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Appendices

Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies

6

The medicinal plants selected for the present research work were

Morinda tinctoria and Nerium indicum. Morinda tinctoria belongs to the family

Rubiaceae that grows widely and distributed throughout Southeast Asia. It is

commercially known as Nunaa, indigenous to tropical countries and considered as

an important folklore medicine. In the traditional system of medicine, leaves and

roots of Morinda tinctoria are used as astringent, deobstruent, emmenagogue and

to relieve pain in the gout (Kumaresan and Saravanan, 2009).

Nerium indicum L. is an evergreen shrub belongs to the family

Apocynaceae, and distributed in tropical Asia (Garima and Amla, 2010). Nerium

indicum is commonly known as Indian oleander (Sikarwar et al., 2009) an

important plant used against various disorders in indigenous system of medicine.

The plant originates from the Mediterranean region and is indigenous to

Indo-Pakistan subcontinent (Govind, 2010a). It is a well known ornamental plant

with leathery evergreen leaves and handsome clusters of red, pink or white flowers

(Jawarkar et al., 2012). Leaves are powerful repellent and the decoction of the

leaves has been applied externally in the treatment of scabies and to reduce

swellings. The leaves and the flowers are cardiotonic, diaphoretic, diuretic,

emetic, expectorant and sternutatory (Shah and Chakraborthy, 2010).

So far, no studies were undertaken to explore the antioxidative, anticancer

and antimicrobial activity of Morinda tinctoria and Nerium incdicum leaves

with an in silico approach. This made us to investigate and examine the

“Anticarcinogenic Effect in DLA Transplanted Mice and Antimicrobial

Efficacy of Morinda tinctoria and Nerium indicum and their Characterization

by in silico Studies”

The present study was carried out in five phases with the following objectives:

• To assess the in vitro antioxidative and anticarcinogenic efficacy of

Morinda tinctoria and Nerium indicum leaves.

• To evaluate the in vivo antioxidative and anticarcinogenic potential of

Morinda tinctoria and Nerium indicum leaves in DLA tumour induced Swiss

albino mice.

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7

• To evaluate the antimicrobial activity of Morinda tinctoria and

Nerium indicum leaves against different microorganisms.

• To characterize the phytochemical constituents of Morinda tinctoria and

Nerium indicum leaves.

• To evaluate the anticarcinogenic and antimicrobial effect of active

compounds of Morinda tinctoria and Nerium indicum leaves against cancer

and microbial target proteins using molecular docking software GLIDE.

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8

RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE

Human history would be incomplete without illustrating the role of plants,

because early man was directly dependent on plants for his needs (Qureshi et al.,

2006 and Rauf et al., 2012). In both developing and developed countries, the

demand for plant-based therapeutics is increasing to a greater extent due to the

growing recognition of their benefits. They are natural products; are neither

sedatives nor tranquilizers; are eco-friendly, producing minimum environmental

hazards; have no adverse side effects; and are reasonably priced (Shariff et al.,

2006).

Cancer is one of the ten leading diseases which cause death and

advancing in rank year by year throughout the world (Sundaram et al., 2012).

Cancer is a group of diseases where cell growth is aggressive, abnormal, invasive

and metastatic many times leading to death (Tongyoo, 2010). Natural products,

especially those from plants, have been a valuable source of new cancer drugs for

many decades. Medicinal plants are the most exclusive source of life saving drugs

for the majority of the world’s population (Thakore et al., 2012).

Oxidative stress is a known mediator of cancer. Medicinal plants are

important source of antioxidants and serve as subcellular messengers of normal

cell function and have a significant protective role against oxidative injury (Upham

and Wagner, 2001). The secondary metabolites from plants have been reported to

be a potent antioxidant and free radical scavengers (Vadnere et al., 2012).

Considering the negative effects of synthetic drugs, people are looking for natural

remedies, which are safe and effective. In this respect, medicinal plants used in

the traditional therapy could be the alternative source for the development of new

therapeutic agents to combat with the resistant organisms. Nowadays, numerous

scientific investigations are going on in isolation of potent phytochemicals as

leading compounds for antimicrobial therapy (Bhattarai and Bhuju, 2011).

2

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9

Molecular docking is a key tool in structural molecular biology and

computer-assisted drug design. The goal of ligand protein docking is to predict

the predominant binding model(s) of a ligand with a protein of known three

dimensional structures (Srivastava et al., 2010).

Information on the Antioxidative, anticancer and antimicrobial role of

Morinda tinctoria and Neriun indicum leaves and its in silico assessment using

glide is still very scanty in literature. The review of literature pertaining to the

present research entitled “Anticarcinogenic Effect in DLA Transplanted Mice

and Antimicrobial Efficacy of Morinda tinctoria and Nerium indicum and their

Characterization by in silico Studies” is appropriately presented under the

following headings:

2.1. Oxidative Stress

2.1.1. Free radicals

2.1.1.1. Reactive oxygen species

2.1.1.2. Reactive nitrogen species

2.1.1.3. Reactive sulphur species

2.1.1.4. Mechanism of formation and damages caused by free radicals

2.1.2. Non-radicals

2.1.2.1. Hydrogen peroxide

2.1.2.2. Nitric oxide

2.1.3. Synthetic free radicals

2.1.3.1. ABTS

2.1.3.2. DPPH

2.1.4. Lipid peroxidation

2.2. Antioxidants

2.2.1. First line defense antioxidants

2.2.1.1. Catalase

2.2.1.2. Glutathione peroxidase

2.2.1.3. Glautathione reductase

2.2.1.4. Superoxide dismutase

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10

2.2.2. Second line defense antioxidants

2.2.2.1. Vitamin A

2.2.2.2. Vitamin E

2.2.2.3. Vitamin C

2.2.2.4. Reduced glutathione

2.3. Anticancer activity of medicinal plants

2.4. Antimicrobial activity of medicinal plants

2.5. Phyochemical constituents of medicinal plants

2.6. In silico drug design and docking for cancer

2.6.1. Histone deacetylase

2.6.2. Tubulin

2.6.3. Aurora kinase A

2.6.4. Protein kinase C

2.7. In silico drug design and docking for microbial infections

2.7.1. Pantothenate Kinase

2.7.2. Deacetoxy C synthase

2.8. Medicinal plants selected for the study

2.8.1. Morinda tinctoria

2.8.2. Nerium indicum

2.1. Oxidative Stress

Oxidative stress is a phenomenon associated with pathogenetic mechanisms

of several diseases including atherosclerosis, neurodegenerative diseases, such

as Alzheimer’s and Parkinson’s disease, cancer, diabetes mellitus, inflammatory

diseases, as well as psychological diseases or aging processes. Oxidative stress

is defined as an imbalance between production of free radicals and reactive

metabolites, so-called oxidants, and their elimination by protective mechanisms,

referred to as antioxidative systems. This imbalance leads to damage of important

biomolecules and organs with potential impact on the whole organism. Oxidative

and antioxidative processes are associated with electron transfer influencing the

redox state of cells and the organism (Durackova, 2010).

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11

Figure 1

Oxidative stress related diseases

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

Uncontrolled generation of ROS can lead to their accumulation causing

oxidative stress in the cells. All forms of life maintain a steady state concentration

of ROS determined by the balance between their rates of production and their

rates of removal by various antioxidants. Thus each cell is characterized by a

particular concentration of reducing species like GSH, NADH, NADPH and FADH

stored in many cellular constituents which determines the redox state of a cell.

By definition redox state is the total reduction potential or the reducing capacity

of all the redox couples such as GSSG/2GSH, NAD+/NADH, Asc•−/AcsH−, NADP+

/ NADPH found in biological fluids, organelles, cells or tissues. Redox state not

only describes the state of a redox pair, but also the redox environment of a cell.

Under normal conditions, the redox state of a biological system is maintained

towards more negative redox potential values. However, with increase in

ROS generation or decrease in antioxidant protection within cells, it is shifted

towards less negative values resulting in the oxidizing environment. This shift from

Appendices


12

reducing status to oxidizing status is referred as oxidative stress (Schafer and

Buettner, 2001; Kohen and Nyska, 2002).

Figure 2

Mutual association between oxidants and antioxidants

(Durackova, 2010) 2.1.1. Free radicals

A free radical has one or more unpaired electrons in its outer orbital. Such

unpaired electrons make these species very unstable and therefore quite reactive

with other molecules. They try to pair their electron(s) and generate a more stable

compound (Shekhawat et al., 2010). Reactive oxygen species (ROS) or free

radicals, formed during physiological and pathological conditions in the body, are

extremely reactive and react with proteins, lipids, carbohydrates and nucleic

acids (Yildiz et al., 2011). The various pathways involved in the generation of

free radicals are given in Figure 3.

The nitrogen derived free radicals are nitric oxide (NO), peroxy nitrite anion

(ONOO), Nitrogen dioxide (NO2) and Dinitrogen trioxide (N2O3). The thiol derived

free radicals include sulphite (SO32-), disulfide S oxide (DSSO), sulfenic acid

(RSOH) and sulfenyl (RS.) radicals (Panchawat et al., 2010 and Mathew et al.,

2011). When an overload of free radicals cannot gradually be destroyed,

their accumulation in the body generates a phenomenon called oxidative stress.

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13

Figure 3

Production of free radicals

(Kunwar and Priyadarsini, 2011)

This process plays a major role in the development of chronic and

degenerative illness such as cancer, autoimmune disorders, aging, cataract,

rheumatoid arthritis, cardiovascular and neurodegenerative diseases (Pham-Huy

et al., 2008).

2.1.1.1. Reactive oxygen species (ROS)

Reactive oxygen species is a collective term that describes the chemical

species that are formed upon incomplete reduction of oxygen. ROS are thought to

mediate the toxicity of oxygen because of their greater chemical reactivity with

regard to oxygen (Autreaux and Toledano, 2007). The sequential reduction of

oxygen through the addition of electrons lead to the formation of a number of

ROS including: superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl

ion, peroxyl and alkoxyl radicals. Most reactive oxygen species are generated as

by-products during mitochondrial electron transport. In addition, ROS are formed

as necessary intermediates of metal catalyzed oxidation reactions (Dolai et al.,

2012 and http://www.biotek.com/resources/articles/reactive-oxygen-species.html).

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14

Figure 4

Generation of reactive oxygen species

(http://www.rndsystems.com/cb_detail_objectname_SP96_FreeRadicalsOxidativeStree.aspx)

The ROS may be very damaging, since they can attack lipids in cell

membranes, proteins or enzymes in tissues, carbohydrates and DNA, to induce

oxidations, which may causes cancer, atherosclerosis, aging, immunosuppression,

inflammation, ischemic heart disease, diabetes, hair loss and neurodegenerative

disorders such as Alzheimer’s disease and Parkinson’s disease (Zadak et al.,

2009 and Naskar et al., 2010). Living cells possess a protective system of

antioxidants which prevents excessive formation and enables the inactivation of

ROS. The antioxidants protect from the potentially damaging oxidative stress,

which is a result of an imbalance between the formation of ROS and the

antioxidant defense of the body (Kratchanova et al., 2010).

The exogenous sources of ROS include electromagnetic radiation,

cosmic radiation, UV-light, ozone, cigarette smoke and low wavelength

electromagnetic radiations; and endogenous sources are mitochondrial

electron transport chain and β-oxidation of fat (Panchawat et al., 2010). ROS

can cause tissue damage by reacting with lipids in cellular membranes,

nucleotides in DNA (Ahsan et al., 2003), sulphydryl groups in protein and cross-

linking/fragmentation of ribonucleoproteins (Knight, 1995; Waris and Alam, 1998).

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15

The role of reactive oxygen species (ROS) in carcinogenesis has been

extensively investigated in several cellular and animal models, and various

connections have been established (Gibellini et al., 2010). In cancer cells, ROS

increase the rate of mutagenicity, which leads to DNA damage and chromosomal

instability, and thereby accelerates cancer progression (Radisky et al., 2005). On

the one hand, appropriate levels of ROS play an important role in the modulation

of several physiologic responses in signalling network regulating cell function. On

the other hand, excessive intracellular levels of ROS, as well as a defective

antioxidant system, can give rise to pathological conditions, and even encourage

the progression of these conditions (Rhee, 2006).

Hydroxyl radicals

Among ROS, hydroxyl radical (OH.) is the most reactive oxidant. Hydroxyl

radical is a highly reactive radical formed in biological systems and capable of

damaging almost every molecule found in living cells. This radical has the capacity

to induce carcinogenesis, mutagenesis and rapidly initiates lipid peroxidation

(Manian et al., 2008). In the Haber-Weiss reaction hydroxyl radicals are generated

in the presence of hydrogen peroxide and iron. The first step involves reduction of

ferric into ferrous ion and the second step is the Fenton reaction. The generation

of hydroxyl radicals catalyzed by ferric ions without any additional redox agent, this

can be considered as a special case of the Fenton reaction. Here, one electron

from the hydroxyl group of water is transferred to the ferric ion with the formation of

a divalent iron and a hydroxyl radical (Lipinski, 2011).

Fe3+ +•O2

- Fe2+ +O2.

Fe2+ +H2O2 Fe3+ +OH− +OH•

Fe3+ +HO− Fe2+ +OH•

Superoxide anions

Superoxide anion radical is known as an initial radical and plays an

important role in the formation of other reactive oxygen species, such as hydrogen

peroxide, or singlet oxygen in living systems (Stief, 2003). It plays an important

role in oxidative stress and related to the pathogenesis of various important

Appendices


16

diseases (Kumar and Kumar, 2009). Superoxide is biologically important since it

can be decomposed to form stronger oxidative species such as singlet oxygen and

hydroxyl radicals (Pari and Amudha, 2011). Superoxide dismutase catalyzes the

dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen

(Sharma et al., 2011).

The most commonly occurring cellular free radical is superoxide radical,

which is produced when an oxygen molecule gains one electron from another

substance (Sherki et al., 2002). The superoxide anion radical is dismutated to

hydrogen peroxide (H2O2). H2O2 is converted to the hydroxyl radical in the

presence of a transition metal such as iron; this is identified as the Fenton reaction

(Reiter, 2000).

(Kyaw et al., 2004)

2.1.1.2. Reactive nitrogen species

Nitrogen-derived free radicals are called reactive nitrogen species (RNS) and

their utmost representative precursors are nitric oxide (NO) and peroxynitrite

(ONOO−). NO is well known to be a product of the catalytic action of the nitric

oxide synthase (NOS) enzyme family on L-arginine (Espey et al., 2002; Li and

Poulos, 2005). However, evidence suggests that it can also be formed by

reduction of nitrite, which can arise in the body by ingestion or from bacterial

metabolism (Lundberg and Weitzberg, 2005). Low levels of both ROS and RNS

are continuously produced in mammalian cells and play important physiological

roles (Gutteridge and Halliwell, 2000). These include processes as diverse as

gene expression (Allen and Tresini, 2000), cell proliferation and survival (Kamata

Appendices


17

and Hirata, 1999), pathogen clearance by the immune system, and blood vessel

permeability. Reactive nitrogen species (RNS) such as nitrogen dioxide (•NO2),

peroxynitrite (ONOO–) and nitrosoperoxycarbonate (ONOOCO2–) are among the

most damaging species present in biological systems due to their ability to cause

modification of key biomolecular systems through oxidation, nitrosylation, and

nitration (Nash et al., 2012).

Figure 5

Generation of reactive nitrogen species

(Eleuteri et al., 2009)

2.1.1.3. Reactive sulfur species (RSS)

Sulfur is an essential and quantitatively important element for living

organisms. Sulfur is a constituent of many organic molecules, for example amino

acids such as cysteine and methionine and the small tripeptide glutathione, but

sulfur is also essential in the form of Fe–S clusters for the activity of many

enzymes, particularly those involved in redox reactions. Sulfur chemistry is

therefore important. In particular, sulfur in the form of thiol groups is central to

manifold aspects of metabolism. Because thiol groups are oxidized and reduced

Appendices


18

easily and reversibly, the redox control of cellular metabolism has become an

increasing focus of research. In the same way that oxygen and nitrogen have

reactive species (ROS and RNS), sulfur too can form reactive molecular species

(RSS), for example when a –SH group is oxidized. Indeed, several redox reactions

occur via RSS intermediates. Furthermore, RSS can also be used as redox-active

pharmacological tools to study cell metabolism (Gruhlke and Slusarenk, 2012).

Figure 6

Generation of reactive sulfur species

(Gruhlke and Slusarenk, 2012)

Free radicals are formed from molecules via the breakage of a chemical

bond such that each fragment keeps one electron, by cleavage of a radical to give

another radical and also via redox reactions (Bahorun et al., 2006). Oxidation is

one of the most important free radical producing processes in food, chemicals and

Appendices


19

even in the living system. Free radicals play an important role in food and chemical

material degradation, and they also contribute to more than one hundred disorders

in humans (Gorghiu et al., 2004; Ye and Song, 2008).

Free radicals are very unstable and react quickly with other compounds,

trying to capture the needed electron for gaining stability. Generally, free radicals

attack the nearest stable molecule, and “steal” its electron. When the “attacked”

molecule loses its electron, it becomes a free radical itself, and begins a chain

reaction. Once the process is started, it can cascade, finally resulting in the

disruption of a living cell. Some free radicals arise normally during metabolism.

Sometimes the body’s immune system’s cells purposefully create them to

neutralize viruses and bacteria. However, environmental factors such as pollution,

radiation, cigarette smoke and herbicides can also spawn free radicals

(http://www.sstwo-mall.com/index.php/free-radicals/).

2.1.1.4. Mechanism of formation and damages caused by free radicals

Free radicals can be formed by three ways.

• By homolytic cleavage of covalent bond of normal molecule, with each

fragment retaining one of paired electrons.

X: Y X* + Y*

• By the loss of single electron from normal molecule.

X: Y X+ + Y-

• By addition of single electron to normal molecule.

X + e- X (Kumar, 2011)

� Oxidative damages to lipids

All of the most important classes of biomolecules may be attacked by free

radicals but lipids are probably the most sensitive. Cell membranes are rich

sources of polyunsaturated fatty acids (PUFAs), which are readily attacked by

oxidising radicals. The oxidative destruction of PUFAs, known as lipid

peroxidation, is particularly damaging because it proceeds as a self-perpetuating

chain-reaction (Palmieri and Sblendorio, 2006).

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20

� Oxidative damages to proteins

Oxidative attack on proteins results in site-specific amino acid modification,

fragmentation of the peptide chain, aggregation of cross linked reaction products,

altered electrical charges and increased susceptibility to proteolysis (Farr and

Kogoma, 1991).

� Oxidative damage to DNA

Activated oxygen and agents that generate oxygen free radicals, such as

ionizing radiations, induce numerous lesions in DNA that causes deletion,

mutations and other lethal genetic effects. Characterization of this damage to DNA

has indicated that both sugar and base moieties are susceptible to oxidation,

causing base degradation, single strand breakage and cross links to proteins

(Imlay and Linn, 1988).

2.1.2. Non radicals

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

configuration in a molecule, include hydrogen peroxide and nitric oxide.

2.1.2.1. Hydrogen peroxide

Hydrogen peroxide is the two electron reduction product of O2. It is

potentially reactive oxygen, but not a free radical (Halliwell et al., 2000). By

comparison with superoxide and certainly by comparison with the hydroxyl radical,

H2O2 is relatively “safe” in the absence of transition metals, it is stable and

unreactive, even at concentrations much higher than a biological system would

ever generate (John, 2007). Hydrogen peroxide is a weak oxidizing agent that

inactivates a few enzymes directly, usually by oxidation of essential thiol (-SH)

groups. It can cross cell membranes rapidly; once inside the cell, it can probably

react with Fe2+ and possibly Cu2+ ions to form hydroxyl radicals and this may be

the origin of many of its toxic effects (Miller et al., 1993). Although hydrogen

peroxide itself is not very reactive, it may convert into more reactive species such

as singlet oxygen and hydroxyl radicals (Bhattacharjee et al., 2011).

Appendices


21

2.1.2.2. Nitric oxide

Nitric oxide (NO) has emerged as one of the most intriguing molecules in

vertebrate biology in recent years (Reeves et al., 2008). NO is lipophilic and highly

diffusible solute forms within the cell and its actions are concentration dependent

(Kim et al., 2001). In addition to reactive oxygen species, nitric oxide is also

implicated in inflammation and other pathological conditions (Nabavi et al., 2008a).

NO has been associated with a variety of physiological processes in the human

body since it was identified as a novel signal molecule. It transmits signals from

vascular endothelial cells to vascular smooth muscle cells and causes vascular

dilation. It also plays an important role in physiological functions in respiratory,

immune, neuromuscular and other systems (Ebrahimzadeh et al., 2010).

2.1.3. Synthetic free radicals

2.1.3.1. 2, 2′-Azinobis (3-Ethylbenzothizoline-6-Sulfonicacid) (ABTS+·)

Bleaching of a preformed solution of the blue-green radical cation 2, 2′-

azinobis (3-ethylbenzothizoline-6-sulfonic acid) (ABTS+·) has been extensively

used to evaluate the antioxidant capacity of complex mixtures and individual

compounds (Henriquez et al., 2002). ABTS-, the oxidant is generated by persulfate

oxidation of 2, 2′-azinobis (3-ethylbenzothizoline-6-sulfonic acid) - (ABTS2-).During

this reaction, the blue ABTS radical cation is converted back to its colorless neutral

form. The reaction may be monitored spectrophotometrically. This assay is often

referred to as the trolox equivalent antioxidant capacity (TEAC) assay (Barclay et

al., 1985 and Baskar et al., 2008). The ABTS radical cation is reactive towards

most antioxidants including phenolics, thiols and Vitamin C (Richard and Jace,

2009).

(Zulueta et al., 2009)

Appendices


22

2.1.3.2. Diphenylpicrylhydrazyl radical (DPPH)

DPPH (1, 1-diphenyl-2-picryl-hydrazyl) is a relatively stable nitrogen centered

free radical that easily accepts an electron by reacting with suitable reducing

agents (Narayanaswamy et al., 2011). It is an excellent tool for determining the

antioxidant activity of hydrogen donating oxidants and of chain breaking

antioxidants (Karthika et al., 2012). DPPH radical is a commonly used substrate

for fast evaluation of antioxidant activity because of its stability in the radical form

and simplicity of the assay (Bozin et al., 2008). This assay is known to give

reliable information concerning the antioxidant ability of the tested compounds

(Huang et al., 2005). The principle behind this assay is the colour change

of DPPH solution from purple to yellow as the radical is quenched by the

antioxidant (Karagozler et al., 2008).

Figure 7

Diphenylpicrylhydrazyl Radical (DPPH)

a) Diphenylpicrylhydrazyl b) Diphenylpicrylhydrazine (non radical) (free radical)

(Molyneux, 2004)

When a solution of DPPH is mixed with substance that can donate a

hydrogen atom, that give rise to the reduced form (Figure 7 b) with the loss of

violet colour (although there may be a residual pale yellow colour from the picryl

group still present). Representing the DPPH radical by Z• and the donor molecule

by AH, the primary reaction is

Z• + AH ZH + A•

Where, ZH is the reduced form and A• is free radical produced in this first

step (Molyneux, 2004).

Appendices


23

2.1.4. Lipid peroxidation

Lipid peroxidation has gained more importance today because of its

involvement in pathogenesis of many diseases like atherosclerosis, cancer,

hepatitis, diabetes mellitus, myocardial infarction and also ageing. Free radicals or

Reactive Oxygen Species (ROS) are produced in vivo from various biochemical

reactions and also from the respiratory chain as a result of occasional leakage.

These free radicals are the main agents in lipid peroxidation (Donfack et al., 2011).

Lipid peroxidation is initiated by the attack on a fatty acid or fatty acyl side chain of

any chemical species. Especially the group of polyunsaturated fatty acids (PUFAs)

is highly susceptible to reactions with free radicals. Peroxidation of fatty acids in

lipids may lead to a radical chain reaction (Rajeshwari and Andallu, 2011).

(Novo and Parola, 2008)

Enhanced production of oxygen free radicals is responsible for peroxidation

of membrane lipids and the degree of peroxides damage of cell was controlled by

the potency of peroxidase enzyme system (Sairam and Tyagi, 2004).

Malondialdehyde (MDA) is a peroxidative decomposition product of polyenoic fatty

acids, and increase in tissue levels indicates an expansive lipid peroxidation (Cavit

et al., 2010).

Unsaturated fatty acids such as those present in cellular membranes are a

common target for free radicals. Reactions typically occur as a chain reaction

where a free radical will capture a hydrogen moiety from an unsaturated carbon to

Appendices


24

form water. This leaves an unpaired electron on the fatty acid and that is

then capable of capturing oxygen, forming a peroxy radical. Lipid peroxides

are unstable and decompose to form a complex series of compounds,

which include reactive carbonyl compounds such as malondialdehyde (MDA)

(http://www.biotek.com/resources /articles/ reactive-oxygen-species.html).

2.2. Antioxidants

Antioxidants are substances that may protect human body cells from the

damages caused by unstable free radicals and they are highly reactive chemicals

that play a part in some forms of cancer. Antioxidants interact with and stabilize

free radicals and may prevent some of the damages created by free radicals.

Propagation and initiation of free radicals chain reaction can be delayed or

minimized by the donation of hydrogen from the antioxidants (Hamid et al., 2010).

Considerable laboratory evidence from chemical, cell culture, and animal studies

indicates that antioxidants may slow or possibly prevent the development of

cancer (http://www.cancer.gov/cancertopics/factsheet/prevention/antioxidants).

Antioxidants are grouped as endogenous or exogenous. The endogenous

group includes metallo enzymes superoxide dismutase (zinc, manganese, and

copper), glutathione peroxidase (selenium) and catalase, and proteins like

albumin, transferrin, ceruloplasmin, metallothionein and haptoglobin. The most

important exogenous antioxidants are dietary phytochemicals (such as

polyphenols, quinones, flavonoids, catechins, coumarins, terpenoids) and the

smaller molecules like ascorbic acid (Vitamin C), alpha-tocopherol, beta-carotene,

Vitamin-E and their supplements (Bizimenyera et al., 2007).

The extracts from number of medicinal plants which are known to have

some biologically active principles are used in ayurvedic preparations and these

extracts are prepared in bulk for commercial purpose (Sathisha et al., 2011).

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 GPx

and GST regulate inner mitochondrial membrane permeability by detoxifying ROS

Appendices


25

produced during electron transport and confer protection against lipid peroxidative

damage (Andreyev et al., 2005).

Antioxidants can be classified into three main types: first line defense

antioxidants, second line defense antioxidants and third line defense antioxidants.

Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT),

Glutathione reductase (GR) and some minerals like Se, Mn, Cu and Zn comes

under first line defense antioxidants (Figure 8). Reduced glutathione (GSH),

Vitamin C, Vitamin E, uric acid, carotenoids, albumin, bilurubin, Vitamin A and

flavonoids come under second line defense antioxidants (Gupta and Sharma,

2006). Lipase, proteases, DNA repair enzymes, transferases and methionine

sulphoxide reductase come under third line defense antioxidants (Irshad and

Chaudhuri, 2002).

2.2.1. First line defense antioxidants

Antioxidant enzymes are able to catalytically remove free radicals and other

reactive species (Isai et al., 2009). Free radical scavenging enzymes such as

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

are the first line of cellular defense against oxidative injury (Burlakova et al., 2010).

Figure 8

Antioxidant enzyme system

(Pandey and Rizvi, 2010)

Appendices


26

2.2.1.1. Catalase (CAT)

Catalase is an antioxidant enzyme widely distributed in all animal tissues.

The enzyme is known to protect the system from highly reactive hydroxyl radicals

through hydrogen peroxide decomposition. Depletion of this enzyme may enhance

the cellular damage caused by assimilation of superoxide and hydrogen peroxide

(Oyedemi and Afolayan, 2011). Among the antioxidant enzymes, CATs are

ubiquitous heme enzymes that are found in aerobic organisms, ranging from

bacteria to higher plants and animals (Lee et al., 2003). CAT has one of the

highest turnover numbers of all enzymes; one molecule of CAT can convert

millions of molecules of hydrogen peroxide to water and oxygen per second

(David, 2004). Therefore reduction in the activity of CAT may result in a number of

deleterious effects due to the assimilation of superoxide radical and hydrogen

peroxide (Palanivel et al., 2008).

2H2O2 CAT O2 + 2H2O (Lenzen, 2008)

2.2.1.2. Glutathione peroxidise (GPx)

Glutathione peroxidases (GPxs) are members of the family of antioxidant

enzymes that scavenge hydrogen peroxide in the presence of reduced glutathione,

and seven isoforms having different substrate specificities and tissue distribution

have been identified (Takebe et al., 2002 and Drevet, 2006). GPx is a selenium-

dependent enzyme that contains a selenium atom incorporated within the

selenocysteine residue (Kryukov et al., 2003). GPx is one of the most important

enzymes in human. The enzyme pays an important role in peroxide detoxification.

GPx utilize the reducing equivalents of glutathione to reduce hydrogen peroxide

and it may be the main mechanism for protection against the deleterious effects of

hydroperoxides (Manjusha et al., 2011).

H2O2 + 2GSH GPx GSSG + 2H2O (Finaud et al., 2006)

2.2.1.3. Glutathione reductase (GR)

Glutathione reductase is a flavine nucleotide dependent enzyme and its

predominant subcellular distribution is in the cytosol and mitochondria (Gururaj

et al., 2004). GR is a key enzyme of the antioxidative system that protects cells

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27

against free radicals. GR inhibition disturbs cellular prooxidant antioxidant balance

and may contribute to the genesis of many diseases (Tandogan and Ulusu, 2006).

It maintains the cellular thiol redox state by catalyzing the reduction of glutathione

disulfide (GSSG) to glutathione (GSH) with NADPH as the reducing cofactor (Qiao

et al., 2007). All GR family members share a similar three dimensional structure in

their FAD binding domain as well as atleast one conserved sequence motif (Dym

and Eisenberg, 2001).

GSSG + NADPH + H+ GR 2GSH + NADP+ (Casao et al., 2010)

2.2.1.4. Superoxide dismutase (SOD)

Superoxide dismutase is a group of metallo-enzymes that play a critical

role in the first line of defense against oxidative stress caused by free radicals in

many organisms. SOD protects cells and cell components against reactive oxygen

species (ROS) by catalysing the conversion of oxygen radicals to hydrogen

peroxide and molecular oxygen, and thus provides a protective role against

oxidative stress (Lester et al., 2009). Three types of SODs have been

characterized according to their metal content; copper-zinc SOD is located in

the cytosol, the manganese SOD is primarily a mitochondrial enzyme, and

extracellular SOD is usually found on the outside of the plasma membrane (Suzy

and Serpil, 2002). Mutations in the cytoplasmic or mitochondrial form of SODs

result in ageing, neurodegenerative diseases and carcinogenesis (Bonatto, 2007).

O2•- +O2

•-+ 2H+ SOD H2O2 +O2 (Lenzen, 2008) 2.2.2. Second line defense antioxidants

Reduced glutathione (GSH), Vitamin C, Vitamin E, uric acid, carotenoids,

albumin, bilurubin, Vitamin A and flavonoids come under second line defense

antioxidants (Gupta and Sharma, 2006).

2.2.2.1. Vitamin A

Vitamin A is a generic term for a group of compounds with similar biologic

activity such as retinol, retinal, and retinoic acid. The term ‘retinoids,’ on its turn,

comprises both these natural forms of Vitamin A and many synthetic analogues to

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retinol. Retinol absorbed may be released directly in extra-hepatic tissues or

captured by the liver or returned to the blood flow to supply the organism’s needs

(Debier and Larondelle, 2005). Vitamin A is a fat-soluble vitamin present in many

lipid substances. β-carotene, present in cell membranes, is converted into vitamin

A when the body needs it (Finaud et al., 2006). Vitamin A also acts a neutralizing

agent for the free radicals (Selvi et al., 2007).It acts as a powerful, free radical

scavenger (Singlet oxygen) and chain breaking antioxidant (Raghavan and

Kumari, 2006).

2.2.2.2. Vitamin E Vitamin E, a component of the total peroxyl radical-trapping antioxidant

system reacts directly with peroxyl and superoxide radicals and singlet oxygen and

protects membranes from lipid peroxidation. The deficiency of Vitamin E is

concurrent with increased peroxides and aldehydes in many tissues (Maritim et al.,

2003). The most well-known function of Vitamin E is that of a chain-breaking

antioxidant that prevents the cyclic propagation of lipid peroxidation (Mustacich

et al., 2007).

Figure 9

Mechanism of action of Vitamin E

(http://www.cnsforum.com/imagebank/item/MOA_VITE/default.aspx)

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29

Vitamin E also plays a role in neurological functions (Muller, 2010) and

inhibition of platelet aggregation (Brigelius and Davies, 2007; Atkinson et al.,

2008). Antioxidants, such as Vitamin E, prevent cell damage by binding to the free

radical and neutralising its unpaired electron. For example, when Vitamin E

binds to OO· or O2· they form an intermediate structure that is converted to

alpha tocopherylquinone as seen in the Figure 9 (http://www.cnsforum.com/

imagebank /item/MOA_VITE/default.aspx).

2.2.2.3. Vitamin C

Vitamin C (ascorbic acid) is a six-carbon lactone that is synthesized from

glucose in the liver. Vitamin C is an electron donor and therefore it is the reducing

agent. All known physiological and biochemical actions of Vitamin C are due to its

action as an electron donor. Ascorbic acid donates two electrons from a double

bond between the second and third carbons of the 6-carbon molecule. Vitamin C is

called an antioxidant because, by donating its electrons, it prevents other

compounds from being oxidized (Padayatty et al., 2003).

Vitamin C has also been used as a dietary supplement intended to prevent

oxidative stress–mediated chronic diseases such as cancer and cardiovascular

diseases (Khaw et al., 2001) and neurodegenerative disorders (Engelhart et al.,

2002). Although it has generally been acknowledged that Vitamin C protects cells

from oxidative DNA damage, thereby block the initiation of carcinogenesis.

Moreover, the chemopreventive mechanism of Vitamin C may be linked to the

inhibition of other processes particularly tumour promotion rather than to that of

tumour initiation (Neeraj et al., 2010).

2.2.2.4. Reduced glutathione (GSH)

Reduced glutathione (GSH) a sulphydryl containing tripeptide (gamma

glutamyl cystenyl glycine) is an important endogenous antioxidant in human which

plays a central role in the defense against oxidative damage and toxins. It serves

as a co-factor for GPx and glutathione S- transferase and can react directly with

hydrogen peroxide, super oxide anion, hydroxyl and alkoxyl radicals by its free

sulphydryl groups (Chaudhari et al., 2008). When present extracellularly, GSH is

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30

able to react directly with cytotoxic aldehydes produced during lipid peroxidation

(Eskiocak et al., 2005).

Figure 10

Non enzymic antioxidant system

(http:// www.biochem.arizona.edu/classes/bioc460/summer/.../pentose.ppt)

2.3. Anticancer activity of medicinal plants

Cancer is the second leading cause of death in the Western world

(Madhusudan and Middleton, 2005). Cancer is one of the life threatening diseases

with more than 200 different types. A tumour, or mass of cells, formed the

abnormal cells may remain within the tissue in which it originated (a condition

called in situ cancer), or it may begin to invade nearby tissues (a condition called

invasive cancer). An invasive tumour is said to be malignant, and cells shed into

the blood or lymph from a malignant tumour are likely to establish new tumours

(metastases) throughout the body. Tumours threaten an individual's life when their

growth disrupts the tissues and organs needed for survival (Jena et al., 2012).

Due to lack of effective drugs, expensive cost of chemotherapeutic agents

and side effects of anticancer drugs, cancer can be a cause of death. Therefore

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31

efforts are still being made for the search of effective naturally occurring

anticarcinogen that would prevent, slow or reverse cancer development. Plants

have a special place in the treatment of cancer (Deepa et al., 2011). Plant

materials have been used for the treatment of malignant diseases for centuries.

Recent phytochemical examination of plants which have a suitable history of use

in folklore for the treatment of cancer had induced and often resulted in the

isolation of principles with antitumour activity (Dhanamani et al., 2011).

Ancient herbal medicines may have some advantages over single purified

chemicals (Vickers, 2002). Often the different components in a herb have

synergistic activities or buffer toxic effects. Mixtures of herbs are even more

complex and so might have more therapeutic or preventive activity than single

products alone. In fact, several studies have demonstrated that extracts from

several herbal medicines or mixtures had an anticancer potential in vitro or in vivo

(Bonham et al., 2002; Hu et al., 2002; Lee et al., 2002 and El-Shemy et al., 2007).

Higher plants have been one of the largest sources of new compounds with

pharmacological activity. For example, the species Catharanthus roseus (L.)

G. Don (Apocynaceae) produces several alkaloids, two of which, vincristine and

vinblastine have anticancer activity (Santos and Elisabetsky, 1999).

Antitumour activity of Mylabris cichorii extracts against murine Ascites

Dalton’s Lymphoma was studied by Prasad et al. (2010). Terminalia arjuna,

Dillenia indica and Oroxylum indicum were screened for anticancer efficacy

against Dalton’s lymphoma (Brahma et al., 2011). Antiproliferative and antioxidant

activity of Aegle marmelos (Linn.) leaves in Dalton's Lymphoma Ascites

transplanted mice was reported by Chockalingam et al. (2012). The methanol

extract of stem bark of Dillenia pentagons appears to be more active against

Dalton’s Lymphoma (Rosangkenia and Prasad, 2004). The Saponins from the

plant of china, Clematis manshrica has obvious antitumour effects against

various transplanted tumour on mice (Zhao et al., 2005). The antineoplastic activity

of methanolic extracts of five medicinal plants that are native to Iran including

Galium mite, Ferula Angulata, Stachys obtuscrena, Grsium bracteosum, and

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Echinophora Cinerea was investigated and proved to have antitumour activity

(Amirghofran et al., 2006). The anti-neoplastic activity of guduchi (Tinospora

cordifolia) on Ehrlich ascites carcinoma was proved by Jagetia and Rao (2006).

Methanol extract of Ledum groelandicum Retzius (Labrador tea) leaf twig extract

showed anticancer activity (Dufour et al., 2007).The methanolic extracts of

Dendrosicyos Socotrana, Withania aduensis, Withania riebeckii, Dracena

Cinnabari and Buxus hildebrandlii exhibited the highest toxicity on all tumour cell

lines (Mothana et al., 2007).

The methanol extract of Bauhinia racemosa stem bark exhibited antitumour

effect in EAC bearing mice (Gupta et al., 2004a). An alcoholic extract of

Biorhythms sensitivum showed antitumour activity by inhibiting the solid tumour

development in mice induced with Dalton’s lymphoma ascites (DLA) cells and

increase the life span of mice bearing Ehrlich ascites carcinoma (EAC) tumours

(Guruvayoorappan and Kuttan, 2007). The Careya arborea bark significantly

reduced the solid tumour volume induced by DLA cells (Natesan et al., 2007).

2.4. Antimicrobial activity of medicinal plants

In the worldwide as well as in the developing countries, the most human

died due to infectious bacterial diseases (Nathan, 2004). The bacterial organisms

including Gram positive and Gram negative like different species of Bacillus,

Staphylococcus, Salmonella and Pseudomonas are the main source to cause

severe infections in humans. Bacterial diseases include any type of illness caused

by bacteria. Bacteria are a type of microorganism, which are tiny forms of life that

can only be seen with a microscope. Millions of bacteria normally live on the skin,

in the intestines, and on the genitalia. The vast majority of bacteria do not cause

disease, and many bacteria are actually helpful and even necessary for good

health. These bacteria are sometimes referred to as “good bacteria” or “healthy

bacteria’’. Common pathogenic bacteria and the types of bacterial diseases they

cause include: Escherichia coli and Salmonella that cause food poisoning,

Staphylococcus aureus causes a variety of infections in the body, including boils,

cellulitis, abscesses, wound infections, toxic shock syndrome, pneumonia, and

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food poisoning (http://www.localhealth.com/article/bacterial-diseases) and Shigella

causes diarrhea and fever (Uyigue and Anukam, 2011).

Proteus vulgaris is a rod-shaped, Gram negative bacterium that inhabits the

intestinal tracts of humans and animals. It can be found in soil, water and fecal

matter. It is grouped with the enterobacteriaceae and is an opportunistic pathogen

of humans. It is known to cause urinary tract infections and wound infections

(http://en.wikipedia.org/wiki/Proteus_vulgaris). It may also cause respiratory

infections that persist even after antibiotic treatment (http://thejediknight4.

iwarp.com/ Proteus%20vulgaris.pdf). Pseudomonas aeruginosa is a Gram-

negative, rod-shaped, asporogenous, and monoflagellated bacterium that has an

incredible nutritional versatility. P. aeruginosa a very ubiquitous microorganism, for

it has been found in environments such as soil, water, humans, animals, plants,

sewage, and hospitals (http://microbewiki.kenyon.edu/index.php/Pseudomonas_

aeruginosa). It is a frequent cause of nosocomial infections such as pneumonia,

urinary tract infections, and bacteremia. Pseudomonal infections are complicated

and can be life threatening (http://emedicine.medscape.com/article/226748-

overview).

Shigella flexneri is a human intestinal pathogen, causing dysentery by

invading the epithelium of the colon and is responsible, worldwide, for an

estimated 165 million episodes of shigellosis and 1.5 million deaths per year. The

bacterium is commonly found in water polluted with human faeces. It is transmitted

in contaminated food or water and through contact between people. Upon

infection, humans develop severe abdominal cramps, fever, and frequent

passage of bloody stools. Shigellosis is not only a significant cause of infant

mortality in developing nations but maintains endemic levels of infection worldwide

(http://www.ebi.ac.uk/2can/genomes/bacteria/Shigella_flexneri.html). Klebsiella

pneumoniae is among the most common Gram-negative bacteria. It is a common

hospital-acquired pathogen, causing urinary tract infections, nosocomial

pneumonia, and intra abdominal infections. K. pneumoniae is also a potential

community-acquired pathogen (http://www.phagetherapycenter.com/pii/Patient

Servlet? command= static_klebsiella).

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These organisms have the ability to survive in harsh condition due to their

multiple environmental habitats (Ahameethunisa and Hoper, 2010). Herbal

treatment would promise a greater viable solution for effective treatment of

diseases caused by bacteria (Khan et al., 2007; Rahman and Hossain, 2010).

Mohamed et al. (2010) reported antimicrobial activity of methanol extracts of

Andrographis paniculata (leaves), Eugenia jambolana (kernel), Cassia auriculata

(flowers), Murraya koenigii (leaves), Salvadora persica (stem) and Ipomoea

batatas (leaves) against two Gram-positive bacteria (Staphylococcus aureus and

Staphylococcus epidermitis) and three Gram-negative bacteria (Escherichia coli,

Klebsiella pneumoniae and Pseudomonas aeruginosa). Antimicrobial activities

have been studied with the methanolic plant extracts of Abultilon indicum,

Adenocalymma alliaceum, Carica papaya, Crotolaria laburnifilia, Croton

bonplandianum, Derris scandens, Eichornia crassipes, Iopomea hispida, Moringa

heterohylla and Peltophorum pterocarpum (Vadlapudi, 2010).

2.5. Phytochemical constituents of medicinal plants

The medicinal value of the plants lies in some chemical substances that

produce a definite physiological action on the human body (Edeoga et al., 2005).

Phytochemicals, the natural bioactive compounds of plants are divided into two

groups, which are primary and secondary constituents according to their functions

in plant metabolism. Primary constituents comprise common sugars, amino acids,

proteins and chlorophyll while secondary constituents consist of alkaloids,

terpenoids, phenolic compounds, tannins and so on (Krishnaiah et al., 2007).

These phytoconstituents work with nutrients and fibres to form an integrated part

of defense system against various diseases and stress conditions (Koche et al.,

2010).

Phytochemicals have complementary and overlapping mechanisms of

action, including gene expression in cell proliferation, cell differentiation,

oncogenes and tumour suppressor genes, induction of cell-cycle arrest and

apoptosis, modulation of enzyme activities in detoxification, oxidation and

reduction, stimulation of the immune system and regulation of hormone

metabolism. They also have antimicrobial effects (Sun et al., 2002).

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35

All plants produce an amazing diversity of secondary metabolites. One of the

most important groups of these metabolites is phenolic compounds. Phenolics are

characterized by at least one aromatic ring (C6) bearing one or more hydroxyl

groups. They are mainly synthetized from cinnamic acid, which is formed from

phenylalanine by the action of L-phenylalanine ammonia-lyase (EC 4.3.1.5), the

branch point enzyme between primary (shikimate pathway) and secondary

(phenylpropanoid) metabolism (Dixon and Paiva, 1995). The conception of

antioxidant action of phenolic compounds is not novel (Bors et al., 1990).

Antioxidant action of phenolic compounds is due to their high tendency to chelate

metals. Phenolics possess hydroxyl and carboxyl groups, able to bind particularly

iron and copper (Jun et al., 2003).

Phenolics are also a kind of natural product and antioxidant substance

capable of scavenging free superoxide radicals, anti-aging and reducing the risk of

cancer (Ghasemzadeh and Ghasemzadeh, 2011). The antioxidant properties of

phenolic and flavonoid compounds are mediated by scavenging radical species

such as ROS/ RNS; suppressing ROS/RNS formation by inhibiting some enzymes

or chelating trace metals involved in free radical production and up regulating or

protecting antioxidant defense (Cotelle, 2001). The reduction activity of phenolic

and flavonoid compounds depends on the number of free hydroxyl groups in the

molecular structure (Rice-Evans et al., 1996). Isolated polyphenols from different

plants have been considered in a number of cancer cell lines at different stages of

cancer growth. For example, the isolated polyphenols from strawberry including

kaempferol, quercetin, anthocyanins, coumaric acid and ellagic acid, were shown

to inhibit the growth of human breast (MCF-7), oral (KB, CAL-27), colon (HT-29,

HCT-116), and prostate (LNCaP, DU-145) tumour cell lines (Damianaki et al.,

2000 and Zhang et al., 2008).

Silymarin is the bioactive extract from Silybum marianum L. seeds

(Asteraceae) and contains 65-85% flavonolignans like silychristin, isosilychristin,

silydianin, silybin A and B, isosilybin A and B, and also 20- 35% fatty acids,

flavonoids, and other polyphenolics. The major source of silymarin is fruits and

seeds from this plant, but traces of these compounds can occur in all plants

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(Ramasamy and Agarwal, 2008). Silymarin has been used medicinally to treat liver

disorders, including acute and chronic viral hepatitis, toxin/drug-induced hepatitis,

and cirrhosis and alcoholic liver diseases. It has been reported to be effective in

certain cancers. Its mechanism of action includes inhibition of hepatotoxin binding

to receptor sites on the hepatocyte membrane; reduction of glutathione oxidation

to enhance its level in the liver and intestine; antioxidant activity; and stimulation of

ribosomal RNA polymerase and subsequent protein synthesis, leading to

enhanced hepatocyte regeneration (Dixit et al., 2007).

Silymarin’s hepatoprotective effects are accomplished via several

mechanisms. These include: antioxidation (Mirguez et al., 1994), Inhibition of lipid

peroxidation (Bosisio et al., 1992), enhanced liver detoxification via inhibition of

phase detoxification (Halim et al., 1997 and Baer-Dubowska et al., 1998),

protection from glutathione depletion (Campos et al., 1989), anticarcinogenesis by

inhibition of cyclin dependent kinases and arrest of cancer cell growth. Silymarin

also found to have immunomodulatory effects on the diseased liver (Deak et al.,

1990 and Lang et al., 1990).

Figure 11

Anticancer mechanism of silymarin

(Ramasamy and Agarwal, 2008)

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37

Numbers of studies have established the cancer chemopreventive role of

silymarin in both in vivo and in vitro models. Silymarin modulates imbalance

between cell survival and apoptosis through interference with the expressions

of cell cycle regulators and proteins involved in apoptosis. In addition, silymarin

also showed anti-inflammatory as well as antimetastatic activity (Figure 11)

(Ramasamy and Agarwal, 2008).

Hiremath and Urmila (2012) explored that methanol and aqueous extract of

Amaranthus caudatus leaves contain carbohydrates, proteins, amino acids,

saponins, glycosides, phenoilcs and tannins; chloroform and acetone extracts

contains glycosides and saponins and petroleum ether extract of leaves contain

saponins. Kondongala et al. (2012) found that phytochemical investigation of

methanolic extract of stem and bark of Bauhinia purpurea showed the presence

of carbohydrates, glycosides, saponins, sterols and triterpinoids. Sheela et al.

(2012) reported the presence of alkaloids, flavonoids, saponins, tannins,

carotinoids and phytates in Sanseiveria roxburghiana leaf extract.

Venkateshwaralu et al. (2012) reported that leaves of methanolic extract of

Ximenia americana Linn. showed the presence of alkaloids, steroids, sugars,

saponins, tannins and terpenoids.

2.6. In silico drug design and docking for cancer In silico methods can help in identifying drug targets via bioinformatics

tools. They can also be used to analyze the target structures for possible binding/

active sites, generate candidate molecules, check for their drug likeness, dock

these molecules with the target, rank them according to their binding affinities,

further optimize the molecules to improve binding characteristics. The use of

computers and computational methods permeates all aspects of drug discovery

today and forms the core of structure-based drug design. The use of

complementary experimental and informatics techniques increases the chance of

success in many stages of the discovery process, from the identification of novel

targets and elucidation of their functions to the discovery and development of lead

compounds with desired properties. Computational tools offer the advantage of

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delivering new drug candidates more quickly at a lower cost. Major roles of

computation in drug discovery are; (1) Virtual screening and de novo design, (2) in

silico Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME/T)

prediction and (3) Advanced methods for determining protein-ligand binding

(http://www.scfbio-iitd.res.in/tutorial/drugdiscovery.htm).

Bioinformatics is seen as an emerging field with the potential to significantly

prove how drugs are found, brought to the clinical trials and eventually released to

the marketplace. Computer Aided Drug Design (CADD) is a specialized discipline

that uses computational methods to simulate drug – receptor interactions. One of

those methods is called docking. The site of drug action, which is ultimately

responsible for the pharmaceutical effect, is a receptor. Docking allows the

scientist to virtually screen a database of compounds and predict the strongest

binders based on various scoring functions (Virupakshaiah et al., 2007).

Docking explores the ways in which two molecules, such as drugs and an

enzyme receptor fit together and dock to each other well. The molecules binding to

a receptor, inhibit its function, and thus act as drug. Complexes were identified via

docking and their relative stabilities were evaluated using molecular dynamics and

their binding affinities, using free energy simulations (Babu et al., 2008).

2.6.1. Histone deacetylase (HDAC)

For the last four decades, a number of potential approaches have been

proposed for the treatment of cancer. One of the recent targets is histone

deacetylase (Saha et al., 2010). It has been widely recognized in recent years that

HDACs are promising targets for therapeutic interventions intended to reverse

aberrant epigenetic states associated with cancer (Pandolfi, 2001; Baylin and

Ohm, 2006). HDAC is an enzyme that removes an acetyl group from histones,

which allows them to bind DNA and inhibit gene transcription (Elaut et al., 2007

and Santini et al., 2007). Acetylation and deacetylation of chromatin histone

protein by HDAC alters chromatin structure and dynamically affects transcriptional

regulation (Liu et al., 2006a).

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Histone deacetylase inhibitors (HDACIs) are emerging as a new class of

anticancer agents. HDACIs have shown activity against diverse cancer types and

notable effects on tumour cell proliferation, programmed cell death, differentiation

and angiogenesis in vitro and in vivo. Currently, there are more than a dozen of

phase I and II clinical trials involving the use of HDACIs in patients with

haematological and solid malignancies (Marks et al., 2004).

Figure 12

Role of Histone deacetylase inhibitors

(http://englishclass.jp/reading/topic/Histone_deacetylase)

In preclinical studies several classes of HDACIs have been found to

have potent anticancer activities, with remarkable tumour specificity, and some

have demonstrated promising therapeutic potential in early-phase clinical trials

for haematological malignancies such as cutaneous T-cell lymphoma,

myelodysplastic syndromes and diffuse B-cell lymphoma (Lindemann et al., 2004;

Jabbour and Giles, 2005; Marks and Jiang, 2005).

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2.6.2. Tubulin

Microtubules the key components of the cytoskeleton are long, filamentous,

tube-shaped protein polymers that are essential in all eukaryotic cells. They are

crucial in the development and maintenance of cell shape, in the transport of

vesicles, mitochondria and other components throughout cells, in cell signalling,

and in cell division and mitosis. Their importance in mitosis and cell division makes

microtubules an important target for anticancer drugs (Jordan and Wilson, 2004).

Figure 13

Role of Tubulin in the inhibition of mitosis

(http://www.photobiology.info/Christensen.html).

Microtubule inhibitors disrupt microtubule dynamics of tubulin

polymerization and depolymerization, which results in the inhibition of

chromosome segregation in mitosis and consequently the inhibition of cell division

(Mulligan et al., 2006). In normal mitosis, the chromosomes are pulled by

microtubules, formed from tubulin, towards the two centrioles, marking the location

where the nuclei of the two daughter cells will be formed. After that, the cell

membrane is pinched off, and the chromosomes decondense in the newly formed

cells. If the chromosomes are not separated by the "ropes" formed from tubulin,

the normal process of mitosis will not be completed, and cell death may result. An

inhibition of the tubulin function leads to an arrest of the cells in mitosis, and no

further cell division as long as the chromosomes are not transported to the two

poles. A large number of cells have been shown to die while they are arrested in

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this phase of cell division. A mitotic arrest is the mechanism behind the

function of mitotic inhibitors commonly used in cancer therapy (Figure 13)

(http://www.photobiology. info/ Christensen.html).

2.6.3. Aurora kinase A

Aurora-A kinase (officially known as Serine/threonine-protein kinase 6) is an

important regulator of cell division and acts in several aspects of spindle formation

and function (Barr and Gergely, 2007). Aurora family kinases play roles in several

mitotic processes, including the G2/M transition, mitotic spindle organization,

chromosome segregation and cytokinesis (Andrews et al., 2003; Crane et al.,

2003; Katayama et al., 2003 and Meraldi et al., 2004). Aurora A is found in the

cytoplasm and at centrosomes during interphase; during mitosis, it also localizes

to microtubules near the spindle poles. Aurora A interacts with several different

proteins that are required for proper centrosome maturation and spindle function

(Gadea and Ruderman, 2005).

Figure 14

Mechanism of action of Aurora kinase

(http://cancergrace.org/cancer-treatments/tag/targeted-therapy/)

The levels of Aurora A are abnormally high in many tumour types, and

altered abundance is thought to be of relevance for oncogenic transformation

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(Gautschi et al., 2008; Vader and Lens, 2008). Its abundance in cell cycle

regulated, with a peak in G2 and M phases, followed by regulated proteolysis at

the end of mitosis (Giubettini et al., 2010). Indeed, Aurora-A has recently been

proposed as a potential target in anticancer therapy; inhibitors of its activity have

been synthesised, some of which are currently being tested in clinical trials

(Castro et al., 2008 and Karthigeyan et al., 2010).

2.6.4. Protein kinase C

Protein kinase C (PKC) was originally discovered by Yasutomi Nishizuka in

1977 as a histone protein kinase activated by calcium and diacylglycerol (DAG),

phospholipids and/or phorbol esters (Takai et al., 1977).It is known that the PKC

family consists of serine/threonine-specific protein kinases that differ in their

structure, cofactor requirement and substrate specificity (Takai et al., 1979).Due to

biochemical properties and sequence homologies, PKCs are divided into three

subfamilies: firstly, classical or conventional, secondly, novel and finally, atypical

(Nishizuka, 1992; Newton, 1995; Schenk and Snaar-Jagalska, 1999).

Figure 15

Mechanism of action of kinase inhibitors

(Kondapalli et al., 2005)

Pre-clinical and clinical data has suggested that protein kinase C (PKC)

may represent an attractive target for cancer therapy (Marengo et al., 2011). PKC

regulates tumour promotion and cell growth by inducing activation of

transcriptional factors, such as activator protein-1 (AP-1) and nuclear factor- kappa

B (NF- kappa B), and by increasing the expression of key enzymes, such as

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ornithine decarboxylase, inducible nitric oxide synthase, and cyclooxygenase-2

(Amstad et al., 1992; Fischer et al., 1993; Stauble et al., 1994 and Klein et al.,

2000). PKC has unique structural aspects that render it susceptible to activation by

oxidant tumour promoters, such as H2O2, periodate, and tobacco related tumour

promoters (Gopalakrishna and Anderson, 1989; Gopalakrishna and Anderson,

1991; Gopalakrishna et al., 1994).

2.7. In silico drug design and docking for microbial infections

2.7.1. Pantothenate Kinase

Coenzyme A (CoA) is a key component of cellular metabolism and is

essential for bacterial viability since disruption of the CoA biosynthetic pathway is

lethal. CoA synthesis begins with the phosphorylation of pantothenate (Vitamin B5)

by pantothenate kinase (Dunster et al., 2002). Pantothenate kinase, which triggers

the first step in the production of coenzyme A (CoA), a molecule that is

indispensable to all forms of life. CoA plays a pivotal role in the cells' ability to

extract energy from fatty acids and carbohydrates; bacteria need CoA to make

their cell walls. The job of pantothenate kinase is to grab a molecule of pantothenic

acid (Vitamin B5) and another molecule that contains a chemical group called

"phosphate." The enzyme then removes the phosphate group from that molecule

and sticks it onto pantothenic acid. In humans, certain mutations in this enzyme

block its ability to put the phosphate group onto pantothenic acid. That diminishes

the production of CoA and causes the pantothenate kinase associated

neurodegenerative disease (Hong et al., 2006).

The pantothenate kinase is a key rate-determining enzyme of this pathway

and become a prime target for its inhibition. By doing so, we are not only inhibiting

this CoA biosynthetic pathway, but also inhibiting the microbial growth. It should

therefore be possible to develop selective small molecule inhibitors for the

pantothenate kinases that are expressed by the pathogenic microorganisms of

interest (Leonardi et al., 2005a).

2.7.2. Deacetoxy C synthase

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from

Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the

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44

ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation

of the latter to deacetylcephalosporin C (Wu et al., 2011). Deacetoxycephalosporin

C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring

expansion of the penicillin nucleus into the nucleus of cephalosporins. The

reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a

reactive iron-oxygen intermediate from a ferrous iron in the active site (Oster et al.,

2004).

2.8. Medicinal plants selected for the study

2.8.1. Morinda tinctoria

The genus Morinda, belonging to the family Rubiaceae, grows wild and is

distributed throughout Southeast Asia, commercially known as Nunaa, is

indigenous to tropical countries and is considered an important traditional folk

medicine (Sivaraman and Muralidharan, 2011). Morinda tinctoria also commonly

known as Aal or Indian Mulberry is a species of flowering plant. Its common name

also refers to Morinda citrifolia (http://en.wikipedia.org/wiki/Morinda_tinctoria). It is

an evergreen shrub or small tree growing to 5-10 m tall. The Leaves are glabrous

of slightly pubescent or only hairy in the axils underneath, from broad-ovate to

elliptic-lanceolate, acuminated at the apex, generally about 4-5 in. long by 1 1/4 - 2

1/4 broad, but sometimes larger; petioles 1/2-1 in. long; stipules membranous

broad, entire or bifid, variable in size. The fruit is a green of each flower-head

united in a compound succulent berry (syncarpium) forming a pulpy mass, about 1

in. diameter., including a number of hard, 1-seeded pyrenes, orbicular, flattened,

usually 2-4 from each flower (Nisha et al., 2011). All parts of Morinda tinctoria have

medicinal properties (Shanthi et al., 2012).

In India Morinda is widely grown under natural conditions in Andaman and

Nicobar Islands. It is seen throughout the coastal region along fences and road

sides due to its wider adaptability to hardy environment. In the main land of India it

is found along the coastal areas of Kerala, Karnataka, Tamil Nadu and many other

places. Survey of Morinda in south India indicated that 12 different species or

varieties of Morinda are distributed throughout Tamil Nadu and Kerala. However,

the species Morinda tinctoria is present abundantly in most parts of Tamil Nadu

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45

and in some parts of Kerala. An unidentified Morinda species with large and

leathery leaves was reported in the Dhandakaranya forest area of Malkanagiri

district in Orissa (Singh et al., 2007).

There is a greater demand for fruit extract of Morinda species in treatment

for different kinds of illness such as arthritis, cancer, gastric ulcer and other heart

diseases (Narayanasamy et al., 2006). Leaves are useful as tonic, febrifuge,

deostruent and emmenagogue. It is also used for curing dyspepsia, diarrhoea,

ulceration, stomatitis, digestion, wound and fever. The leaf juice is useful as a local

application. Root is used to cure inflammation and boils. Unripe fruit is used to

cure rheumatism. Ash of the fruit prevents dysentery, vomiting, diarrhoea and

cholera (Kanchanapoom, 2001).The major components have been identified in the

Nunaa plant which includes octoanic acid, potassium, Vitamin C, terpenoids,

scopoletin, flavonesglycosides, lineoleicacid, anthraquinones, morindone, rubiadin

and alizarin (Moorthy and Reddy, 1970; Singh and Tiwari, 1976; Duduku et al.,

2007).

Many species of Morinda are available in India, of which Morinda tinctoria

predominantly grows as a weed tree in vacant agricultural land and especially on

uncultivated lands and along the boundaries of the cultivated fields. Ancient

writings reveal that Morinda has long been cultivated in different parts of Tamil

Nadu state in India. Although the south Indian ancestors realized the therapeutic

value of M. tinctoria and used it in the traditional Indian medicinal systems like

siddha, lack of proper documentation resulted in loss of that knowledge (Jeyabalan

and Palayan, 2009).

Fruit extract of Morinda tinctoria was found to accelerate wound healing in

rats (Mathivanan et al., 2006). The fruit extracts of Morinda tinctoria showed

effective antidiabetic activity in alloxan induced experimental rats (Mathivanan and

Surendiran, 2006). Ethanol extract of Morinda tinctoria Roxb leaves found to

possess antiulcer activity in rats (Vadivu et al., 2008). Jeyabalan and Palayan

(2009) have reported the analgesic and anti-inflammatory activity of leaves of

Morinda tinctoria Roxb. Anticonvulsant activity of Morinda tinctoria has been

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46

reported by Kumaresan and Saravanan (2009). Fruit extract of Morinda tinctoria

showed antihyperglycemic and antidiabetic effects in streptozotocin (STZ)-induced

diabetic rats (Muralidharan and Sivaraman, 2009). Antidiabetic and antioxidant

activity of Morinda tinctoria roxb fruit extract in streptozotocin induced diabetic rats

were also reported (Pattabiraman and Muthukumaran, 2011). Cytoprotective effect

of Morinda tinctoria Roxb. against surgical and chemical factor induced gastric and

duodenal ulcers in rats were studied by Sivaraman and Muralidharan (2011).

Pharmacognostical studies on Morinda tinctoria (Roxb) was reported by Shanthi

et al. (2012).

2.8.2. Nerium indicum

Nerium indicum (family: Apocynaceae) has been traditionally attributed

with several medicinal properties (Banerjee et al., 2011). It is commonly known as

Indian oleander (http://www.flower-knowledge.com/2011/10/nerium-indicum-

flower.html), soland, lorier bol, rosebay, and rose laurel and kaner (Ansford and

Morris, 1981). Nerium indicum is an erect, smooth shrub, 1.5 to 3 meters height

with a cream colored sticky resinous juice. Leaves are in whorls of 3 or 4, linear-

lanceolate, 10-15cm long, with numerous horizontal nerves. Flowers are showy,

sweet-scented, single or double, 4-5 cm in diameter, white, pink or red, borne in

terminal inflorescence (cymes). Fruit is cylindrical, paired, with deep linear

striations, 1.5-2.0 cm long. Seeds are numerous and compressed, with a tuft of

fine, shining, white, silky hairs (Vijayvergia and Kumar, 2007).

The flowers of this plant are hermaphrodite (Pendse and Dutt, 1934). All

parts of the plant are reputed therapeutic agents and have been used in folklore in

a variety of disease (Simon, 1942). Decoction of leaves has been applied

externally in the treatment of scabies and to reduce swellings (Wasif et al., 2008).

The leaves and flowers are cardiotonic, diaphoretic, diuretic (promotes excretion),

emetic, expectorant, sternutatory as well as for treatment of malaria and

abortifacient (Govind, 2010a).

Antiinflammatory and antinociceptive activity was reported by Erdemoglu

et al. (2003). Ahmed et al. (2006) reported the analgesic activity of Nerium indicum

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47

Mill. Primary Metabolites of Nerium indicum Mill was quantified by Vijayvergia and

Kumar (2007). Protective potential of Nerium indicum extract on lipid profile, body

growth rate, and renal function in streptozotocin induced diabetic rats were

reported (Yassin and Mwafy, 2007). It also possess to have antiulcer (Govind and

Saurabh, 2010), neuroprotective (Man-shan et al., 2007), molluscicidal (Zhang et

al., 2009), piscicidal (Sudhanshu and Ajay, 2009) and antiviral (Rajbhandari et al.,

2001) activities. Methanolic flowers extract of Nerium indicum was evaluated for its

hepatoprotective effect in rats (Govind, 2010b). Antihyperlipidemic activity of

Nerium indicum leaves extracts in hyperlipidimic rats has been studied by Sikarwar

and Patil (2011).

The molluscicidal activity of Nerium indicum bark against Lymnaea

acuminata snails was studied (Singh and Singh, 1998). Polysaccharides from the

flowers of Nerium indicum showed neuroprotective effects (Yu et al., 2004).

Insecticidal potentialities of Nerium indicum leaves extracts against Epilachna

28-punctata (F.) was reported by Ranjana and Anil (2005). N-butanol extracts

and water extracts of Nerium indicum also shown to possess molluscicidal

activity (Wang et al., 2006). New polysaccharide from Nerium indicum

protected neurons via stress kinase signaling pathway (Yu et al., 2007). Nerium

indicum leaf extract showed antidiabetic activity in alloxan induced diabetic rats

(Sikarwar et al., 2009).Cardiac glycosides from fresh leaves of Nerium indicum

were also evaluated for its molluscicidal activity against Pomacea canaliculata

(Dai et al., 2011).

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