2.0 REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5560/9/09... ·...

Chapter 2 Review Of Literature

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2.0 REVIEW OF LITERATURE

Plants have been an essential part of human society since the

civilization started. A medicinal plant is any plant which, in one or more of its

parts contain substances that can be used for therapeutic purposes or which

are precursors for the synthesis of useful drugs (Perumal et al., 2004;

Oladunmoye, 2007). Recent awareness of therapeutic potential of several

traditionally used plants has opened a new dimension for the study and

research of medicinal plants (Bhandari et al., 2007).

Cancer is one of the most dreaded diseases of the 20th century and

spreading further with continuance of increasing incidence in 21st century

(Balachandran and Govindarajan, 2005). In recent years, a considerable

attention has been placed to identify naturally occurring chemopreventive

substances capable of inhibiting, retarding or reversing the process of

carcinogenesis (Shukla and Kalra, 2007).

Cancer is a hyperproliferative disorder that involves transformation,

dysregulation of apoptosis, proliferation, invasion, angiogenesis and

metastasis (Aggarwal et al., 2006). Chemoprevention by the use of naturally

occurring substances is becoming a promising strategy to prevent cancer

(Chen et al., 2007). Cancer chemopreventive agents are classified as blocking

or suppressing agents if they inhibit initiation or promotion / progression phase

of carcinogenesis, respectively (Moreno et al., 2007).

India is well known for a plethora of medicinal plants. The traditional

Indian medicinal plants act as antiradicals and DNA cleavage protectors.

These plants have also been considered to protect health, longevity,

intelligence, immunosurveillance and body resistance against different

infections and diseases (Manna et al., 2006).

To the possible extent sincere efforts have been made to collect the

relevant literature of the study. After thorough reviewing of all possible sources, it

was observed that very few studies have been conducted earlier on certain

dimensions of the present study in our laboratories. Information on the

Antioxidative, antitumor and immunomodulatory role of C. dactylon and

T. catappa leaves is still very scanty in literature. The review of literature


9

pertaining to the present research entitled “Antioxidative, antitumor and

immunomodulatory efficacy of protein fraction of Cynodon dactylon and

Terminalia catappa leaves on experimentally implanted ELA cells in Swiss

albino mice” is appropriately presented under the following headings:

2.1 Reactive oxygen species

2.1.1 Free radicals

Formation of free radicals

Superoxide anion

Perhydroxyl radical

Hydroxyl radical

DPPH

2.1.2 Non-radicals

Singlet Oxygen

Hydrogen peroxide

Hyphochlorous acid

Nitric oxide

2.2 Lipid peroxidation

2.3 Antioxidants

2.3.1 Enzymic antioxidants

2.3.2 Nonenzymic antioxidants

2.4 Antitumor activity of medicinal plants

2.5 Immunomodulatory activity of medicinal plants

2.6 Medicinal plants selected for the study

2.6.1 Cynodon dactylon

2.6.2 Terminalia catappa

2.1 REACTIVE OXYGEN SPECIES

Reactive Oxygen Species are usually highly reactive and short-lived,

known to cause damage to cellular components including lipid, DNA, protein,

carbohydrate, and other biological molecules. They consequently lead to many

pathological processes such as aging, cancer, cardiovascular diseases, diabetes,

inflammation, neurodegenerative diseases and infertility (Piconi et al., 2003;

Junqueira et al., 2004; Casetta et al., 2005; Singh et al., 2005; Haidara et al.,

2006; Valko et al., 2006; Reddy, 2006; Gupta et al., 2006; Grossman, 2008;


10

Cheung et al., 2008). Fortunately, biological systems can protect themselves

against harmful effects of ROS and free radicals by designing an optimal

nutritional countermeasure and formation of antioxidants (Fang et al., 2002).

The ROS play a major role in tumor promotion by cellular damage. The

biological importance of these radicals arises from the fact that they occur

during the normal metabolic process. The ultimate target of free radicals is the

genome, ie., usually DNA and RNA molecules or it may cause strand breakage

in both nuclear and mitochondrial organelles. The irreversible nature of these

an alteration ultimately leads to malignant and mutagenic states toppling the

whole cellular metabolism (Yu et al., 2007).

Mammalian life depends upon oxygen as the final acceptor of electrons

in mitochondrial electron transport, but the process also generates toxic

metabolites (Phillips et al., 2003). Reactive oxygen species leak from

mitochondria into the cytoplasm where they cause cellular damage by oxidizing

a variety of biologically important molecules, including DNA, proteins, lipids

and carbohydrates (Przekwas et al., 2003). Reactive Oxygen Species produce

cellular injury and necrosis via several mechanisms including peroxidation of

membrane lipids, protein denaturation and DNA damage (Hemnani and

Parihar, 1998; Cuzzocrea et al., 2002). Reactive Oxygen Species mediated

DNA damage has long been thought to play a role in carcinogenesis initiation

and malignant transformation (Valko et al., 2006).

The ROS include both free radicals and non-radicals. Most biologic

molecules are non-radicals containing two electrons per orbital, which is a

stable configuration in a molecule (http://www.cellscience.com). A free radical

is a molecule that can exist independently and contains one or more unpaired

electrons. An unpaired electron means that there is only one electron in an

orbital (shown in red), which is an unstable configuration and makes free

radicals highly reactive (Figure 1). ROS encompasses other reactive species

which are not true radicals but are nevertheless capable of radical formation in the

intra and extra cellular environment. Eg., Hydrogen peroxide, Hypochlorous acid

and singlet oxygen (Chapple and Matthews, 2007).


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FIGURE 1: REACTIVE OXYGEN SPECIES (ROS)

(http://www.cellscience.com)

2.1.1 Free radicals

A free radical can be defined as any atom or molecule possessing one

or more unpaired electrons. A major source of free radicals in biological

systems is molecular oxygen. They are formed when oxygen interacts with

certain molecules. Once formed, these highly reactive radicals can start a

chain reaction hence they have significant biological importance. They are

generally unstable and very reactive. The biologically relevant free radicals

derived from oxygen are the superoxide anion (O2-), the perhydroxyl radical

(protonated superoxide, HO2), the hydroxyl radical (HO.) and free radical nitric

oxide (Cuzzocrea et al., 2001).

Formation of free radicals

Living cells are exposed to oxidants originating from a large variety of

exogenous or endogenous sources. Exogenous sources - air pollutants,

ozone, radiation, chemicals, toxins, pathogenic microorganisms. Endogenous

sources-due to leaks in electron transport chain in mitochondria during

oxidation of food stuffs and inflammatory cells produce free radicals by a

process of respiratory burst (Figure 2).


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FIGURE 2: FORMATION OF FREE RADICALS

(http://www.smokersrx.com)

Free radicals cause tissue damage by a variety of different mechanisms

which include DNA damage, protein damage, lipid peroxidation, enzyme oxidation

and stimulation of proinflammatory cytokines release (Pauwels et al., 2007).

The role of free radicals has been implicated in the causation of several

pathophysiological disorders such as liver cirrhosis, atherosclerosis, cancer,

aging, rheumatoid arthritis, diabetes and neuro degeneration (Finkel and


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Holbrook, 2000; Droge, 2002) and the compounds that scavenge free radicals

have great potential in ameliorating these disease processes. The human body

has inherent mechanisms to reduced free radical induced injury by glutathione,

arginine, citrulline, taurine, creatine, selenium, zinc, vitamin E, vitamin C, vitamin A

and tea polyphenols and endogenous enzymes such as superoxide dismutase.

Sometimes these protective mechanisms are found not to be sufficient when

compared to the insult produced to the body; hence the search for exogenous

antioxidants is continued (Shirwaikar et al., 2004).

Reactive Oxygen Species are emerging as critical signaling molecules

(Pouyssegur et al., 2006). Redox balance, the ratio between oxidizing and

reducing species within the cell, plays a significant role in the regulation of signaling

pathways, including kinase and phosphatase activity and gene expression through

modulation of transcription factor function (Biswas et al., 2006).

Free radicals do their damage with a sequence of changes resulting

from an injury (burn or thermal shock) and ultimately oxidative stress from the

depletion of antioxidant defense mechanisms. Oxidative stress can damage

many biological molecules. Proteins and DNA appear to be some significant

targets of cellular injury (Gradelet et al., 1998; Glei et al., 2000; Collins, 2001).

Condition of oxidative stress arises either from the overproduction of free

radicals of oxygen or from the deficiency of antioxidant defenses or repair

mechanisms and results in reversible or irreversible tissue injury (Athar, 2002;

Dey and Cederbaum, 2006). It was also showed that oxidative stress from

chronic inflammation favours cancer development in many organs

(Valko et al., 2007). Evidences have accumulated to suggest that ROS play an

important role in tumor initiation by enhancing or facilitating the metabolic

activation and / or initiating effect of carcinogens. It also occurs due to an

imbalance in prooxidant and antioxidant levels (Zablocka and Janusz, 2008). The

formation of excessive amounts of ROS including superoxide anions is toxic to the

cell. Hence, metabolizing and scavenging systems to remove them are

functionally critical and tightly controlled in the cells (Mullineaux and Creissen,

1996).


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Superoxide anion (O2-)

The superoxide free radical anion is formed when oxygen is reduced by

the transfer of a single electron to its outer shell. The major source of

superoxide in vivo is the electron leakage that results from the electron transfer

chain of the mitochondria. Superoxide anion plays an important role in the

formation of more reactive species such as hydrogen peroxide, hydroxyl

radical, and singlet oxygen, which induce oxidative damage in lipids, proteins

and DNA (Wu and Cederbaum, 2004).

A major portion of the biological consumption of molecular oxygen

occurs during reduction to water via oxidative phosphorylation in mitochondria.

However a small portion of the total oxygen consumed is reduced in a specific

pathway yielding superoxide and hydroxyl free radical, all of which can be

potentially damaging to respiring cells (Vuillaume, 1987).

O2-

(+H+) e-

O2 (+H+) e- O2- (+H+) e- O2

- (+H+) e- OH (+H+) e- H2O

hv or other energy

Perhydroxyl radical (OOH.)

Perhydroxyl radical, the conjugate acid of O2- play an important role in

oxidant damage. Protonation of O2- gives rise to HOO' radical which can

initiate peroxidation. It is also known as hydroperoxyl radical. Perhydroxyl

radical [HOO', the conjugate acid of superoxide (O2-)], initiates fatty acid

peroxidation (a model for biological lipid peroxidation) by two parallel

pathways: fatty acid hydroperoxide (LOOH)-independent and LOOH-

dependent. The superoxide anion and the perhydroxyl radical are in

equilibrium in aqueous solution (http://en.wikipedia.org/wiki/Hydroperoxyl).

Hydroxyl radical (OH.)

The hydroxyl radical is an extremely reactive oxidizing radical that will react

to most biomolecules at diffusion controlled rates, which means that reactions will

occur immediately with biomolecules. The hydroxyl radical is important in

radiobiological damage and is more reactive towards cellular constituents than


15

superoxide radicals. When superoxide and hydrogen peroxide react together they

produce hydroxyl radicals (Coyle and Puttfarcken, 1993).

_22

_2 OHOHOHO _

DPPH

A stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical has long been

used as a convenient method for the antioxidant assay of biological materials

such as cysteine, glutathione, ascorbic acid, tocopherol and polyhydroxy

aromatic compounds (Suja et al., 2004; Nishizawa et al., 2005). DPPH is a

commercially available free radical which is soluble in ethanol

(Summa et al., 2006). This method is based on the reduction of alcoholic

DPPH solution at 517nm in the presence of hydrogen donating antioxidant

(AH) due to the formation of non-radical form DPPH-H by the reaction:

AHDPPHAHDPPH _ (Koleva et al., 2002)

The sensitivity of the method is determined by the strong absorption of

DPPH. This method is rapid, sample analysis takes only 15 minutes and little

manpower, no expensive reagents or sophisticated instruments are required

(Koleva et al., 2002). The oils and aqueous extracts of leaves and flowers of

Bidens pilosa were subjected to screening for their possible antioxidant activities

by using DPPH and β-carotene bleaching methods (Deba et al., 2008).

The DPPH radical scavenging activity of the ethanol and aqueous extracts

of aerial parts of Varthemia iphionoides Boiss (Al- Dabbas et al., 2006), ethanol

extract of aerial parts of herb Poeonia emodi (Khan et al., 2005), aqueous extract

of the herb Thymus fallax Fisch and Mey (Ozgen et al., 2006), D. hamiltonii roots

and Choerospondias axillaries fruits (Wang et al., 2008) were reported.

2.1.2 Non-radicals

Non-radicals containing two electrons per orbital, which is a stable

configuration in a molecule, include singlet oxygen, hydrogen peroxide,

hypochlorous acid and nitric oxide.

Singlet oxygen (O2.)

Singlet oxygen is a nonradical reactive oxygen species often associated

with oxygen free radicals that has strong oxidizing activity. It is an electronically

excited and mutagenic form of oxygen. It is generated by input of energy,


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example radiation, but can also be generated enzymatically by the action of

peroxidases or lipoxygenases or by the reaction of hydrogen peroxide with

hypochlorite or peroxynitrite, thermo-decomposition of dioxetanes or during the

respiratory burst of phagocytes. They are also generated in biological systems in

a number of pigment reactions including chlorophylls, retinal and flavins when

they are illuminated in the presence of oxygen (Prakash et al., 1998).

Hydrogen peroxide (H2O2)

Hydrogen peroxide is not a free radical but falls in the category of reactive

oxygen species. It is an oxidizing agent that is not particularly reactive but its

main significance lies in that it is the main source of hydroxyl radicals in the

presence of transition metal ions. Hydrogen peroxide can be generated from the

two electron reduction of oxygen. In biological systems hydrogen peroxide is

generated by the production of superoxide: two superoxide molecules can react

together to form hydrogen peroxide and oxygen (Winston and Di Giulio, 1991).

222_

2 OOH2H2O

Hypochlorous acid (HOCl.)

Hypochlorous acid is a chemically reactive oxidant. It is an important

component of the inflammatory response and may contribute to carcinogenesis.

Stimulation of phagocytosis triggers a membrane-associated NADPH oxidase,

which reduces molecular oxygen to superoxide. The latter dismutates to

hydrogen peroxide. Phagocytic cell myeloperoxidase catalyzes the formation of

HOCl. from hydrogen peroxide and chloride ions. It induces oxidative injury

during phagocytosis (Lakshmi et al., 2000).

Nitric oxide (NO)

Saha et al. (2008), stated that NO is an important chemical mediator

generated by endothelial cells, macrophages, neurons and involved in the

regulation of various physiological processes. Overproduction of NO can mediate

toxic effects such as DNA fragmentation, cell damage and neuronal cell death.

NO does not interact with the bioorganic macromolecules such as the DNA or

proteins directly. During infections and inflammations, formation of NO is elevated

and may bring about some undesired deleterious effects like tumor growth. The

peroxynitrite produced during the reaction of NO with O2- is probably responsible

for genetic damage (Roberfroid and Calderon, 2008).


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2.2 LIPID PEROXIDATION

Lipid peroxidation has been defined as “Oxidative deterioration of

polyunsaturated fatty acids”, i.e. more than one carbon-carbon double bonds. It

lowers the nutritional quality of food (Gulcin et al., 2005). Initiation of peroxidation

in a membrane or polyunsaturated fatty acid is due to the attack of any species

that can “pull off” a hydrogen atom from one of the -CH2- groups in the carbon

chain. Many observations support the notion that lipid peroxidation plays an

important role in carcinogenesis (Gulcin, 2006).

Lipid peroxidation is usually initiated by the interaction of a ROS or other

free radical with polyunsaturated fatty acids and exacerbated by the presence

of divalent metal ions. During lipid peroxidation, polyunsaturated fatty acids

are oxidized to produce lipid peroxyl radicals that in turn lead to further

oxidation of polyunsaturated fatty acids in a perpetuating chain reaction as

follows (Novo and Parola, 2008):

RHLRadicalLH radicalperoxyllipidLOOOL 2

npropagatiolipidtoleadingLOOHLLOOLH nterminatiochaintantioxidanLOOHtantioxidanLOO

Lipid peroxides are potentially toxic and possess the capacity to damage

most cells (Halliwell, 1994) by inactivating membrane enzymes and receptors,

depolymerising polysaccharide and cross-linking and fragmenting protein. As a

result, the structure and fluidity of the membrane are damaged and normal cell

function is lost (Ng et al., 2005). Membranes and lipids are particularly

susceptible to the oxidant process and to the peroxidative reaction induced by free

radicals (Rahman, 2003). Great emphasis has recently been placed on the

significant contribution of lipid peroxidation to the development of cancer (Dreher

and Junod, 1996) and atherosclerosis. Lipid peroxidation may be prevented at

the initiation stage by free radical scavengers, while the chain propagation

reaction can be intercepted by peroxy-radical scavengers such as phenolic

antioxidants (Magnani et al., 2000; Sun et al., 2000; Dangles et al., 2000).


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

major mechanisms of cell injury in aerobic organisms subjected to oxidation

stress. Lipid peroxidation products such as malondialdehyde (MDA) and

4-hydroxy-2-nonenal (HNE), are closely related to carcinogenesis as they are

potent mutagens and they have been suggested as modulators of signal

pathways related to proliferation and apoptosis, two processes implicated in

cancer development (Olalye and Rocha, 2007).

2.3 ANTIOXIDANTS

The body is usually under a dynamic equilibrium between free radical

generation and quenching. The physiological defense systems to counteract

free radicals encompass endogenous antioxidant enzyme systems such as

catalase, glutathione reductase and superoxide dismutase as well as

glutathione, urate and coenzyme Q or exogenous factors such as β-carotene,

vitamin C, vitamin E and selenium. All these molecules have an antioxidant

effect due to their ability to transform ROS into stable and harmless

compounds or by scavenging both ROS and reactive nitrogen species (RNS)

with a redox based mechanism (Valko et al., 2006).

Under normal physiological conditions, the highly toxic ROS are quenched by

the mitochondrial antioxidant defense systems. In particular, mitochondrial catalase,

manganese superoxide dismutase, as well as glutathione in conjunction with

glutathione peroxidase and glutathione S-transferase regulate inner mitochondrial

membrane permeability by detoxifying ROS produced during electron transport and

confer protection against lipid peroxidative damage (Andreyev et al., 2005).

The plants are susceptible to damage caused by active oxygen and thus

develop numerous antioxidant defense systems resulting in the formation of

numerous potent antioxidants. Many aromatic and spice plants contain compounds

that possess confirmed strong antioxidative components. The essential oils derived

from aromatic plants not only serve as fragrance and flavor agents but also as

dietary antioxidant expected to prevent several diseases caused by free radicals

(Mishra et al., 2007).

Plant extracts with antioxidant activity are traditionally used to

strengthen the natural immune defenses. Many studies have focused on the

antioxidant effects of flavonoids resulting in their identification as potential


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antioxidants and anticancer agents (Lee et al., 2004). Antioxidant properties of

water and ethanol extract of Day lily flowers were evaluated in terms of total

antioxidant activity, reducing capacity and metal chelating activity and the

ethanolic extract showed strong antioxidant activity which is further evaluated

by feeding mice for 60 days which significantly increased superoxide

dismutase activity and decreased lipid peroxidation in both blood and liver of

mice (Que et al., 2007).

The ethanolic extract of the plant Cytisus scoparius L., showed potent

free radical scavenging and antioxidant activity which might be helpful in

preventing or slowing the progress of various oxidative stress related diseases

(Sundararajan et al., 2006). The methanolic extract of the plants Cassia

spectabilis and Cassia fatula were identified as potentially novel sources of the

radical scavenging compounds (Nehru et al., 2008).

Silymarin

Silymarin, a standardized extract obtained from seeds of Silybum

marianum (Asteraceae or Compositae) is widely used in treatment of liver

diseases of varying origins and cancer (El-Samaligy et al., 2006; Dixit et al.,

2007). It consists of a mixture of three bioflavonoids found in the fruit, seeds and

leaves of this plant namely Silybin, Silydianin and Silychristine (Khan et al.,

2006). Seeds of S. marianum have been used to treat liver and gall bladder

disorders, including hepatitis, cirrhosis and jaundice and to protect the liver

against poisoning from chemicals, environmental toxins, snake bites, insect

stings, mushroom poisoning and alcohols (Ball and Kowdley, 2005; Kren and

Walterova, 2005). It also protects liver cells directly by stabilizing the membrane

permeability through inhibiting lipid peroxidation and preventing liver glutathione

depletion (Skottova et al., 2003).

Silymarin is an important bioactive principle having anticancer,

antiinflammatory, antioxidant and immunomodulatory effects (Okawa et al., 2001;

Lebedev et al., 2001; Tyagi et al., 2002; Kohno et al., 2002; Johnson et al., 2003;

Katiyar, 2005). It is also useful to treat alcoholic DNA damage, in addition to

alcoholic liver injury (Saravanan and Pugalendi, 2005). Silymarin promoted UV-

irradiated A375-S2 cell survival partly. It also modulated the distribution of the cell

cycle to allow more time for the damaged cells to repair. This result may broaden


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silymarin's potential therapeutic use for many diseases in the future (Li et al., 2007).

The dietary silymarin exerts a chemopreventive effect on 4-Nitroquinoline 1-oxide-

induced rat tongue carcinogenesis, when fed during the promotion phase. This

cancer protective effect of silymarin might relate to the control of carcinogen-

induced hyper-cell proliferation and/or alteration of the amino acid metabolic

pathway (Yanaida et al., 2002).

Recent studies suggested that silymarin and its polyphenolic fraction could

have beneficial effects on some risk factors of atherosclerosis. The results have

shown that silymarin has hypolipidemic effect (Sobolova et al., 2006) and

preventive effect on low density lipoprotein peroxidation in vitro. It also has

protective effects against stress-induced gastric ulcers and induces recovery of

pancreatic function after alloxan damage in rats (Soto et al., 2004).

It has been introduced fairly recently that silymarin can be used as a

hepatoprotectant for acute viral hepatitis, poisoning by Amanita phalloides,

ethanol, paracetamol and carbon tetrachloride (Fraschini et al., 2002). The

silymarin, a single herbal drug formulation which is mostly used in liver disease

amounts to about 180 million dollars in Germany alone (Thakur et al., 2007).

The protection to immune system provided by silymarin appears to rest

on four properties (Figure 3):

activity against lipid peroxidation as a result of free radical

scavenging and the ability to increase the cellular content of GSH

ability to regulate membrane permeability and to increase membrane

stability in the presence of xenobiotic damage

capacity to regulate nuclear expression by means of a steroid-like

effect and

inhibition of the transformation of stellate hepatocytes into

myofibroblasts, which are responsible for the deposition of collagen

fibres leading to cirrhosis.


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FIGURE 3: MECHANISM OF ACTION OF SILYMARIN

(Valenzuela and Garrido, 1994)

Silymarin and silibinin inhibit the absorption of toxins such as phalloidin or

amanitin, preventing them from binding to the cell surface and inhibiting

membrane transport systems. Furthermore, silymarin and silibinin, by interacting

with the lipid component of cell membranes, can influence their chemical and

physical properties. Studies in erythrocytes, mast cells, leucocytes, macrophages

and hepatocytes have shown that silymarin renders cell membranes more

resistant to lesions (Mourelle et al., 1989).

Furthermore, the well documented scavenging activity of silymarin and

silibinin can explain the protection afforded by these substances against


22

hepatotoxic agents. Silymarin and silibinin may exert their action by acting as free

radical scavengers and interrupting the lipid peroxidation processes involved in

the hepatic injury produced by toxic agents.

Types of antioxidants

Antioxidants are chemical substances that donate an electron to the free

radical and convert it to harmless molecule. Antioxidants are molecules, which

can safely interact with free radicals and terminate the chain reaction before

vital molecules are damaged. Plants have evolved different phytochemicals

and enzymes as antioxidant defense to maintain growth and metabolism.

Antioxidants are produced in leaves and protect the plants from damage by

quenching free radicals (Pandhair and Sekhon, 2006). As a defensive

strategy, cells are capable of inducing antioxidant enzymes such as superoxide

dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), to

remove harmful ROS. Although there are several nonenzymic systems in the

body that scavenge free radicals, the principle micronutrient antioxidants are

vitamin E, vitamin C and beta-carotene (Huang et al., 2005).

Antioxidants of plants experimentally proved to have effective protective

agents against oxidative stress (Rekha et al., 2001). The extracts of Withania

somnifera increase the action of antioxidants CAT, SOD and GPx (Kaur et al., 2004).

Aglaia roxburghiana increase the level of antioxidants by scavenging oxide free

radicals (Chakrabarty et al., 2004). Calotropis species has reported to have very

high antioxidant activity (Mueen et al., 2003). Andrographis paniculata activate

antioxidant enzymes thereby protecting the tissues from free radicals. Leaf

extracts of Aloe vera, Allium sativum, Azadirachta indica, Emblica officinals and

Tinospora cordifolia are some other plants which have been reported to have

antioxidant activity (Govindarajan et al., 2005).

2.3.1 Enzymic antioxidants

The first line of defense is the preventive antioxidants, which suppress

formation of free radical (enzymes such as Catalase, Superoxide dismutase

and Glutathione peroxidase). The first line of defense against and hydrogen

peroxide mediated injury (Figure 4) are antioxidant enzymes like CAT, SOD

and GPx (Bukan et al., 2003).


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FIGURE 4: ACTION OF ENZYMIC ANTIOXIDANTS

(http://www.redlabs.be)

Catalase (CAT)

Catalase is an enzymatic antioxidant widely distributed in all animal tissues

and the highest activity is found in the red cells and in liver and localized mainly in

peroxisome (Prakash et al., 2009). CAT decomposes H2O2 and protects the

tissue from highly reactive hydroxyl radicals (Darlington and Stone, 2001; Edwin

and Jarald, 2005). Therefore, the reduction in the activity of this enzyme may

result in a number of deleterious effects due to the accumulation of superoxide

radicals and hydrogen radicals (Szymonik et al., 2003). Overexpression of

catalase targeted to mitochondria showed extension of murine life span (Schriner

et al., 2005). Catalase contains heme as prosthetic group and can act as

peroxidase when the concentration of H2O2 is low and the concentration of electron

donors is high. They are tetramers each subunit possessing a protoporphyrin IX as

prosthetic group with one Fe (III). It is involved in β-oxidation of fatty acids,

glyoxylate cycle and purine metabolism (Munne-Bosch and Falk, 2004).


24

Catalase has one of the highest turnover rates for all enzymes i.e., one molecule

of CAT can convert 6 million molecules of H2O2 to water and oxygen each minute.

Catalase has a double function, because it catalyses the following reactions.

1) Decomposition of H2O2 to give

2222 OO2HO2H

2) Oxidation of H donors, for example, methanol, ethanol, formic acid,

phenol with the consumption of 1 mole of peroxide.

AROHOHAHROOH 22

Superoxide dismutase (SOD)

Superoxide dismutase is one of the important intracellular antioxidant enzymes;

present in all aerobic cells (Arteel, 2003) has an antitoxic effect against superoxide

anion and forms hydrogen peroxide and molecular oxygen (Loki and Rajamohan, 2003;

Kerksick and Willoughby, 2005; Jyothi et al., 2008; Garg et al., 2008).

22222 OOH2HOO

It is considered to be stress protein, which is synthesized in response to

oxidative stress (Oberley and Oberely, 2006). Mutations in the cytoplasmic or

mitochondrial form of SODs result in aging, neurodegenerative diseases and

carcinogenesis (Bonatto, 2007).

Glutathione peroxidase (Gpx)

The Gpx competes with CAT for H2O2 as a substrate and is the major

source of protection against low levels of oxidative stress. It converts reduced

glutathione into oxidized glutathione and also removes lipid peroxides and

H2O2 along with catalase (Kashyap et al., 2005) leading to the maintenance of

membrane integrity and increased tolerance to oxidative stress caused by

various stress conditions (Yoshimura et al., 2004).

O2HGSSG2GSHOH 222

NADP2GSHHNADPHGSSG

This system forms an excellent protection against lipid peroxidation by

scavenging the lipid peroxides (Figure 5).

OHROHGSSG2GSHROOH 2


25

FIGURE 5: ACTION OF GLUTATHIONE PEROXIDASE

(http://www.google.co.in/images)

It also scavenges H2O2, which is responsible for the initiation of lipid

peroxidation (Silva and Jerald, 2005). H2O2 formed by the enzymatic processes

of SOD and peroxidase is reduced to H2O by the reaction of glutathione

peroxidase (GPx) with GSH, which is oxidized to GSSG (Dringen, 2000).

GSSG is then reduced back to GSH; a step catalyzed by glutathione reductase

(GSSG - R) with NADPH, and is then reused as a GPx substrate. This ensures a

steady supply of the reductive substrate (NADPH) to glutathione Peroxidase

(Agarwal and Prabakaran, 2005).

2.3.2 Nonenzymic antioxidants

The antioxidant enzymes are complemented by small molecule

antioxidants. The small molecule antioxidants are present extra and

intracellularly and include Vitamin A, E and Glutathione.


26

Vitamin A

Vitamin A is a fat soluble vitamin is essential for the normal functioning of

the visual system, epithelial cell integrity and growth, immunity and reproduction

(Maciel et al., 2007). It also plays an important role in respiratory diseases.

Vitamin A maintains the integrity of the epithelial tissue and retinoids, either

topically or orally administered, were able to induce complete remission in a high

proportion of patients with basal cell or advanced squamous cell carcinoma. It is

also involved in the regulation of lipid peroxidation in plasma (Zobali et al., 2002).

Retinoic acid is an active metabolite of vitamin A (Ren et al., 2007) and serves as

a hormone like nutrient in cellular differentiation and proliferation in various tissues

including the small intestine (Ogura et al., 2005).

Liver contain the highest concentrations of vitamin A, followed by epidermis

and serum. Vitamin A is an essential micronutrient to the normal brain function.

However, there is an increasing concern regarding the use of Vitamin A high

doses even therapeutically (Oliveira and Moreira, 2007). This enzyme catalyses

the decomposition of H2O2 to water and oxygen and protects the cell oxidative

damage by H2O2 and OH (Schunemann et al., 2002).

Vitamin E

Vitamin E (á-tocopherol) is a major lipid-soluble antioxidant found in

cells. Vitamin E is the most effective chain-breaking antioxidant within the cell

membranes and lipoproteins. Its main antioxidative function is protection

against lipid peroxidation by scavenging peroxyl radical intermediates in the

chain reaction (Ganapathi and Jagetia, 1995; Prior and Cao, 2000). Vitamin E

protects the cells against the effect of dangerous free radicals and potentially

damaging product of our body metabolism (Pryor, 2000; Dwivedi et al., 2005).

Many studies have suggested that high intake of Vitamin E may slow down the

development and progression of atherosclerosis. Some clinical trials also

reported beneficial effects of Vitamin E supplementation in the secondary

prevention of cardiovascular events (Meydani, 2004). It prevents the attack of

ROS of the membrane PUFA (Kamal and Appelqvist, 1996). It protects the

membranes, lipids and lipoprotein (Van Bakl et al., 2000).


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Reduced Glutathione

Reduced glutathione (GSH) found in most tissues, and is present in

millimeter concentrations in some tissues (Townsend and Tew, 2003) cells and

sub cellular compartments, scavenges H2O2, reacts non-enzymatically with singlet

oxygen, superoxide radical and hydroxyl radical (Gomez et al., 2004). It is the

most abundant non protein thiol (Guven and Gulmez, 2004). Halliwell and

Gutteridge (1984) identified that GSH is important in redox regulation of

transcription factors and enzymes for signal transduction. Polyphenols mediated

regulation of GSH alters the cellular processes. GSH is probably the most

important antioxidant present in cells. It is an essential cofactor for GST, which

helps to remove reactive molecules from the cells. Moreover, GSH can interact

directly with certain hydroxyl radical to detoxify them. Mahakunakorn et al. (2004)

stated that GSH functions as a catalyst in disulfide exchange reactions. During

oxidative stress, -SH groups become oxidized to form disulfide links known as

GSSG (Sinha et al., 2007). Gupta et al. (2008) found that glutathione is one of

the most abundant tripeptide L-glutamyl-L-cysteinyl glycine, a non-enzymatic

biological antioxidant present in the liver. Its functions are concerned with the

removal of free radical species and maintenance of thiol proteins and as a

substrate for GPx and GST.

2.4 ANTITUMOR ACTIVITY OF MEDICINAL PLANTS

Isolation and identification of some potent antitumor compounds from plants

has encouraged scientists to screen different parts of plant species against cancer

cell lines (Emami et al., 2005). Increasing DNA repair (folic acid); changing

immunological response (carotenoids, vitamins C and E, selenium and zinc);

inhibition of cyclooxygenase (resveratrol); restriction of caloric intake and

absorption; decreasing time for transit of intestinal bulk, avoiding carcinogen

formation and absorption (fibers); inhibition of angiogenesis and abrogation of tumor

cells proliferation (by suppressing telomerase or induction of apoptosis) also

constitute important antitumor properties of functional foods (Kelloff et al., 2000;

Ferrari and Torres, 2003).

Numerous drugs and compounds have been reported to have antitumor

effects on different organ cancer such as lung, liver, breast and ovarian

(Llovet et al., 2003). Qin et al. (2006) reported that vaccination with


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pSLC-3P-Fc (DNA vaccine) by gene gun inoculation induced a strong

antitumor response in a mouse tumor model, which significantly inhibited tumor

growth and prolonged the survival time of the tumor-bearing mice.

Derivatisation of diospyrin, a bisnaphthoquinonoid isolated from Diospyros

montana Roxb., led to the modification of its inhibitory activity towards a murine

tumor model, Ehrlich ascites carcinoma (ELA) and two human cancer cell

lines: (A375) malignant skin melanoma and (Hep2) epidermoid laryngeal

carcinoma (Sarma et al., 2007).

The effects of the anticancer drug irinotecan combined with ethanolic

extract of propolis, a water-soluble derivative of propolis, quercetin and

naringin on the growth of Ehrlich ascites tumor and the life span of tumor-

bearing Swiss albino mice may be beneficial in maximizing antitumor activity

and minimizing post-chemotherapeutic reactions to the cytostatic drug

(Benkovic et al., 2007). Similar antitumor properties of Zanthoxylum rhoifolium

Lam leaves was investigated in vitro and in vivo using the Ehrlich ascites tumor

model (Silva et al., 2007). The extract of Tinospora cordifolia (Guduchi)

against Ehrlich ascites tumor (ELA) in mice resulted in growth inhibition and

induction of apoptosis in a dose-dependent manner (Thippeswamy and

Salimath, 2007). The aqueous extract from the roots of Glycyrrhiza glabra

inhibits the in vivo and in vitro proliferation of Ehrlich ascites tumor cells and

may be used as a potential supplemental source for cancer therapy

(Sheela et al., 2006).

2.5 IMMUNOMODULATORY ACTIVITY OF MEDICINAL PLANTS

An immunomodulator is a substance which has an effect on the immune

system. There are two types of effects-immunostimulation and

immunosuppression. Immunostimulants primarily have the stimulant effect and

immunosuppressants primarily have the suppressant effect. Immunomodulation is

a process that can alter the immune system of an organism by interfering with its

functions (Shivaprasad et al., 2006). The immune system may be the last line of

defense against cancer development. According to the most recent point of view

about cancer immunology, the key issue is whether recognition of tumor antigens

by the immune system leads to activation (i.e. surveillance) or tolerance

(Pardoll, 2003).


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The natural resistance of the body against infection can be enhanced by

the use of herbal drugs. Several herbal preparations that can enhance the body’s

immune status are extensively being used in the indigenous system of medicines.

There is an upsurge in the clinical usage of indigenous drugs as they are free from

serious side effects. The term immunomodulatory means regulation of the

immune system by suppression and stimulation of the cells and organs of the

immune system (Bafna and Mishra, 2005). Immunostimulation in a drug-induced

immunosuppression model and immunosuppression in an experimental hyper-

reactivity model by the same preparation can be said to be true

immunomodulation (Patwardhan et al., 1990). Certain agents have been shown

to possess activity to normalize or modulate pathophysiological processes and

are hence called immunomodulatory agents. A number of plant products are

being investigated for immune response modifying activity. Modification of

immune functions by pharmacological agents is emerging as a major area of

therapeutics (Upadhyay, 1997).

Immunomodulators are used as an adjuvant in conditions of

immunodeficiency in cancer and to a limited extent in acquired immunodeficiency

syndrome (Malfitano et al., 2006). Immunomodulatory agents of plant and animal

origin enhance the immune response of an organism against a pathogen by

activating the immune system. However these agents should be subjected to

systematic studies to substantiate the therapeutic claims made with regard to their

clinical utility (Fulzele et al., 2003). The immune system is known to be involved in

the aetiology as well as pathophysiologic mechanism of many diseases.

Immunology is thus probably one of the most rapidly developing areas of biomedical

research and has great promises with regard to prevention and treatment of a wide

range of disorders (Ziauddin et al., 1996). Immunomodulators of herbal origin appear

to be a better alternative to overcome the above problem. Herbal drugs are known to

possess immunomodulatory properties and generally act by stimulating both specific

and non-specific immunity (Patwardhan et al., 1991).

Many plants used in traditional medicine have immunomodulating

activities. Some of these stimulate both humoral and cell-mediated immunity,

while others activate only the cellular components of the immune system. Some

of these plants also suppress both humoral and cell-mediated immunity. The


30

great majority of chemicals identified as cytotoxic to cancer cells are generally

also toxic to normal cells (Kim et al., 1996). Nevertheless, the potentiation of host

defense mechanisms has been recognized as a possible means of inhibiting

tumor growth without harming the host (Ameho et al., 1997). Therefore,

searching for immunomodulatory materials from natural herbs and characterizing

the immune enhancement effects may have great potential in cancer treatment,

based on a combination of time honored traditional usage and ongoing scientific

research (Rivera et al., 2003).

Acidic polysaccharides isolated from Tanacetum vulgare L. suggesting the

modulation of immune system (Xie et al., 2007). The administration of crude

extracts of Nyctanthes arbortristis to Swiss albino rats showed significant increase

in RBC and WBC counts. The ethanolic extract of Nyctanthes arbortristis

screened in rats for humoral and cell mediated immunity showed enhanced

circulating antibody titer and delayed type hypersensitivity reactions (Kannan

et al., 2007). Immunostimulatory activities of Chlorophytum borvilionum was

evident when ethanolic extract of roots were administered to the wistar strain

albino rats to asses their immunomodulatory activity. The studies showed that

there is improved survival against Candidia albicans and showed increase in

delayed type hypersensitivity response, percentage neutrophil adhesion and in

vivo phagocytosis (Thakur et al., 2007).

The methanolic extract of Haussknechtia elymatica showed inhibitory

effects on both humoral and cell mediated immune response in a dose dependent

manner (Amirghofran et al., 2007). The aqueous extract of Actinidia

macrosperma on the growth of ascite tumor showed it exerts in vivo

immunomodulatory activities on S180-bearing mice in a dose dependent manner.

The assessment of immunomodulatory activity against tumor was carried out by

testing the humoral, cellular and non-specific immune response to the antigenic

challenge by sheep RBC, lymphocyte proliferation and natural killer cell

cytotoxicity and by macrophages function test showed significant increase in the

immune functions (Lu et al., 2007).

The immunomodulatory activity determined by administration of

Kalpaamrutha which is composed of Semecarpus anacardium nut milk, Phyllanthus

emblica and honey into Wistar albino rats and the humoral, cell mediated and non-


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specific immunity assessed by haemagglutination titer, delayed type

hypersensitivity and phagocytic index respectively showed immunostimulatory role

(Sujatha and Sachdanadam, 2004; Arulkumaran et al., 2007). Butanol and ethyl

acetate soluble fractions of methanolic extracts of Lagenaria sinceraria fruits

significantly inhibited sheep RBC induced delayed type hypersensitivity response.

Both these fractions significantly increased haemagglutination antibody titer in a

dose dependent fashion. Both the fractions significantly increased total WBC,

neutrophil and lymphocyte counts, while insignificant change was observed in

monocyte, eosinophil and basophil counts (Gangwal et al., 2008).

Immunomodulatory function of Tinospora cordifolia has been reported

on increased total WBC count, using Balb/C mice and enhancement in

antibody titre, macrophage activation (Kuttan, 2000). The antibody titre,

spleenocytes and peripheral immune cells showed a significant result of

Nigella sativa immunomodulatory effect against thyroid antigen

(Newaz et al., 2005). The aqueous extract of tea, Camellia sinensis possesses

the immunomodulatory effect (Zvetkova et al., 2001).

2.6 MEDICINAL PLANTS SELECTED FOR THE STUDY

2.6.1 Cynodon dactylon

Cynodon dactylon (Poaceae), a hardy perennial grass, forming thick

mats by means of stolons and rhizomes (Gibbs Russell et al., 1991), eight to

40 culms (stems), rarely to 90 cm high; leaves hairy or glabrous, three to seven

spikes (rarely two), usually 3-6 cm long and in one whorl, or in robust forms up

to ten spikes and is one of the most commonly occurring weeds in India.

Although a problem for farmers, dhoub grass is a valuable herbal medicinal

plant and used as first aid for minor injuries (Oudhia, 1999a and Oudhia,

1999b). Bermuda grass is a perennial weed growing largely from root stocks

and stolons (Oudhia, 2001; Oudhia, 2002).

The aqueous fluid extract of the rhizome is used as anti-inflammatory,

diuretic, anti-emetic, purifying agent and also in dysentery (Ahmed et al., 1994;

Singh et al., 2008). As medicine Cynodon dactylon holds a reputed position in all

systems of medicine in India. According to Ayurveda, India’s traditional

pharmacopoeia, Cynodon dactylon plant is pungent, bitter, fragrant, heating,

appetizer, vulnary, antihelminthic and antipyretic. It destroys foulness of breath,


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useful in leucoderma, bronchitis, piles, asthma, tumors and enlargement of the

spleen (Mahesh and Brahatheeswarn, 2007). According to Unani system of

medicine, Cynodon dactylon is bitterish, vulnerary, expectorant and useful in

vomiting, diarrhoea, burning sensation, blood disorders, stomatitis, biliousness

and hiccup (Oudhia, 2003).

The ethanol extract of aerial parts of Cynodon dactylon showed marked

CNS depressant activities compared to other extracts of it in preliminary

pharmacological screening. The plant Cynodon dactylon has been reported for

antiatherosclerotic, antioxidant, Helicobacter pylori activities and traditionally, for

management of neurodegenerative diseases (Surendra et al., 2008).

Singh et al. (2007) revealed that the Cynodon dactylon aqueous extract has

remarkable effects on blood glucose level and marked improvement on

hyperlipidemia due to diabetes. Its specific effect on HDL has additional

advantage in checking coronary risks. Virus-affected discolored leaves of

Cynodon are used for the treatment of liver complaints. In Homoeopathic

systems of medicine, it is used to treat all types of bleeding and skin troubles

(Oudhia et al., 1998). Farmers traditionally apply crushed leaves to minor wounds

as a styptic to stop bleeding similar to Tridax procumbens, Achyranthes aspera

and Blumea iacera

(Oudhia and Pal, 2000).

2.6.2 Terminalia catappa

Terminalia catappa is a large deciduous tree, originally from India, growing

up to 9.0 feet tall with horizontal whorls of branches offering clusters of foot long,

obovate leaves. Its bark is brownish black and its fresh leaves are green in colors

which turn pink-red to red-yellow before falling. The leaves turn brown before

decaying. This changing of leaf’s color is caused by the present of pigments like

violaxanthin, lutein and zeaxanthin. The leaves are quickly replaced by new

growth so the tree is bare for only a short period of time (Gilman and Watson,

2007). It alternates fragrant white tomentose flower where its female and male

flowers are on the same tree (Lopez-Hernandez et al., 2001; Chen and Nicholas,

2007). These flowers are not very showy. The tree has large nutty edible fruits

that taste very much like commercially grown almonds and the color of the fruit is

green, yellow or reddish.


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The best Terminalia catappa Linn habitat is at an area that receives full

sun, moist, well drained soil, has salt and drought tolerance. The more specific

habitats are at sandy seashores, beaches with humid climate, village and grassy

village commonly. It can shadow large area underneath it till many people

consider T. catappa as a desirable shade tree. This tree is quite popular in

medical world whether in modern or traditional one. The most important and

useful part is definitely its leaves. In Taiwan, the fallen leaves are used as an

herbal drug in the treatment of liver related diseases.

The extract of T. catappa leaves inhibit Lewis lung carcinoma cells that

contribute to lung cancer (Chu et al., 2007). It means T. catappa is an anticancer

agent. In another study conducted by Ahmed et al. (2005) showed that the

extracts of T. catappa produced a significant antidiabetic activity. The various

extract of leaves is also reported to be anticarcinogenic and antioxidant. The

antioxidant in T. catappa gives anticlastogenic effect - a process which causes

breaks in chromosomes. While in traditional treatment, a tea from the leaves is

used against diarrhea and dysentery. The extract of T. catappa showed

antimicrobial activities against Escherichia coli, Staphylococcus aureus,

Pseudomonas aeruginosa, Candida albicans, Trichophyton mentagrophytes,

Pityrospon ovale and Microspoum gypseum (Suganda et al., 2004).

In view of its chemical composition, antioxidative potential of the plant has

been evaluated and proved by many workers (Wang et al., 2000; Ko et al., 2002).

The plant is very well known for its therapeutic values since long and has proved

by many researchers to be useful as antiinflammatory (Fan et al., 2004),

anticancer (Kandil et al., 1999), antihepatotoxic (Lin et al., 2001), antigenotoxic

(Chen et al., 2000), anticlastogenic (Liu et al., 1996), for the treatment of skin

aging, irritation, hyperpigmentation, allergy (Renimel et al., 1998) and bronchial

asthma in children (Prazeres, 1995). It also exhibits antimicrobial (Pawar and Pal,

2002; Naz et al., 2007; Nair and Chanda, 2008), insecticidal and molluscicidal

activities (Jayasinghe et al., 2000).

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