial Institute (RPMI) 1640 (supplemented with 100

IU/ml of penicillin and 100 µg/ml of streptomycin) containing 10% (v/v) heat inactivated

Fetal Bovine Serum (FBS) [RPMI-FBS] at 370C at 5% CO2 in air in tissue culture

petridishes of 65 mm diameter (Tarson India Ltd), following which culture medium was

washed off and fresh RPMI-FBS was added. About 1x106 MΦs were cultured on 22 mm

x 22 mm cover glass placed in plastic petridishes.


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4.3.5. Measurement of Reactive Oxygen Species (ROS)

Oxidative activity was determined by measuring Reactive oxygen species (ROS)

and nitric oxide (NO) in macrophages derived from Peritoneal Exudate cells (PEC) of

BALB/c mice in vitro with or without treatment.

To measure the level of Reactive Oxygen Species (ROS) (includes superoxide,

hydrogen peroxide (H2O2) and other reactive oxygen intermediates) produced within the

cells, the cell permeable nonpolar H2O2 sensitive probe 2´, 7´- Dichloro

dihydrofluorescein diacetate (DCFDA) was used (Schreck and Baeuerle, 1994). Extent of

H2O2 generation was defined as ROS generation. The cells were harvested in Hanks

buffer saline solution (HBSS). Finally, 1x106 cells were taken in 1ml of HBSS, pulsed

with DCFDA (5 µM) for 20-30 min at 370C in dark and then washed twice with HBSS;

the relative fluorescence was measured by fluorimetry using a fluorimeter (Hitachi)

(excitation at 505 nm and emission at 525 nm at slit size1 nm). For each experimental

sample, fluorimetric measurements were performed in triplicate and expressed as Mean

Fluorescent Intensity (MFI)/106cells.

4.3.6. Measurement of Nitric Oxide (NO)

Nitric Oxide (NO) generation in normal MΦs was monitored by colorimetric

assay for nitrite by Griess reaction (Green et al., 1982). 0.5x106 cells/ ml were taken for

nitrite assay. The amount of nitrite accumulated was calculated from a standard curve

constructed with different concentrations of sodium nitrite, and the concentration of

nitrite accumulated was expressed as µM nitrite (Mookerjee Basu et al., 2006).


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4.3.7. Cytotoxicity Assay

Cell viability was evaluated microscopically by trypan blue exclusion method and

by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Trypan

Blue is a blue acid dye that has two azo chromophores group. Trypan Blue is an essential

dye, use in estimating the number of viable cells present in a population (Phillips and

Terryberry, 1957). Equal volume of cell culture and equal volume of 10% diluted trypan

blue was taken together. The solution was mixed well using a pipette. Some of the cell

suspension: trypan blue mixture was transferred to a hemocytometer and covered with a

cover slip. Count was taken under an inverted microscope. After staining with trypan

blue solution counting should commence


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2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in

living cells (Mosmann, 1983). A solubilization solution dimethyl sulfoxide (DMSO) is

added to dissolve the insoluble purple formazan product into a colored solution. The

absorbance of this colored solution can be quantified by measuring at 550 nm wavelength

by a spectrophotometer. 0.5x106 cells were seeded in 24 well tissue culture plates and

after 24 hr, the cells exposed to different concentration of drug. After 48hrs, the cells

were exposed to 100 µl of MTT reagent from stock (5 mg/ ml MTT in PBS/ HANKS)

and incubated for 4 – 5 hrs at 370C. The viable cells convert the tetrazolium into a

coloured product by the action of an enzyme mitochondrial dehydrogenase. The medium

together with MTT was aspirated off from the wells. The formed dye formazan was

dissolved in 500 μl of DMSO and the plates shaken for 5 minutes. The absorbance was

measured at 550 nm in a spectrophotometer and the result was expressed as Cytotoxicity

Index (CI).

Cytotoxicity Index (CI) = OD at 550nm/106 cells

4.4. RESULTS

4.4.1. Measurement of Reactive Oxygen Species (ROS) in normal Macrophages in

response to P. javanica and E. nummularius methanol extracts

Results of ROS generation in P. javanica and E. nummularius treated/ untreated

(control) macrophages (MΦs) are presented in (Table 4.2, Fig. 4.3A & 1B). ROS

generation increased in P. javanica and decreased in E. nummularius (Table 4.2, Fig.


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4.3A & 1B) treated macrophages to about two fold compared to untreated control. P.

javanica showed pro-oxidant activity in ROS generation. Dose dependent response was

observed, however the response was inversely related with the dose. When compared to

control, approximately three fold increase in ROS was observed at 1µg/ml dose and

approximately two fold increase was observed at 100µg/ml dose (Table 4.2, Fig. 4.3A).

E. nummularius showed anti-oxidative activity regarding ROS generation and the

response was increased with increase of the dose. Approximately four fold decrease was

at 100µg/ml dose. At 1µg/ml dose more than two fold decrease was observed (Table 4.2,

Fig. 4.3B).

4.4.2. Measurement of Nitric Oxide (NO) in normal Macrophages in response to P.

javanica and E. nummularius methanol extracts

Results of Nitric Oxide (NO) generation in P. javanica and E. nummularius

treated/ untreated (control) macrophages (MΦs) are presented in (Table 4.3, Fig. 4.3C &

4.3D). NO generation was measured at two time intervals i.e., after 24hr and after 48hr of

incubation. Results show a significant increase in NO generation (Table 4.3, Fig. 4.3C &

4.3D) in response to both the plant methanol extracts. In P. javanica treated MΦs

maximum increase was observed at 1 µg/ml dose (about eight fold increase after 24 hr

and about four fold increase after 48 hr of incubation compared to control). In E.

nummularius treated MΦs maximum increase was observed at 10 µg/ml dose (about nine

fold increase after 24 hr and about five fold increase after 48 hr of incubation compared

to control).


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4.4.3. Cytotoxicity test

In order to test the safety of extracts on mammalian cells, murine macrophages

were treated with the extracts, and their cytotoxicity was evaluated by using MTT and

trypan blue. The results of MTT assay shows that there is increase in reading in P.

javanica and E. nummularius extract treated sample upto 100 µg/ml dose compared to

control. Maximum reading was observed at 1 µg/ml dose in case of P. javanica and 50

µg/ml dose in case of E. nummularius. Increase in reading in extract treated samples

means more number of cells are viable in treated samples which indicate that P. javanica

and E. nummularius extracts are not toxic for normal macrophages up to the dose of 100

μg/ ml concentration (Table 4.4, 4.5 & Fig. 4.4A, 4.4B). In other words the plant extracts

are safe for normal tissue cells upto the dose of 100 μg/ ml.

The viability of uninfected MΦ cells upon treatment with methanol extracts was

checked by trypan blue dye exclusion method. When murine MΦs were treated with 10,

50 and 100μg/ ml of E. nummularius for 24 hour, 95, 93 and 83% of cells respectively,

remained viable (Table 4.6). When MΦs were treated with P. javanica at the above-

mentioned doses for the same duration, 97, 95, 89% of cells respectively remained viable.

MΦs were treated with the maximum dose (100μg/ ml) of the extracts, 11% and 17% of

dead cells were found at maximum dose (Table 4.6) for P. javanica and E. nummularius

respectively.


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Table 4.2. Reactive Oxygen Species production in normal macrophages in response

to in vitro methanol extract treatment of P. javanica and E. nummularius.

Plant extract Concentration/Dose

(μg/ml)

ROS in normal Mφ (MFI ±

SD) at 6 hr

P. javanica extract 0 233 ± 3.5

1 717 ± 5***

10 631 ± 1.5***

100 499 ± 6***

E. nummularius

extract

0 251 ± 2

1 113 ± 5***

10 93 ± 4.8***

100 56 ± 3***

Statistical analysis was done using student’s T- test. *, ** and *** represent significant

differences of the extract treated compared to untreated control at the level of p= 0.05, p=

0.01 and p= 0.001 respectively.


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Table 4.3. Nitric Oxide (NO) Induction by P. javanica and E. nummularius in

normal macrophages.

Plant extract Concentration/Dose

(μg/ml)

Mean μM nitrite in

normal Mφ ± SD at

24 hr

Mean μM nitrite in

normal Mφ ± SD at

48 hr

P. javanica 0 4.5 ± 0.5 6 ± 1

1 32.25 ± 1*** 23 ± 2***

10 31 ± 1.5** 21.5 ± 1.5***

100 26.8 ± 1*** 19 ± 1***

E.

nummularius

0 4.5 ± 0.5 6 ± 0.5

1 31.5 ± 0.8*** 19.5 ± 1***

10 39.5 ± 2** 29.5 ± 2**

100 39 ± 1*** 19.5 ± 0.5***

Statistical analysis was done using student’s T- test. ** and *** represent significant

differences of the extract treated compared to untreated control at the level of p= 0.01 and

p= 0.001 respectively.


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Table 4.4. Cytotoxicity assay for P. javanica methanol extract on normal Mφ cells

derived from peritoneum of BALB/c mice by using MTT.

Concentration/ Dose (μg/ml) C.I at 24 hour ± SD C.I. at 48 hour ± SD

0 2.443 ± 0.301 2.067 ± 0.374

0.1 7.1 ± 1** 5.967 ± 0.802**

1 13.973 ± 0.57*** 7.062 ± 0.411***

10 10.383 ± 0.58*** 6.111 ± 0.253***

50 9.983 ± 0.88** 5.578 ± 0.984**

100 7.2 ± 0.529** 4.99 ± 0.642**

C.I. = Cytotoxicity Index

Statistical analysis was done using student’s T- test. ** and *** represent significant

differences of the extract treated compared to untreated control at the level of p= 0.01 and

p= 0.001 respectively.


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Table 4.5. Cytotoxicity assay for E. nummularius methanol extract on normal Mφ

cells derived from peritoneum of BALB/c mice by using MTT.

Concentration/ Dose

(μg/ml)

C.I at 24 hour ± SD C.I. at 48 hour ± SD

0 2.443 ± 0.301 2.067 ± 0.374

1 5.11 ± 0.441*** 3.933 ± 0.653*

10 8.293 ± 0.566*** 6.18 ± 0.407***

50 9.833 ± 0.807*** 5.85 ± 0.541***

100 6.617 ± 0.333*** 4.987 ± 0.023**

C.I. = Cytotoxicity Index

Statistical analysis was done using student’s T- test. *, ** and *** represent significant

differences of the extract treated compared to untreated control at the level of p=0.5, p=

0.01 and p= 0.001 respectively.


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Table 4.6. Cytotoxicity assay of P. javanica and E. nummularius methanol extract on

normal Mφ cells derived from peritoneum of BALB/c mice by using Trypan blue.

Plant extracts Concentration/ Dose (μg/ml) % Viable cells

P. javanica

10 97%

50 95%

100 89%

E. nummularius

10 95%

50 93%

100 83%


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Fig. 4.3. P. javanica extract (1A, 1C) and E. nummularius extract (1B, 1D) induced

generation of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) in

macrophage.


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Fig. 4.4. Toxicity of (A) P. javanica and (B) E. nummularius extract in macrophages.

Cytotoxicity was measured using MTT as described in the methods section. Data

points are mean and SD values obtained from six replicates.


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4.5. DISCUSSION

In order to study the anti/pro-oxidative properties, generation of Reactive Oxygen

Species (ROS) and nitric oxide (NO) in macrophage cells were estimated. Reactive

Oxygen Species in terms of hydrogen peroxide generation was estimated using H2O2

sensitive fluorescent dye, DCFDA and nitric oxide generation was estimated by Griess

reagent in macrophage cells in in vitro condition.

In connection to ROS generation, P. javanica and E. nummularius acted

differently, being pro-oxidative and anti-oxidative respectively. P. javanica treated cells

showed approximately three fold higher (p


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oxidant capacity. Dual effects of tea extracts are also reported (Yen et al., 1997). Plants

contain active components, namely phenolic and poly phenolic, that are known to act as

anti-oxidants (Cai et al., 2003). However studies have also revealed pro-oxidant effects

of several classes of plant-derived polyphenols such as flavonoids (Rahman et al., 1990),

tannins (Singh et al., 2001) and cucurmin (Ahsan and Hadi, 1998).

Reactive oxygen species are key actors of non-specific immune defense and the

toxic potential of ROS is used by the innate immune defense as a powerful weapon

against pathogens (Manda et al., 2009). One of the most beneficial functions of NO is

also its implication in host defense against intracellular pathogens (viz., Salmonella and

Leishmania) (Gautam and Jain, 2007). Its derivatives such as per-oxynitrite are strong

bactericidal in nature (Gautam and Jain, 2007). ROS appear to activate and modulate

apoptosis when cells are under stress (Benhar, 2002). It is reported that ROS levels are

increased in cells exposed to various stress agents, including anticancer drugs (Jabs,

1999) and they promote apoptosis by stimulating pro-apoptotic signaling molecules, such

as ASK1, JNK and p38 (Benhar et al., 2001, Davis et al., 2001; Tobiume et al., 2001).

ROS also play a pivotal role in p53 induced apoptosis (Polyak et al., 1997). Reactive

nitrogen intermediates also play a central role in apoptotic cell death (Gautam and Jain,

2007) and the role of NO in tumor cytotoxicity is well documented (Chang, 2001).

Therefore, pro-oxidant nature of the extracts, especially in respect to NO generation, may

have clinical and therapeutic proposition in tumor cytotoxicity and intracellular pathogen

killing.

chapter 4 anti/ pro-oxidant and cytotoxicity...

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