CHAPTER 1shodhganga.inflibnet.ac.in/bitstream/10603/35283/11/11_chapter1.pdf · Introduction and Review of LiteratureIntroduction and Review of Literature 11 Fig. 1.3 Organs affected

CHAPTER 1

INTRODUCTION AND

REVIEW OF LITERATURE

Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature

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1.1. INTRODUCTION

India has a unique wealth of biota which includes a large number of

medicinal and aromatic plants. A medicinal plant is a plant which contain substance

that can be used for therapeutic purposes or which are precursors for chemico-

pharmaceutical semi-synthesis. Medicinal plants may therefore be defined as a

group of plants that possess some special properties or virtues that qualify them as

articles of drugs and therapeutic agents, and are used for medicinal purposes. Thus it

is not unreasonable to believe that plant kingdom should yield safe and effective

drugs for most of the human ailments.

Nowadays plants are still important sources of medicines, especially in

developing countries that still use plant-based traditional medicine for their

healthcare. It was estimated in the Bulletin of the World Health Organization

(WHO) that around 80% of the world’s population relied on medicinal plants as

their primary healthcare source.

Plants have formed the basis of sophisticated traditional medicine (TM)

practices that have been used for thousands of years by people in China, India, and

many other countries. Some of the earliest records of the usage of plants as drugs are

found in the Artharvaveda, which is the basis for ayurvedic medicine in India.

Ayurveda has a long tradition in treating various diseases including liver diseases

using herbal medicines. Apart from timely cure the ayurvedic herbs give a

permanent relief from the diseases by removing the metabolic toxins from our body.

Herbal drugs play an important role in healthcare programmes worldwide,

mainly due to the general belief that they are without any side effects, besides being

Chapter 1Chapter 1Chapter 1Chapter 1

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cheap and locally available. Ayurveda is a perfect science of life; it works

effectively fighting against various infections and diseases and thereby gaining

quick recovery. Lately there is a resurgence of interest in herbal medicines for

treatment of various ailments including liver disorders.

Liver, the largest organ in the vertebrate body - is the major site of intense

metabolic activities such as drug and xenobiotic metabolism. Liver injury caused by

toxic chemicals and certain drugs has been recognized as a crucial toxicological

problem. In the present world a large number of toxins are introduced daily. So it is

more important than ever to keep the liver healthy and potent. The most important

metabolic function of liver is the detoxification and excretion of toxic chemicals,

drugs and hormones. Liver tissue has the capacity to regenerate, so a moderate cell

injury is not reflected by measurable change in its metabolic function. Due to the

high tolerance of liver, liver disease is seldom detected at the early stage and once

detected treatment faces a poor prognosis in most cases.

Till date, there is no effective medicine for hepatic disorders, such as hepatic

fibrosis and hepatocellular carcinoma. Many plants have been reported for their

antioxidant and hepatoprotective activity and are used in ayurvedic system of

medicine for the treatment of liver disorders. Woodfordia fruticosa is a traditional

medicinal plant and its flowers are used for the preparation of fermented drugs and

for the treatment of various disorders such as dysentery, sprue, rheumatism,

hematuria, hemorrhoids, derangement of liver, disorders of mucous membrane etc.

All parts of the plant possess valuable medicinal properties viz anti inflammatory,

anti tumour, hepatoprotective and free radical scavenging activity but flowers are in

maximum demand. But still there is a paucity of information regarding the potential


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of Woodfordia fruticosa flowers in resisting oxidative stress, hepatic fibrosis and

hepatocellular carcinoma. Hence, this study was undertaken with the following

Objectives.

1. To explore the phytochemical constituents and in vitro antioxidant activity of

Woodfordia fruticosa flowers.

2. To prove the antioxidant effect of this medicinal plant against thioacetamide

induced oxidative stress.

3. To study the effect of W. fruticosa flowers against CCl4 induced hepatic

fibrosis.

4. To explore the anticancer properties of W. fruticosa in combating

hepatocellular carcinoma induced by N-nitrosodiethylamine.

5. To study the chemopreventive effect of the methanolic extract of

W. fruticosa and its sub-fractions on human hepatoma cell line.

1.2. REVIEW OF RELATED LITERATURE

1.2.1. The Liver – Structure and functions

The liver is the largest organ and is the central chemical laboratory in the

body. It is an organ of paramount importance and plays a pivotal role not only in the

metabolism and disposition of exogenous toxins and therapeutic agents responsible

for metabolic derangement, but also in the biochemical regulation of fats,

carbohydrates, amino acids, proteins, blood coagulation and immuno-modulation.

The liver is a major target organ for toxicity of xenobiotics and drugs, because most

of the orally ingested chemicals and drugs first go to liver where they are


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metabolized into toxic intermediates. A large number of xenobiotics are reported to

be potentially hepatotoxic (Ajith et al., 2007).

1.2.1.1. The structure of liver

The liver consists of four sections, or lobes. There are two main lobes - the

right lobe, which is by far the larger, and the left lobe. Two small lobes lie behind

the right lobe.

Each lobe is made up of multisided units called lobules. Most livers have

between 50,000 and 100,000 lobules. Each lobule consists of a central vein

surrounded by tiny liver cells grouped in sheets or bundles. These cells perform the

work of the liver. Cavities known as sinusoids separate the groups of cells within a

lobule. The sinusoids give the liver a spongy texture and enable it to hold large

amounts of blood.

The liver is composed of a number of cell types that function independently.

The most abundant cell is the hepatocyte, comprising approximately 70 percent of

the liver volume and performing the bulk of liver functions. Each hepatocyte is

supplied with nutrient rich portal blood and oxygen rich aortic blood, supporting its

high metabolic and secreatory activity. Another unique feature of the hepatocyte is

its unusually high capacity for proliferation when a portion of the liver is removed or

damaged, which underlies the liver’s regenerative properties.


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Fig. 1.1. Anatomy of liver


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1.2.1.2. Liver Functions

This complex organ performs multiple interrelated functions that are

essential for life, including: (1) uptake, storage, metabolism and release of nutrients

(e.g., amino acids, carbohydrates, lipids, vitamins and minerals); (2) synthesis of bile

salts from cholesterol and their secretion to assist with fat absorption and digestion;

(3) synthesis and secretion of plasma proteins necessary for blood clotting and

transport of molecules through the circulation; (4) detoxification of drugs, hormones

and the end products of metabolism and distribution to the bile or urine for

excretion; and (5) removal of bacteria and dying red blood cells from the circulation.

During the detoxification of xenobiotics, reactive oxygen species (ROS) are

generated which cause oxidative stress (Kohen and Nyska, 2002) and which leads to

the hepatic damage.

1.2.2. Reactive oxygen species and oxidative stress

Oxygen is thought to have been responsible for the expansion of life on

Earth, there are two sides to this molecule: life giving and life taking. Oxygen in the

air we breathe is a relatively nonreactive chemical. However, when oxygen is

exposed to high-energy or electron-transferring chemical reactions, it can be

converted to various highly reactive chemical forms (Fig. 1.2) collectively

designated “reactive oxygen species” (ROS) (Rodriguez and Redman, 2005)

Reactive oxygen species (ROS) known as free radicals, are oxidizing agents

generated as a result of metabolism of oxygen and have at least one unpaired

electron that make them very reactive species. Normally, free radicals attack the

nearest stable molecule, which becomes a free radical itself, beginning a cascade of


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chain reaction. These can very rapidly oxidize biomolecules that they encounter in

their vicinity thus exerting either a positive or a negative influence on normal cell

function (Warren et al., 1987)

Fig. 1.2. Generation of different ROS by energy transfer or sequential univalent reduction of ground-state triplet oxygen

Normal aerobic metabolism is related to optimal levels of ROS because a

balance exists between ROS production and antioxidant activity. Oxidative stress

(OS) is the term applied when oxidants outnumber the antioxidants due to excessive

generation of reactive oxygen species and when antioxidants cannot scavenge these

free radicals (Sharma et al., 1999). Such phenomena cause pathological effects,

damaging cells, tissues and organs (Aitken and Baker 1995).

Reactive oxygen and nitrogen species are physiologically produced during

metabolic processes and especially during electron transport chain reactions (Di

Meo and Venditti, 2001). Another internal source of reactive species is peroxisomes,

small membrane-enclosed organelles containing enzymes important for oxidation

reactions. Furthermore, the enzymes of the Ρ-450 complex generate reactive species

during the detoxification of xenobiotics, such as drugs. There are also external

sources of reactive species related to UV radiation, air pollution, smoking, alcohol

consumption, and exercise (Halliwell and Gutteridge, 2007).


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ROS are potential carcinogens because of their roles in mutagenesis, tumour

promotion, and progression (Dröge, 2003). If not regulated properly, the excess ROS

can damage lipids, protein or DNA, inhibiting normal function (Perry et al., 2000).

Proteins are one of the major targets of reactive species which induce the formation

of carbonyl groups (aldehydes and ketones) in those amino acids that are susceptible

to oxidation, such as histidine, arginine, lysine, and proline. The carbonyl groups are

not metabolized in proteasomes and lysosomes, but are accumulated (Levine, 2002).

Furthermore, the thiol groups (−SH) present in protein molecules are oxidized in

thiol radicals (RS.). Protein oxidation leads to conformational changes which result

in the modification or loss of protein function (Halliwell and Gutteridge, 2007).

Apart from proteins, lipids are vulnerable in reactive species-induced oxidative

damage (Halliwell and Chirico, 1993). Lipid peroxidation increases the permeability

of cellular membranes, resulting in cell death. Reactive species also affect DNA by

causing chain breaks and damaging its repair mechanism (Jenkins, 1988). DNA, and

especially guanine, oxidation results in the production of 8-hydroxy-2-

deoxyguanosine. This by-product, if not repaired, induces DNA mutations that may

cause aging and carcinogenesis (Radak et al., 1999). In addition, excessive

production of reactive species has been implicated in immune system dysfunction

(Schneider and Tiidus, 2007), muscle damage (Nikolaidis et al., 2007a, 2007b), and

fatigue (Betters et al., 2004).


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Fig. 1.3 Organs affected by oxidative stress

1.2.3. Antioxidant defense mechanism

Antioxidants are substances that delay or prevent the oxidation of cellular

oxidizable substrates (Halliwell and Gutteridge, 2007). When the generation of the

active oxygen-free radical exceeds the scavenging ability many degenerative

diseases such as brain dysfunction, cancer, heart diseases, age-related degenerative

conditions, declination of the immune system, gastric ulcer and DNA damage will

arise. Antioxidants can be divided into two categories according to specific

characteristics: - endogenous antioxidants and exogenous antioxidants.

1.2.3.1. Endogenous antioxidants

Endogenous antioxidant systems possess enzymatic and non-enzymatic

antioxidative mechanisms which minimizes the generation of reactive oxygen species.

They include enzymes such as superoxide dismutase (SOD), catalase, glutathione

reductase (GR), and glutathione peroxidase (GPx) and non-enzymatic metabolites

such as glutathione, uric acid, vitamins and polyphenols. Regarding their origin,


12

various antioxidants such as glutathione, uric acid, catalase and SOD can be

synthesized in vivo, whereas others namely, polyphenols and β-carotene, are obtained

from food. Based on their physical properties, antioxidants can be divided into water

soluble antioxidants such as uric acid, glutathione and polyphenols or lipid-soluble

antioxidants such as vitamins A, vitamin E and lipoic acid (Veskoukis et al., 2012).

The SOD enzyme catalyzes the dismutation of Ο2. to Η2Ο2. It exists in

mitochondrial form (MnSOD) and in cytoplasmic form (Cu/ZnSOD) that is

primarily found in muscle cells (Das et al., 1997). Catalase is present in almost every

kind of cell, but its concentration is higher in the erythrocytes and liver (Masters et

al., 1986). Its subcellular localization is in peroxisomes, mitochondria and in the

nucleus. It catalyzes the conversion of Η2Ο2, which is produced by SOD to Η2Ο and

Ο2. The antioxidant activity of catalase is of great significance as it prevents the

conversion of Η2Ο2 to the very harmfulOH.. Furthermore, it has been demonstrated

that catalase and SOD activities exhibit a linear correlation with life span in

mammals (Cutler, 1984). GPx, which requires selenium as a cofactor is present in

the cytoplasm and mitochondria and is an alternative route of Η2Ο2 degradation.

Specifically, Η2Ο2 is converted to Η2Ο and Ο2 and oxidizes GSH (reduced form of

glutathione) to GSSG (oxidized form of glutathione).

Glutathione is considered one of the most important antioxidant metabolites

and is the first line of defense against reactive species. At rest, glutathione is usually

present in the reduced state. GSH is a tripeptide consisting of glutamic acid, cysteine

and glycine. It is the most abundant low-molecular-weight thiol-containing

compound in biological fluids and tissues of mammals. In eukaryotic cells, 90% of


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the intracellular GSH pool resides in the cytoplasm, and the remaining 10% is found

in the mitochondria, endoplasmic reticulum and the nucleus. However, the

biosynthesis of GSH appears to occur exclusively in the cytoplasm (Barycki, 2007).

GSH possess potent antioxidant properties, maintaining the intracellular redox

homeostasis due to the thiol group of cysteine which serves as a substrate of GPX

and contributes to xenobiotic detoxification (Halliwell and Gutteridge, 2007). In

physiological conditions, GSH is in a dynamic equilibrium with GSSG. However, in

the context of oxidative stress GSH works with GPX to efficiently remove

intracellular H2O2. This process protects biomolecules from oxidative modifications,

and GSH is converted to GSSG. Glutathione reductase reduces GSSG to GSH using

NADPH as an electron donor, thus replenishing the GSH pool (Barycki, 2007).

Fig. 1.4. Generation of free radicals exceed the cellular antioxidants leads to cancer

1.2.3.2. Exogenous antioxidants

The cellular antioxidants may not be capable of neutralizing all the free

radicals produced in the body as well as those derived from the environment, so

therefore a need for an external source of antioxidants to neutralize the free radical

load in the body. A large number of antioxidants, both nutritive and nonnutritive,


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occur in foods. Besides β-carotene, vitamin C and vitamin E (which are nutrients),

a number of carotenoids, phenols and flavonoids also occur naturally in foods and

can act as antioxidants. β-carotene is an excellent scavenger of singlet oxygen.

Vitamin C interacts directly with radicals like 2O . and OH. (hydroxyl). Vitamin C

and vitamin E prevent formation of nitrosamine, which is carcinogenic. Vitamin E

also protects selenium against reduction and protects polyunsaturated fatty acids

(PUFA) in the membrane against oxidative damage (Rao, 2003).

1.2.3.3. Natural antioxidants

Natural antioxidants present in fruits, vegetables, cereals and medicinal

plants act as effective free radical scavengers, by donating hydrogen to highly

reactive radicals. They are converted into relatively harmless free radicals, which

may react with other free radicals and inactivate them. Studies reveal that increased

consumption of fruits rich in antioxidant polyphenols lower the risk of degenerative

diseases such as cancer (Patel et al., 2011) and increase the concentration of β-

carotene in the blood. Polyphenolic compounds are plant secondary metabolites

which have at least one aromatic ring in their molecule and usually exist in the form

of glycosides. More than 8,000 different polyphenolic compounds have been

described. They are subdivided into nonflavonoids (e.g., hydrobenzoic acids,

hydroxycinnamic acids, and stilbenes) and flavonoids (e.g., flavonols, flavanals,

isoflavones, and anthocyanins). Flavonoids are composed of more than 4,000

different species that have two aromatic benzene rings linked through three carbons

forming an oxygenated heterocycle.


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Synthetic antioxidants such as propyl gallate, butylated hydroxyanisol

(BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) are

commonly used to control lipid oxidation in foods but are suspected to be

responsible for liver damage and carcinogenesis (Ito et al, 1986; Safer and al-

Nughamish 1999). Recently, interest in finding naturally occurring antioxidants has

increased considerably to replace synthetic antioxidants. All these concerns

regarding the synthetic antioxidants, together with consumers’ preference for natural

food ingredients, have reinforced the current attention toward the development of

alternative natural antioxidants. The use of traditional medicine is widespread, and

plants still present a large source of natural antioxidants. Several medicinal plants

have been screened based on the integrative approaches on drug development from

Ayurveda (Mukherjee and Wahile, 2006). The ability of certain secondary

metabolites present in plants act as scavengers of free radicals, besides their

antioxidant and antimicrobial properties, is raising the possibility of their food and

pharmaceutical applications.

Table 1.1. Selected list of plants with antioxidant activity

SI. No.

Plant Part used Major active compounds

Reference

1. Allium sativum (garlic)

Underground stem

allicin, ajoene, selenium, quercetin

Meriga et al., 2012

2. Avena sativa (oat) endosperm phenolic acids Emmons et al., 1999

3. Cassia sieberiana root polyphenolic compounds

Nartey et al., 2012

4. Cornus capitat adventitious root

ellagic acid derivatives Tanaka et al., 2003


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Table 1.1. (Cont.) Selected list of plants with antioxidant activity

SI. No. Plant Part used Major active compounds Reference

5. Daucus carota (carrot)

root tuber carotenoids Mech-Nowak et al., 2012

6. Fagopyrum esculentum (buckwheat)

endosperm flavonoids Oomah and Mazza, 1996

7. Glycyrrhiza uralensis

licorice root glycyrrhizin Tanaka et al., 2008

8. Malus domestica (apple)

fruit quercetin, catechin, phloridzin, chlorogenic acid

Boyer and Liu, 2004

9. Momordica charantia (bitter melon)

fruit gallic acid, gentisic acid, catechin

Santos et al., 2010; Horaz et al., 2005

10. Moringa oleifera leaf polyphenols, anthocyanin, thiocarbamates

Luqman et al., 2012

11. Oryza sativa (rice)

endosperm quinolone alkaloid Chung and Woo, 2001

12. Piper nigrum (black pepper)

fruit piperine, arbutin, magnoflorine

Singh et al., 2008

13. Psidium guajava (common guava)

fruit carotenoids, polyphenols

Mai et al., 2007

14. Punica granatum (pomegranate)

fruit ellagic acid, ellagitannins, anthocyanins, punicic acid, flavonoids, anthocyanidins, flavones

Karasu et al., 2012

15. Rosmarinus officinalis (rosemary)

- carnosoic acid Kim et al., 2011

16. Rubus idaeus (raspberry)

fruit ellagic acid, phenolic compounds, vitamin C

Liu et al., 2002


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Table 1.1. (Cont.) Selected list of plants with antioxidant activity

SI. No.

Plant Part used Major active compounds

Reference

17. Salvia officinalis (sage)

- carnosol, carnosic acid, rosmanol, apigenin

Walch et al., 2011

18. Solanum lycopersicum (tomato)

fruit lycopene Palozza et al., 2012

19. Solanum tuberosum (potato)

potato peel phenolic compounds

Kanatt et al., 2005

20. Syzygium aromaticum (clove )

Flower bud eugenol, eugenyl acetate

Lee and Shibamoto, 2001

21. Thymus zygis (thyme)

- thymol, carvacrol, terpinene

Youdim et al., 2002

22. Vitis vinifera (grapes)

fruit resveratrol, anthocyanins, catechins

Bunea et al., 2012

23. Zingiber officinale (ginger)

Underground stem (rhizome)

quercetin, catechin, kaempferol

Rahman et al., 2011

1.2.4. Oxidative stress and liver damage

Liver cells possess endogenous antioxidant defense system consisting of

antioxidants such as GSH, GST, ascorbic acid, vitamin E and antioxidant enzymes

such as SOD, Catalase and GPx to protect own cells against oxidative stress, which

causes destruction of cell components and cell death.

The liver is a major target organ for toxicity of xenobiotics and drugs,

because most of the orally ingested chemicals and drugs first go to liver where they

are metabolized into toxic intermediates. A large number of xenobiotics are reported


18

to be potentially hepatotoxic (Ajith et al., 2007). It also handles the excretion of

drugs and other xenobiotics from the body thereby providing protection against

foreign substances by detoxifying and eliminating them (Saleem et al., 2010).

Hepatocytes, which make up the majority of the liver structure, are very active in the

metabolism of exogenous chemicals, and this is one of the major reasons why the

liver is a target for toxic substances (Timbrell, 2001). During the detoxification of

xenobiotics, reactive oxygen species (ROS) are generated which cause oxidative

stress (Kohen and Nyska, 2002) which leads to the hepatic damage.

1.2.5. Liver disorders caused by drugs and toxins

Liver disease is one of the major causes of morbidity and mortality in public,

affecting humans of all ages. About 20,000 deaths occur every year due to liver

disorders. Some of the commonly known disorders are viral hepatitis, alcohol liver

disease, non-alcoholic fatty liver disease, autoimmune liver disease, metabolic liver

disease, drug induced liver injury, liver fibrosis, cirrhosis, hepatocellular carcinoma

etc. According to WHO estimates, globally 170 million people are chronically

infected with hepatitis C alone and every year 3–4 millions are newly added into the

list.

Depending on the duration of the disease the liver diseases are classified as

acute or chronic. If the disease does not exceed to months it is considered as acute

liver disorder while diseases of longer duration are classified as chronic. Acute viral

hepatitis and drug reactions account for the majority of cases of acute liver disease.

Hepatitis A, B and E are the commonest causes of viral hepatitis. Hepatitis C is not

usually recognized as an acute infection because it rarely causes jaundice at this

stage.


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Drug/chemical-mediated hepatic injury is the common sign of drug toxicity

(Lee, 2003) and accounts for greater than 50% of acute liver failure cases. Hepatic

damage is the largest obstacle to the development of drugs and is the major reason

for withdrawal of drugs from the market (Cullen and Miller, 2006). Chronic liver

damage is a worldwide common pathology characterized by inflammation and

fibrosis that can lead to chronic hepatitis, cirrhosis and cancer (Tessitore and Bollito,

2006; Kohle et al., 2008). Chronic hepatitis or long term intoxification can severely

injure hepatic cells. Initially, the damaged cells are denatured, but subsequently

transformed to hypertrophic fibrosis and necrosis, and eventually may progress to

hepatoma. Hepatic fibrosis is usually initiated by hepatocyte damage. Biologic

factors such as hepatitis virus, bile duct obstruction, cholesterol overload,

schistosomiasis, etc; or chemical factors such as CCl4 administration, alcohol intake,

etc. were known to contribute to liver fibrosis. In many patients liver damage

become chronic and eventually progresses to more serious liver pathologies such as

fibrosis, cirrhosis or even carciongenesis, causing devastating economic losses and

mortality.

Liver toxicity mainly occurs due to drugs, alcohol, viruses and by chemicals.

The use of drugs like paracetamol and antibiotics cause acute liver damage. Some of

the drugs causing liver damage are listed below:

1.2.5.1. Non-steroidal anti-inflammatory drugs (NSAIDs)

Non-steroidal anti-inflammatory drugs (NSAIDs) are the centre piece of

pharmacotherapy for most rheumatologic disorders, and are used in large numbers

as analgesics and antipyretics, both as prescription drugs and over the counter

purchases. They are the most frequently used medications for the treatment of


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a variety of common chronic and acute inflammatory conditions (Manoukian and

Carson, 1996). Nearly all of the NSAIDs have been implicated in causing liver

injury (Rabinovitz and Van Thiel, 1992). Diclofenac and particularly sulindac, are

reported to be more commonly associated with hepatotoxicity (Bjorkman, 1998).

Several NSAIDs have been withdrawn from clinical use because of associated

hepatotoxicity (Rabkin et al., 1999). The new more selective COX-2 inhibitors (e.g.

celecoxib, rofecoxib, nimesulide) are also associated with hepatotoxicity (Merlani

et al., 2001). Hepatotoxicity from NSAIDs can occur at any time after drug

administration, but like most adverse drug reactions, most commonly occurs within

6–12 weeks of initiation of therapy (Aithal and Day, 1999).

There are two main clinical patterns of hepatotoxicity due to NSAIDs

(Rabinovitz and Van Thiel, 1992; Aithal and Day, 1999). The first is an acute

hepatitis with jaundice, fever, nausea, greatly elevated transaminases and sometimes

eosinophilia. The alternative pattern is with serological and histological (periportal

inflammation with plasma and lymphocyte infiltration and fibrosis extending into

the lobule) features of chronic active hepatitis. Some of the NSAIDs which cause

liver damage are listed below.

a. Diclofenac

Diclofenac sodium has antipyretic, analgesic and anti-inflammatory effects

but significant incidence of hepatotoxicity. In many cases clinical and biochemical

features of diclofenac hepatotoxicity suggest the involvement of reactive or toxic

metabolites. These products presumably were formed via the hepatic cytochrome

P450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that

are trapped by GSH (glutathione) conjugation. It is therefore possible that reactive


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benzoquinone imines may be formed and contribute to diclofenac mediated hepatic

injury (Tang et al., 1999).

b. Sulindac

Liver injury from sulindac appears within a few days to six weeks after

therapy is initiated. Fever, rash, eosinophilia, and edema are frequently found in

association with evidence of liver injury.

c. Nimesulide

It is an antiinflamatory drug and is almost exclusively metabolized and

cleared by the liver (Chatterjee and Sil, 2007). The drug can cause several types of

liver damage, ranging from mild abnormal function such as increase in serum amino

transferase activity to severe organ injuries such as hepatocellular necrosis or

intrahepatic cholestasis (Lucena et al., 2001).

d. Bromfenac

This acetic acid derivative was introduced in 1997 as non-narcotic for short

term pain relief, but was removed from the market in 1998 owing to several

instances of fulminant hepatic failure (FHF) leading to death or transplant that

occurred after prolonged administration (Goldkind and Laine, 2006).

e. Indomethacin

Indomethacin has produced hepatocellular necrosis, sometimes accompanied

by microvesicular steatosis and striking cholestasis (Fenech et al., 1967)

f. Ibuprofen

Ibuprofen was withdrawn from use in the 1960s because of fatal

hepatocellular injury.


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1.2.5.2. Acetaminophen

Acetaminophen, a widely used analgesic and antipyretic drug commonly

used for pain and fever relief (Whitcomb, 1994). It is commonly considered as a

“safe drug” when take within the suggested therapeutic dose. But in higher doses it

cause hepatotoxiciy in humans and experimental models. As the ingested dose of

acetaminophen is increased, hepatic glutathione stores become progressively depleted.

Consequently glutathione available for scavenging oxygen radicals are brought down,

resulting in an increase in reactive oxygen. This will be thus associated with

concurrent increase in lipid peroxidation and other hydroperoxides. When the

formation of N-acetyl-p-benzoquinoneimine (NAPQI) is of sufficient magnitude,

glutathione stores will fall below a critical level that is no longer adequate to sustain

detoxification of NAPQI. At this point, the disruption of cellular structure and

function occurs due to the covalent binding of NAPQI to cellular macromolecules

such as proteins and lipids thereby leading to hepatic necrosis (Dahlin et al., 1984).

1.2.5.3. Alcohol

Alcohol consumption causes accumulation of reactive oxygen species, which

in turn causes lipid peroxidation of cellular membranes, proteins and DNA oxidation

resulting in hepatocyte injury (Zhou et al., 2002). Alcohol treatment of rats is known

to cause the translocation of fat from the peripheral adipose tissue to liver, kidney

and brain for accumulation (Nadro et al., 2006).

1.2.5.4. Antibiotics

Tetracycline, Erythromycin, Nitrofurantoin, Ampicillin, Sulphonamides and

Lincomycin


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1.2.5.5. Anti-tubercular drugs

Para amino salicylate, Isoniazid, Rifampicin, Pyrazinamide, Ethionamide and

Ethanobutol

1.2.5.6. Anaesthetics

Halothane and chloroform

1.2.5.7. Anti-convulsants and anti-depressants

Phenobarbitone, Trimethadione, Tricyclic anti-depressants (eg. Amitrypyline),

Chloridiazepoxide, Monamine oxide inhibitors (eg. Iproniazid) and Phenothiazine.

Poisons affecting the liver are of three main classes, namely physical, biological

and chemical.

(i) Physical toxins

Hyperthermia, Burns and Irradiation

(ii)Biological toxins

Aflatoxin, Senecio alkaloid, and Amanita mushrooms.

(iii) Chemical toxins

Carbon tetrachloride, Tetrachloroethane, Chlorophenithone (DDT), Benzene

derivatives, Trinitrotoluene, Tannic acid, Phosphorus, Iron, Beryllium and Arsenic

1.2.6. Hepatic xenobiotic metabolism

On exposure to xenobiotics, the liver of vertebrates manages to eliminate

such foreign compounds as early as possible. This is accomplished by making use of

the normally existing biochemical mechanisms in the tissue. Certain enzymes and

other endogenous biomolecules which are actually meant for the metabolism of

endogenous substrates may be utilized for this purpose. Biotransformation of a


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xenobiotic compound following its exposure can alter its distribution and action

leading to its detoxification and excretion or enhance its toxicity due to the

activation of the compound (Athar et al., 1997).

Biotransformation of xenobiotics usually occurs in two phases.

Phase I: The main drug metabolizing system reside in the microsomal fraction of

the liver cell (smooth endoplasmic reticulum). The enzymes concerned are mixed

function monooxygenase, cytochrome c-reductase, and cytochrome P-450 (Mitchell

et al., 1973). NADPH in the cytosol is a cofactor. The drug is rendered more polar

by hydroxylation or oxidation. Alternative phase I drug metabolizing reactions

include the conversion of alcohol to aldehyde by alcohol dehydrogenase found

mainly in the cytosolic fraction.

Phase II: These biotransformations involve conjugation of the drug or drug

metabolite with a small endogenous molecule. The enzymes concerned are usually

not confined to the liver, but are present there in high concentration. An active

transport system is located at the biliary pole of the hepatocytes and this system

regulates the transport of drug molecules in and out of the hepatocytes.

1.2.7. Hepatotoxins and their effect

A number of pharmacological and chemical agents act as hepatotoxins and

produce a variety of liver ailments. They include industrial toxins, the heat-stable

toxic bicyclic octapeptides of certain species (Amanita and Galerina), chemicals and

various pharmacological agents. In general there are two major types of chemical

hepatotoxins, namely direct and indirect hepatotoxins. The most common direct

hepatotoxins are carbon tetrachloride, thioacetamide, paracetamol, galactosamine,


25

fulvine, phalloidin, ethyl alcohol, aflatoxins etc. Some examples of indirect

hepatotoxins are methyl testosterone, chlorpropamide, tetracycline, halothane,

phenytoin, methyldopa, amanita phalloides, acetaminophen, sulphonamides,

allopurinol, rifampicin etc. Thioacetamide, carbon tetrachloride and

N-nitrosodiethylamine were the hepatotoxins used in this study.

1.2.7.1. Thioacetamide

Thioacetamide is a compound endowed with liver damaging and

carcinogenic activity. Shortly after its administration thioacetamide is metabolized

to acetamide and thioacetamide-5-oxide. The latter binds to tissue macromolecules

responsible for the change in cell permeability, increased intracellular concentration

of Ca++, increase in nuclear volume and enlargement of nucleoli and inhibits

mitochondrial activity eventually leading to hepatic necrosis (Bautista et al 2010).

Thioacetamide on prolonged exposures causes cirrhosis (Bruck et al., 2001) by

inhibiting the respiratory metabolism of the liver through uncontrolled entry of Ca++

into hepatocytes. The final result is the inhibition of oxidative phosphorylation.

1.2.7.2. Carbon tetrachloride (CCl4)

Carbon tetrachloride (CCl4) has been one of the most intensively studied

hepatotoxicants to date and provides a relevant model for other halogenated

hydrocarbons that are used widely (Dahm et al., 1996; Weber et al., 2003). It

consistently produces liver injury in many species, including non-human primates

and man (Kumar et al., 1972). Acute poisoning with CCl4 becomes manifested as a

multisystem disorder, involving the liver, kidneys, brain, lungs, adrenal glands, and

the myocardium (Gitlin, 1996). CCl4 is a potent hepatotoxin and a single exposure

can rapidly lead to severe centrizonal necrosis and steatosis (Recknagel et al., 1973).


26

Mechanism of CCl4 toxicity

Carbon tetracholoride is lipophilic and because of this property CC14 is

absorbed from the skin and gastrointestinal tract as well as the lungs, although the

rate of absorption by the separate routes is different. Nowadays, CCl4 is used as a

classical hepatotoxicant for experimental liver functions.

CC14 is first metabolised by cytochrome P-450 in the liver endoplasmic

reticulum to the highly reactive 3CCl . radical (Recknagal, 1967). This free radical

may react again with oxygen to form trichloromethylperoxy radical 3(CCl OO). . The

free radicals thus formed can attack lipids on the membrane of endoplasmic

reticulum more readily than the 3CCl . free radical. The trichloromethylperoxy free

radical leads to elicit lipid peroxidation, the distruption of Ca2+ homeostasis and

finally results in cell death (Recknagal, 1983). As a consequence of this necrotic

behavior, leakage of large quantities of enzymes into the blood stream is often

associated with CCl4 toxicity.

1.2.7.3. N-nitrosodiethylamine (NDEA)

N-nitrosodiethylamine (NDEA) is a potent carcinogenic dialkylnitrosoamine

present in tobacco smoke, cheddar cheese, cured and fried meals and in a number of

alcoholic beverages. It is a hepatocarcinogen producing reproducible HCC after

repeated administration and is the most important environmental carcinogen among

N-niroso compounds (Singh et al., 2009). Administration of NDEA to animals

causes cancer in liver and at low incidence in other organs also. The formation of

reactive oxygen species (ROS) during the metabolism of NDEA may be one of the

key factors in the etiology of cancer (Bansal et al., 2005).


27

The mechanism of action is due to metabolism of NDEA to alkylating agents

and reactive oxygen species and further interaction with DNA molecule, forming

various DNA adducts that can lead to mutations (Jeena et al.,1999). The O4 – ethyl

deoxythymidine adduct (O4- Etdt) accumulates in hepatocyte DNA following NDEA

administration which is thought to be important in tumor initiation (Sivalokanathan

et al., 2006).

1.2.8. Types of hepatic diseases

Mainly there are four types of hepatic diseases:

1. Hepatitis (an inflammatory liver disease)

2. Hepatosis (non inflammatory disease)

3. Cirrhosis (a degenerative disease)

4. Hepatocellular carcinoma

1.2.9. Management of Hepatic diseases

Drugs that stimulates liver function offers protection to the liver from

damage or helps regeneration of hepatic cells. As it is the function of

hepatoprotective agents to interfere with these pathological processes by blocking

their evolution and helping recovery, the development of new antihepatotoxic drugs

is the need of the hour.

Since increase in the use of synthetic drugs in therapy leads to many side

effects and undesirable hazards, there is a worldwide trend to go back to natural

resources (mainly traditional plants) which is both culturally acceptable and

economically viable. Thousands of plants are used in this world to prevent or cure


28

diseases, but the biochemical basis of protective action which is necessary for the

rational development of safe and potent drugs, is lacking in most of the cases.

Ayurveda, an indigenous system of medicine in India, has a long tradition of

treating liver disorders with plant drugs. This ancient system of medicine makes use

of active principles present in plants for treating diseases. Medicinal herbs provide

protection against hepatotoxins in various ways: by enhancing the functioning of the

hepatic glutathione antioxidant system, inhibiting cytochrome P450, promoting

glucuronidation, stimulating hepatic regeneration, activating functions of reticulo-

endothelial systems, inhibiting biosynthesis of cytochrome P450 (Rao and Mishra,

1998) preventing lipid peroxidation, stabilizing hepatocellular membrane, enhancing

protein biosynthesis (Lin et al., 1997); accelerating the regeneration of parenchymal

cells and thus protecting against membrane fragility, decreasing the leakage of

marker enzymes into the circulation, interfering with the microsomal activation of

CC14 and/or accelerating detoxification (Bishayee et al., 1995); counteracting the

hepatic lysosomal enzymes (Saxena et al., 1993).

1.2.9.1. Drugs for liver diseases

Conventional and synthetic drugs used in the treatment of liver diseases are

sometimes inadequate and can have serious adverse effects. Steroids, vaccines, and

antiviral drugs, have been used as therapies for liver pathologies, have potential

adverse side-effects, especially if administered chronically or sub-chronically.

Current medical treatments for these liver diseases are often ineffective, and

therefore efforts are being made to seek new effective medications (Seeff et al.,

2001). Developing pharmacologically effective agents from natural products has


29

become a new trend by virtue of their little toxicity or few side effects. There are

few plant derived drugs in the market which are used for the liver disorders.

(a) Silymarin

Silymarin, derived from the seeds of Silybum marianum L. (Family:

Asteraceae or Compositae), commonly known as milk thistle, has been used for

centuries as a natural remedy for liver and biliary tract diseases (Saller et al., 2001).

Milk thistle protects and regenerates the liver in most liver diseases such as

cirrhosis, jaundice, and hepatitis (Flora et al., 1998). Silymarin offers good

protection in various models of experimental liver disease. It has antioxidative,

antilipid peroxidative (Pascual et al., 1993), antifibrotic (Mourelle et al., 1989),

membrane stabilizing, immunomodulatory and liver regenerating mechanisms

(Pradhan and Girish, 2006).

Limitations

Silymarin is insoluble in water and typically administered as a sugar coated

tablet (Thakur, 2002) or as an encapsulated standardized extract. The absorption by

oral route is as low as 2-3 percent of the silybin recovered from rat bile in 24 h.

About 20- 40 percent of the administered dose of silymarin is excreted in bile as

sulphates and glucuronide conjugates in human beings (Saller et al., 2001).

Side Effects

Silymarin has low level of toxicity. Although, silymarin has a good safety

record, there are few reports of gastrointestinal disturbances and allergic skin rashes

(Negi et al., 2008).


30

(b) Liv – 52

Liv-52 was introduced in 1954 as a specially formulated Ayurvedic herbal

remedy for the treatment of viral hepatitis and has been widely prescribed for

infective hepatitis since then (Mukerjee and Dasgupta, 1970). Experimentally, Liv-

52 prevented injurious effects of carbon tetrachloride and other toxic substances on

the liver.

Liv.52 is available as tablets and syrup containing the following herbs:

Capparis spinosa; Cichorium intybus; Solanum nigrum; Terminalia arjuna; Cassia

occidentalis, Achillea millefolium; Tamarix galica and Phyllanthus amarus. These

herbs are processed and formulated according to the principles of Ayurveda, which

are aimed at enhancing efficacy and avoiding toxicity (Charak and vimanasthan,

1981).

1.2.9.2. Herbal medicine

India is a rich source of medicinal plants and a number of plant derived

extracts are used against diseases in various systems of medicine such as Ayurveda,

Unani and Siddha. Use of herbal medicines can be traced back as far as 2100 B.C. in

ancient China (Xia dynasty) and India (Vedic period). The first written reports date

back to 600 B.C. with the Charaka Samhita of India and the early notes of the

Eastern Zhou dynasty of China that became systematized around 400 B.C. The

recipes, once formulated, were usually expanded rather than abandoned during

subsequent centuries.

The use of medicinal plants in curing diseases is as old as man. The World

Health organization (WHO) has long recognized and drawn the attention of many


31

countries to the ever increasing interest of the public in the use of medicinal plants

and their products in the treatment of various ailments. It is estimated that 70-80% of

people worldwide rely on traditional herbal medicine to meet their primary health

care needs (Uprety et al., 2012). The use of herbal medicines presents unique clinical

and pharmacological challenges that are not encountered with conventional single-

compound medicines. These medicines are usually complex mixtures of many

bioactive compounds and conventional “indications and uses” criteria devised for

single compound entities may not be applicable to this system in a significant number

of ways. Compared to single-agent pharmaceuticals, phytomedicines may differ in the

different mechanisms of action of bioactive constituents, in their dose-response

relationships, and in the synergistic/combinatorial effects of the many bioactive

compounds found in herbal extracts” (Yong et al., 2004).

India has been identified as one of the top twelve mega bio-diversity center

of the world. This is because India has a vast area with wide variation in climate,

soil, altitude and latitude (Tiwari, 2008). India is rich in all the three levels of

biodiversity, namely species diversity, genetic diversity and habitat diversity. In

India thousands of species are known to have medicinal value and the use of

different parts of several medicinal plants to cure specific ailments has been in

vogue since ancient times (Parekh et al., 2005). India with its biggest repository of

medicinal plants in the world may maintain an important position in the production

of raw materials either directly for crude drugs or as the bioactive compounds in the

formulation of pharmaceuticals and cosmetics etc (Tiwari, 2008). Extraction of

bioactive compounds from medicinal plants permits the demonstration of their


32

physiological activity. It also facilitates pharmacology studies leading to synthesis of

a more potent drug with reduced toxicity.

Plant derived natural products such as flavonoids, terpenes and alkaloids

(Shukla et al., 2010) have received considerable attention due to their diverse

pharmacological properties including inflammatory, antipyretic and analgesic

activities. Consumption of natural products reduce the risk of developing

pathological conditions, including cancer, nervous system disorders, hepatic

damage, cardiovascular, genetic, and inflammatory diseases (Newman and Cragg,

2007; Jurenka, 2009). Plants contain numerous bioactive molecules that can improve

the body’s resistance to cellular stress and prevent the cytotoxicity of various agents.

Many of the active ingredients for health care products are directly or indirectly

derived from plants (Newman et al., 2000). However, many high value plant-derived

natural products remain undiscovered or unexplored for their pharmacological

activity (Raskin et al., 2002).

1.2.10. Hepatoprotective Study

The Ayurveda has a long tradition of treating liver diseases using herbal

medicines, and the control of liver diseases has become a major goal of modern

medicine. As it is the function of hepatoprotective agents to interfere with these

pathological processes by blocking their evolution and helping recovery, the

development of new antihepatotoxic drugs is the need of the hour. Traditional

medicine all over the world is nowadays being re-evaluated by extensive research on

different plant species with reference to their therapeutic principles. The 21st century

has seen a paradigm shift towards therapeutic evaluation of herbal products in liver

disease models by carefully synergizing the strength of the traditional systems of


33

medicine with that of the modern concept of evidence based medicinal evaluation,

standardization and randomized placebo controlled clinical trials to support clinical

efficacy (Thyagarajan et al., 2002).

A large number of compounds are capable of causing liver injury. To

evaluate the hepatoprotective potential of medicinal plants in animal models some

chemical compounds like acetaminophen, carbon tetrachloride, galactosamine and

thioacetamide are used as a toxicants to induce liver damage (Subramoniam and

Pushpangadan, 1999).

Hepatotoxins are of two major groups namely direct and indirect hepatotoxins.

Direct hepatotoxic agents damage the membrane of hepatocytes directly resulting

interference in cell metabolism. The indirect hepatotoxins cause hepatic injury as a

result of selective interference with metabolic pathways or selective binding to or

alteration of a specific component. The degree of injury varies from alteration of

only one metabolic function without structural change. The liver injury may

progress to chronic liver disease, fulminant hepatic failure, cirrhosis or malignancy

in due course.

Table 1.2. List of hepatoprotective medicinal plants

Sl.No Plant (family) Part used Solvent used

Hepatotoxicant used References

1. Acalypha racemosa Wall. (Euphorbiaceae)

Leaves Methanol CCl4 Iniaghe et al., 2008

2. Actinidia deliciosa Chev. Actinidiaceae)

Roots Ethanol CCl4 Bai et al., 2007

3. Adhatoda vasica Nees (Acanthaceae)

Leaves Water Galactosamine Bhattacharyya et al., 2005


34

Table 1.2. (Cont.) List of hepatoprotective medicinal plants



4. Aloe barbadensis

Mill. (Liliaceae)

Aerial parts Water CCl4 Chandan et al., 2007

5. Andrographis lineate Fam. (Acanthaceae)

Leaves Methanol, Water

CCl4 Sangameswaran

et al., 2008

6. Anoectochilus formosanus Hayata. (Orchidaceae)

Whole plant Water CCl4 Fang et al.,

2008

7. Apium graveolens Linn. (Apiaceae) Croton oblongifolius Roxb. (Euphorbiaceae)

Seeds Petroleum ether, Acetone, Methanol

CCl4 Ahmed et al.,

2002

8. Artemisia absinthium L. (Asteraceae)

Aerial part Water CCl4 Amat et al., 2010

9. Azadirachta indica Juss. (Meliaceae)

Leaves Fresh juice

Acetaminophen Yanpallewar

et al., 2002

10. Bauhinia variegate L. (Leguminosae)

Stem bark Alcohol CCl4 Bodakhe and

Ram, 2007

11. Berberis tinctoria Lisch. (Berberidaceae)

Leaves Methanol Acetaminophen Murugesh et al., 2005

12. Boerhaavia diffusa Linn. (Nynctaginaceae)

Leaves Ethanol Acetaminophen Olaleye et al.,

2010

13. Boswellia serrata Roxb. (Burseraceae)

Oleo gum resin

n- Hexane CCl4,

Thioacetamide Jyothi et al., 2006

14. Camellia sinensis

Linn. (Theaceae)

Leaves Water Sodium oxalate Oyejide and

Olushola, 2005

15. Cassia tora L.

(Caesalpiniaceae)

Ononitol

Monohydrate from Leaves

- CCl4 Dhanasekaran

et al., 2009


35




16. Cleome viscosa Linn.(Capparidaceae)

Leaves Ethanol Thioacetamide Gupta and Dixit, 2009

17. Cistus laurifolius

L. (Cistaceae)

Isolation of flavonoid from Leaves

Ethanol Acetaminophen Kupeli et al.,

2006

18. Citrus limon L.

Burm. (Rutaceae)

Fruits 70%

Ethanol

CCl4 Bhavsar et al.,

2007

19. Curculigo orchioides Gaertn. (Amaryllidaceae)

Rhizome methanol CCl4 Venukumar and Latha, 2002

20. Curcuma longa Linn. (Zingiberaceae)

Rhizome Ethanol,

Water

Diclofenac Hamza, 2007

21. Cuscuta chinensis Lam. (Convolvulaceae)

Seeds Ethanol,

Water

Acetaminophen Yen et al.,

2007

22. Cytisus scoparius L. (Leguminosae)

Aerial part Ethanol: Water (7:3)

CCl4 Raja et al., 2007

23. Decalepis

hamiltonii Wight.

(Asclepiadaceae)

Root Water CCl4 Srivastava and

Shivanandappa,

2010

24. Diospyros

malabarica Kostel.

(Ebenaceae)

Bark Methanol CCl4 Mondal et al.,

2005

25. Enicostemma

axillare Raynal.

(Gentianaceae)

Swertiamarin

from

Whole plant

Ethyl

acetate

D-galactosamine Jaishree and

Badami, 2010

26. Epaltes divaricata

L. (Compositae)

Whole plant Water CCl4 Hewawasam

et al., 2004


36




27. Euphorbia

fusiformis D.Don.

(Euphorbiaceae)

Tubers Ethanol Rifampicin Anusuya et al., 2010

28. Ginkgo biloba Linn. (Ginkgoaceae)

Leaves Water CCl4 Shenoy et al., 2001

29. Glycyrrhiza glabra

L. (Leguminosae)

Glycyrrhizin

from Root

Methanol CCl4 Lee et al.,

2007

30. Halenia elliptica

(Gentianaceae)

Whole plant 70%

Methanol

CCl4 Huang et al.,

2010a

31. Hibiscus sabdariffa

L. (Malvaceae)

Flowers

Water Azathioprine Amin and

Hamza, 2005

32 Rosmarinus

officinalis L.

(Lamiaceae)

Leaves

Water Azathioprine Amin and

Hamza, 2005

33 Salvia officinalis

L. (Lamiaceae)

Leaves Water Azathioprine Amin and

Hamza, 2005

34. Hybrophila

auriculata Heine.

(Acanthaceae)

Root Water CCl4 Shanmugasund

aram and

Venkataraman,

2006

35. Indigofera pinnatifida Cass. (Asteraceae)

Leaves and roots

Chloroform

Methanol

Paracetamol Kumar et al., 2008

36. Justicia simplex D.

Don. (Acanthaceae)

Whole plant

(Isolated

lignans)

Petroleum

ether

CCl4 Jasemine et al., 2007

37. Kyllinga nemoralis

L. (Cyperaceae)

Rhizome Petroleum ether, Ethanol

CCl4 Somasundaram

et al., 2010


37




38. Ligustrum

robustum Roxb.

(Oleaceae)

Leaves Water CCl4 Lau et al.,

2002

39. Lygodium

flexuosum (L.) Sw.

(Lygodiaceae)

Whole plant Hexane CCl4 Wills and

Asha, 2006a

40. Moringa oleifera

Lam. (Moringeaceae)

Seed 70%

Ethanol

CCl4 Hamza, 2010

41. Nelumbo nucifera

Gaertn.

(Nelumbonaceae)

Leaves 60%

Ethanol

CCl4 Huang et al.,

2010b

42. Operculina

turpethum L.

(Convolvulaceae)

Root Ethanol Acetaminophen Suresh kumar

et al., 2006

43. Phyllanthus niruri

L.(Euphorbiaceae)

Leaves,

Stem

PO4 buffer

Nimesulide Chatterjee and

Sil, 2007

44. Phyllanthus

urinaria L.

(Euphorbiaceae)

Whole plant 80%

Ethanol

Acetaminophen Hau et al.,

2009

45. Physalis peruviana

L. (Solanaceae)

Whole plant Water Acetaminophen Chang et al.,

2008

46. Polyalthia longifolia

var. pendula.

(Annonaceae)

Leaves Methanol Diclofenac Tanna et al.,

2009

47. Punica granatum

Linn. (Punicaceae)

Water CCl4 Celik et al.,

2009


38


Sl.No Plant (Family) Part used Solvent used


48. Rhoicissus tridentata

Wild. (Vitaceae)

Root Water CCl4 Opoku et al.,

2007

49. Sida acuta Burm. f.

(Malvaceae)

Root Methanol Acetaminophen Sreedevi et al., 2009

50. Syzygium cumini

L. (Myrtaceae)

Leaves Water CCl4 Moresco et al., 2007

51. Terminalia arjuna

Bedd.

(Combretaceae)

Bark Water CCl4 Manna et al.,

2006

52. Terminalia belerica Roxb. (Combretaceae)

Fruit Ethanol CCl4 Jodan et al. 2007

53. Terminalia

catappa L.

(Combretaceae)

Leaves Ethanol CCl4 Gao et al.,

2004

54. Vernonia

amygdalina Delile.

(Astereaceae)

Leaves 90%

Methanol

CCl4 Adesanoye

and Farombi,

2010

55. Vitis vinifera L.

(Vitaceae)

Leaves 80%

Ethanol

CCl4 Orhan et al.,

2007

56. Zanthoxylum

armatum DC.

(Rutaceae)

Bark 70%

Ethanol

CCl4 Ranawat et al., 2010

57. Zingiber officinale

Roscoe.

(Zingiberaceae)

Rhizome 50%

Ethanol

Acetaminophen Ajith et al.,

2007


39

1.2.11. Hepatic Fibrosis and Cirrhosis

Hepatic fibrosis is a precursor of cirrhosis, developing in response to chronic

hepatocellular injury show general features of a wound repair process characterized by

specific cellular reactions. Hepatic fibrosis is usually initiated by hepatocyte damage.

Biologic factors such as hepatitis virus, bile duct obstruction, cholesterol overload,

schistosomiasis, etc; or chemical factors such as CCl4 administration, alcohol intake,

etc. were known to contribute to liver fibrosis. Hepatic fibrosis is major features of a

wide range of chronic liver injuries including metabolic, viral, cholestatic and

genetic disease. The failure of bile salt excretion in cholestasis leads to retention of

hydrophobic bile salts within the hepatocytes and causes apoptosis and/or necrosis

(Miyoshi et al., 1999).

Hepatic fibrosis is characterized by the excessive deposition of extracellular

matrix (ECM) proteins including collagen, fibronectin, laminin and proteoglycans

(Wills and Asha, 2006b). Activated hepatic stellate cells, portal fibroblasts and

myofibroblasts of bone marrow origin have been identified as major collagen-

producing cells in the injured liver. Excess depositions of ECM proteins disrupt the

normal functioning of the liver, ultimately leading to patho-physiological damage to

the organ, which has high mortality rate (Wills and Asha, 2007). Reactive oxygen

free radicals have been known to produce tissue injury through covalent binding and

lipid peroxidation and have been shown to augment fibrosis as seen from increased

collagen synthesis (Geesin et al., 1990). As these processes continue, accompanying

fibrosis interfere with blood flow through the liver resulting in severe

pathophysiological consequences such as portal hypertension, hepatic insufficiency,

jaundice and ascites.


40

Fig. 1.5. Schematic representation of the development of hepatic fibrosis


41

1.2.11.1. Pathogenesis of hepatic fibrosis

Hepatic fibrosis is the result of the wound-healing response of the liver to

repeated injury.

Fig. 1.6. Changes in hepatic architecture associated with advanced hepatic fibrosis

After an acute liver injury (e.g., viral hepatitis), parenchymal cells regenerate

and replace the necrotic or apoptotic cells. This process is associated with an

inflammatory response and a limited deposition of ECM. If the hepatic injury

persists, then eventually the liver regeneration fails, and hepatocytes are substituted

with abundant ECM, including fibrillar collagen. In chronic liver injury,

inflammatory lymphocytes infiltrate the hepatic parenchyma. Some hepatocytes

undergo apoptosis, and Kupffer cells activate, releasing fibrogenic mediators. HSCs

proliferate and undergo a dramatic phenotypical activation, secreting large amounts

of extracellular matrix proteins. The distribution of this fibrous material depends on


42

the origin of the liver injury. In chronic viral hepatitis and chronic cholestatic

disorders, the fibrotic tissue is initially located around portal tracts, while in alcohol-

induced liver disease; it locates in pericentral and perisinusoidal areas (Pinzani,

1999). Sinusoidal endothelial cells lose their fenestrations, and the tonic contraction

of HSCs causes increased resistance to blood flow in the hepatic sinusoid. As

fibrotic liver diseases advance, disease progression from collagen bands to bridging

fibrosis to frank cirrhosis occurs.

Liver fibrosis is associated with major alterations in both the quantity and

composition of ECM. In advanced stages, the liver contains approximately 6 times

more ECM than normal, including collagens (I, III, and IV), fibronectin, undulin,

elastin, laminin, hyaluronan, and proteoglycans. HSCs are the main ECM-producing

cells in the injured liver (Gabele, 2003). In the normal liver, HSCs reside in the

space of Disse and are the major storage sites of vitamin A. Following chronic

injury, HSCs activate or transdifferentiate into myofibroblast-like cells. Activated

HSCs migrate and accumulate at the sites of tissue repair, secreting large amounts of

ECM and regulating ECM degradation. PDGF, mainly produced by kupffer cells, is

the predominant mitogen for activated HSCs. Collagen synthesis in HSCs is

regulated at the transcriptional and post-transcriptional levels (Lindquist, 2000).

Although fibrosis and cirrhosis are of high incidence worldwide, therapeutic

management of these diseases still remains insufficient. These therapeutic concepts

focus mainly on symptoms rather than on blocking central fibrogenic mechanisms.

Progress in the understanding of the pathological mechanisms may open new

strategies with which to interfere, at early steps, in the development of these diseases


43

(Gebhardt, 2002; Tsukada et al., 2006). Detection of the expression of liver cytokines

is useful in exploring the probable mechanisms of anti-fibrotic drugs.

Fig.1.7. Cellular mechanism of hepatic fibrosis

The CCl4-treated rat is frequently used as an experimental model to study

hepatic fibrosis (Oh et al., 2003; Inao et al., 2004). Reversibility of advanced liver

fibrosis in patients has been recently documented, which has stimulated to develop

antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the

accumulation of fibrogenic cells and/or preventing the deposition of extracellular

matrix proteins in the liver (Bataller and Brenner, 2005).

Several herbal drugs have been investigated for their antifibrotic effects on

chemically induced hepatic fibrosis in rats. Some of them are listed in the Table 1.4.

Traditional plant drugs have been found to be effective in preventing fibrogenesis

and other chronic liver injury which develops a more hopeful future in controlling

liver fibrosis, cirrhosis and hepatocarcinogenesis (Lee et al., 2003; Yao et al., 2005).


44

Table 1.3. List of medicinal plants with antifibrotic activity

Sl. No Plant Part used/compound

References

1. Artemisia capillaries Entire plant Wang et al., 2012

2. Artemisia iwayomogi Entire plant Wang et al., 2012

3. Curcuma longa Curcumin Bruck et al., 2007

4. Ginkgo biloba - Luo et al., 2004

5. Lygodium flexuosam entire plant Wills and Asha, 2006b

6. Regimen (combination of Salvia miltiorrhiza, Ligusticum chuanxiong, Glycyrrhiza glabra

Entire plant Lin et al., 2008

7. Rheum palmantum L Emodin Hu et al., 2009

8. Rheum officinale Rhein Guo et al., 2002

9. Rhus verniciflua Butein Lee et al., 2003

10. Salvia miltiorrhiza Bge. Salvianolic acid B

Hu et al., 2009; Hsu et al., 2005

11. Scutellaria baicalensis Georgi

Baicalin Hu et al., 2009

12. Tinospora crispa stem Kadir et al., 2011

1.2.12. Cancer

Cancer is a class of disease or disorder characterized by uncontrolled

division of cells and has the ability to invade other tissues, either by direct growth

into adjacent tissue through invasion or by implantation into distant sites by

metastasis. If the spread is not controlled, it can result in death (American Cancer

Society, 2011). Occasionaly, dividing and differentiating cells deviate from their

normal genetic program and give rise to tissues called tumours or neoplasm. This


45

process by which a cell loses its ability to remain constrained in its growth

properties is called transformation. If the transformed cells stay together in a single

mass, the tumour is said to be benign and the cells of a tumour can invade and

disrupt surrounding tissues, the tumour is said to be malignant and is identified as a

cancer. Cells from malignant tumours can break off and move through the blood

and lymphatic system, forming new tumours at other locations in the body.

Malignancy can result in death due to damage to critical organs, starvation,

secondary infection, metabolic problems or haemorrhage (Karp, 1996). Metastasis is

defined as the stage in which cancer cells are transported through the blood or

lymphatic system. It is the sum of all processes which transform normal healthy

alive cells into abnormal damaged denatured cells. Genetic alteration is the

fundamental underlying process that allows a normal cell to evolve into cancerous

one. Critical events in the evolution of the neoplastic disease include the loss of

proliferative control, the failure to undergo programmed cell death (apoptosis), the

onset of neoangiogenesis, tissue remodeling, invasion of tumour cells into

surrounding tissue and finally metastatic distribution of tumour cells to distant

organs (Herzig and Christofori, 2002).

1.2.12.1. Etiology of cancer

The causes of cancer have been determined to be the result of genetic

predisposition, environmental exposure, infection by suitable agent or a combination

of these. The agents which cause cancer are called carcinogens. Carcinogenesis in

multicellular organism can result from anyone or a combination of genetic (DNA

damage or gene mutation), chemical (drugs, tobacco, alcohol, diet etc.), physical

(radiation- X rays, UV rays) and/or biological (infections due to DNA and RNA


46

viruses) insults to cells. These obviously are implicated as causal agents of

mammalian cancers (Sakarkar and Deshmukh, 2011).

(a). Genetic factors

The majority of recognized carcinogens cause genetic mutations. Changes in

gene expression in somatic cells, mostly due to mutation, are thought to be the basis

for malignant transformation; there may be one or more, rare, dominantly inherited

susceptibilities to every type of cancer. The contribution made by these highly

penetrant, dominant susceptibilities to the total incidence of cancer has been

estimated at 2–5% of fatal cancers. Genetic variation in susceptibility to cancer may

also arise because of genetic polymorphism affecting the absorption, transport,

metabolic activation, or detoxification of environmental carcinogens.

(b). Tobacco

Tobacco smoking is the largest single avoidable cause of premature death

and the most important known carcinogen. Based on proportions of cancers of lung,

larynx, oral cavity and pharynx, oesophagus, pancreas, kidney, and bladder due to

smoking, 15% (1.1 million new cases per year) of all cancer cases worldwide are

attributed to smoking (25% of cases worldwide in men, 4% in women).

(c). Alcohol

Free radicals generated as a result of the metabolism of alcohol are shown to

be responsible for augmentation of hepatic lipid peroxidation and ethanol mediated

liver carcinogenesis. The main effect of alcohol is a joint effect with tobacco

smoking in cancers of the oral cavity, pharynx, larynx, and oesophagus.


47

(d). Diet

High intake of vegetables and fruit show a consistent inverse relationship

with cancer of the larynx, lung, oesophagus, and stomach, and there is weaker

evidence that this is the case also for cancer of the mouth and pharynx, pancreas, and

colon. Low levels of consumption of fruit and vegetables, high levels of meat

consumption, obesity, and lack of regular physical activity tend to be aspects of a

lifestyle more typical of developed than of developing countries. Nowadays majority

of our vegetables, fruits and rice are contaminated with pesticides and toxic

chemicals. All these orally ingested chemicals first go to liver because liver is the

major target organ for toxicity of xenobiotics. A large number of xenobiotics are

reported to be potentially hepatotoxic, this increases the risk of liver cancer.

(e). Drugs

Intake of acetaminophen like drugs and certain chemicals may also lead to

hepatocellular carcinoma. Long term use of analgesics and antipyretics cause hepatic

injury and on prolonged conditions it leads to cancer.

(f). Infections

Viruses may be the cause of at least 15% of all human cancers. Human

papillomavirus (HPV) of any type accounts for 82% of cervical cancers in

developed countries and 91% in developing countries. The human papilloma viruses

occur in 70 different types. The strongest evidence for carcinogenicity is for

HPV types 16 and 18. 81% of cases of liver cancer are attributable to chronic

infection with hepatitis B or hepatitis C.


48

Strong evidence supports a causal relationship between chronic infection

with the bacterium Helicobacter pylori and the development of gastric

adenocarcinoma, and there is some evidence for gastric lymphoma. 60% of cases of

gastric cancer in developed countries, and 53% in developing countries, may be

attributable to Helicobacter pylori.

(f). Environmental factors

The incidence of many types of cancer varies greatly between geographical

areas. There are changes of rates following migration between areas of contrasting

incidence, changes in incidence over time, and variation within populations

according to socio-economic status. Thus environmental factors appear to have a

major role in the aetiology of most types of cancer, accounting for over 80% of

human cancer.

(g). Solar exposure

The 1996 Harvard Report on Cancer Prevention concluded that over 90% of

malignant melanoma is attributable to solar radiation. Malignant melanoma

accounted for just over 1% of the world cancer burden in 1985. Uncertainties

remain, even though it is widely assumed that exposure to solar radiation also

accounts for the great majority of cases of basal cell and squamous cell carcinoma.

(h). Other exposures

Other exposures account for 5% or less of the cancer burden. Occupational

exposures have been linked with lung, bladder, and haematopoietic malignancies.

Breast cancer has consistently been associated with early age at menarche, late age


49

at first birth, and late age at menopause with relative risks of the order of 2.0 or less.

Parity is associated inversely with endometrial and ovarian cancer.

Although most types of cancer are more common in urban than in rural

areas, few causal links with environmental pollutants have been firmly established.

It has been estimated that 1% of lung cancer deaths in the US are attributable to air

pollution. While exposure to ionizing radiation at doses of 500–2000 mSv is known

to be carcinogenic, exposures of this magnitude are unusual—about 1% of the

deaths of the Japanese atomic bomb survivors could be attributed to radiation. The

average per capita dose from all sources of ionizing radiation is about 3.4 mSv per

year, of which about 88% is from natural sources and the remainder primarily from

medical exposures. Extrapolation from data on people exposed to doses of 500 mSv

or more suggests that 1–3% of all cancers might be attributable to radiation arising

largely from natural sources. No clear association with exposure to extremely low

frequency magnetic fields has been established.

Some pharmaceutical agents (e.g. immunosuppressive agents, anti-neoplastic

drugs, and hormonal preparations) are human carcinogens.

1.2.12.2. Stages of Carcinogenesis

The process of carcinogenesis may be divided into at least three stages:

initiation, promotion and progression. Cancer development is now commonly

recognized as a microevolutionary process that requires the cumulative

accumulation of multiple events. These events may occur in a single cell clone and

can be explained by a simplified three stage model. These stages include initiation of

DNA mutation in a somatic cell is known as initiation, stimulation of initiated cell


50

and its clonal expression referred to as promotion and conversion of benign tumor

into malignant termed as progression (Athar, 2002). The initiation phase appears to

be irreversible and relatively easily induced by DNA damaging agents, mutagenesis

has been implied underlying mechanism responsible for this step. The promotion

phase of carcinogenesis, operationally is an interruptible process (and reversible up

to certain point). This implies that the initiated cell can be stimulated to proliferate

but will not terminally differentiate. The promotion process can be implied to be an

epigenetic process. Mitogenesis, rather than mutagenesis, best describe the

promotion process.

Fig. 1.8. Stages of carcinogenesis and the occurrences involved in each one

1.2.12.3 Cancer prevention and treatment

Cancers due to use of tobacco, alcohol, exogenous hormones and exposure of

environmental carcinogen can be effectively prevented through education and social

policies that discourage unhealthy practices. Certain cancers that are related to

infectious agents such hepatitis B (HBV), human immunodeficiency virus (HIV),


51

human papilloma virus (HPV) and Helicobacter pylori could be prevented through

known interventions such as vaccines, antibiotics, improved sanitation and

education. Through regular screening and examination many cancers can be

diagnosed and easily cured at the early stages of their development.

Several methods exist for treatment of cancer in modern medicine, which

include chemotherapy, radiotherapy, surgery, hormonal therapy and immunotherapy.

Selective killing or removal of the cancer cells without affecting the rest of the body

is the goal of cancer treatment.

(a). Chemotherapy

Chemotherapy remains as the treatment choice for most advanced cancers or

as an adjunct to other treatment modalities like surgery and radiotherapy, but severe

toxic effects towards normal tissues also limit its use (Baxevanis et al., 2009).

It involves the use of cytotoxic agents which are transported by the bloodstream to

different parts of the body to destroy cancer cells. The first uses of chemotherapy to

control cancer were reported in the 1940s, and in the decades since, treatment of

patients with broadly toxic chemicals have represented a mainstay of medical

oncology, in spite of the frequent severe side effects associated with such treatments.

Many chemotherapeutic agents used to treat malignant diseases damage

lymphocytes and consequently suppress cell-mediated immunity (Bagnyukova et al.,

2010).

(b). Radiotherapy

Radiotherapy is effectively used to reduce the initial tumour load. It involves

the use of ionizing radiation to kill cancer cells and shrink tumours and also


52

developing as a clinically essential part of cancer therapy for the majority of solid

malignant neoplasm including brain tumours. It has been proved to be a fundamental

tool available in the battlefield against cancer, offering a clear survival benefit in

most cases. However, numerous studies have associated tumour irradiation with

enhanced aggressive phenotype of the remaining cancer cells. A cell population

manages to survive after the exposure, because it either receives sub lethal doses or

it successfully utilizes the repair mechanisms. The biology of irradiated cells is

altered leading to up-regulation of genes that favor cell survival, invasion and

angiogenesis (Kargiotis et al., 2010).

(c). Surgery

Surgery is the oldest treatment of cancer. Surgical interventions can be used

for diagnosis, treatment of precancerous lesions or for removal of normal organs

which are at an elevated risk of developing cancers. As long as the growth of the

tumour remains localized, it can usually be treated and cured by surgical removal of

the tumour and surrounding tissue, but the tendency of cancers to invade adjacent

tissue or to spread to distant sites by metastasis makes complete surgical excision of

the cancer usually impossible (Ozgediz et al., 2008).

(d). Immunotherapy

Historically, the first successful immunotherapy to treat cancer involved the

use of toxins from Streptococcus erysipelatis and Bacillus prodigious by William

Coley in the 1890's (Coley, 1991). Toll-like receptor agonists have been shown to

boost immune responses toward tumours. Also, a wide array of cell based immune

therapies utilizing T cells, NK cells and DC cells have been established.

Furthermore, a rapidly expanding repertoire of monoclonal antibodies is being


53

developed to treat tumours, and many of the available antibodies have demonstrated

impressive clinical responses (Borghaei et al., 2009). More recently, the

development of vaccines to tumour-causing hepatitis B virus and papilloma virus are

contributing significantly to prevent cancer in a large portion of the human

population (Blumberg, 1997; Rogers et al., 2008). In addition to vaccines interferon

and interleukin 2 are also used in immunotherapy.

(e). Hormonal therapy

Hormonal therapy is also used for certain type of cancers. Hormones

commonly used in the cancer therapy are steroids, anti-estrogens, anti-androgens,

LH-RH analogues and anti-aromatase agents.

(f). Complementary and Alternative Medicine

Recently, complementary and alternative medicine (CAM) is becoming a

popular treatment for various cancers. Among the CAMs, herbal medicine is one of

the methods used in cancer therapy (Cassileth, 1999). CAM has been defined as a

group of diverse medical and healthcare systems, practices and products that are not

presently considered to be part of conventional medicine. In the last three decades,

the use of CAM has increased in popularity in both the worldwide general

population and in patients with cancer (Yildirim, 2010). The goals of CAM are to

increase the efficacy of conventional cancer treatment programs, reduce symptoms,

and improve quality of life for patients with cancer (Levine, 2010).

(g). Natural products

The natural products have afforded a rich source of compounds that have

found many applications in the fields of medicine, pharmacy and biology and are


54

being used to treat a wide variety of clinical conditions, with relatively little

knowledge of their modes of action. Within the sphere of cancer, a number of

important new commercialized drugs have been obtained from natural sources, by

structural modification of natural compounds, or by the synthesis of new compounds

and by designing as natural compound as model (Gordaliza, 2007). Currently,

numerous scientific studies support herbal medicine as a potent anticancer drug.

However, herbal remedies are yet to be integrated into main stream medicine mainly

due to lack of experimental and clinical studies on their safety, efficacy, quality

control and pharmacological mechanisms. Careful in vitro and in vivo studies will be

essential and necessary to evaluate their efficacy and safety before clinical trials can

be contemplated (Buchanan et al., 2005; Kwon et al., 2009).

Table 1.4. Selected lists of plants with anticancer activity

SI. No Plant Major active compound Reference

1. Aglaia foveolata Silvestrol Kim et al., 2007

2. Allium sativum (Garlic)

Diallyl sulfide, Diallyl disulfide, Diallyl trisulfide

Choi and Park, 2012

3. Aloe barbadenis

(Aloe vera) Aloe-emodin, Emodin

Chiu et al., 2009

4. Ananas comosus (Pine apple)

Bromelain Hale et al., 2010

5. Berberis amurensis (Berberis)

Berbamine Wei et al., 2009

6. Boswellia serrata Boswellic acid Agrawal et al., 2011

7. Brassica oleracea (Cabbage)

Indole-3-carbinol Wu et al., 2010


55

Table 1.4. (Cont.) Selected lists of plants with anticancer activity


8. Catharanthus roseus (Vinca)

Vinblastine, Vincristine , Alstonine, Ajmalicine.

Van Der Heijden et al., 2004

9. Citrullus lanatus (Watermelon)

Lycopene Ilic et al., 2011

10. Citrus reticulate (Mandarin orange)

Tangeretin, Nobiletin, Hesperetin, hesperidin, Naringenin, Naringin

Meiyanto et al., 2012

11. Curcuma longa (Turmeric)

Curcumin, Tumerone Manikandan et al., 2012

12. Glycine max (Soyabean)

Genistein Li et al., 2012

13. Malus domestica (Apple)

Ursolic acid Gayathri et al., 2009

14. Rheum rhabarbarum (Rhubarb)

Emodin Huang et al., 2009

15. Saussurea lappa Confertin, myricetin, diaminobutryic acid

Thara and Zuhara, 2012

16. Solanum lycopersicum (Tomato)

Lycopene Tang et al., 2009

17. Solanum pseudocapsicum

O-methylsolanocapsine Dongre et al., 2007

18. Taxus brevifolia (Pacific Yew)

Paclitaxel Kingston., 2007

19. Typhonium flagelliforme (Rodent tuber)

Pheophorbide-a, Pheophorbide-a', Pyropheophorbide-a, Methyl pyropheophorbide-a

Lai et al., 2010


56

Table 1.4. (Cont.) Selected lists of plants with anticancer activity


20. Vicia faba (Fava bean)

Diadzein, Genistein Kaufman et al., 1997

21. Vitis vinifera (Grapes)

Resveratrol, Piceatannol Kita et al.,2012

22. Wikstroemia indica (Indian stringbush)

Daphnoretin Lu et al., 2011

23. Zingiber officinale (Ginger)

Curcumin, gingerenone A, gingerols, 6-shogaol, 10-gingerol, enone-diarylheptanoid

Peng et al.,2012

1.2.13. Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common ubiquitous

deadliest cancer worldwide with poor diagnosis and accounts for approximately

about 500,000 to 1,000,000 new cases per year and accounting for more than

600,000 deaths each year (American Cancer Society, 2012). Generally, HCC is more

frequent in men than in women and the incidence increases with age (Levrero,

2006). Liver is often abused by environmental and biological toxins, poor eating

habits, consumption of alcohol, prescription and over-the-counter drug use, and

viruses which can damage and weaken the liver. These factors eventually lead to

hepatitis, cirrhosis, alcoholic liver disease and hepatocellular carcinoma (Gitlin, 1996).

1.2.13.1. Risk factors of hepatocellular carcinoma

There are multiple etiological agents that are associated with the

development of HCC, the most frequent being chronic hepatitis B virus (HBV),

hepatitis C virus (HCV) infections, long-term exposure to the mycotoxin and

aflatoxin B1.


57

Fig.1.9. Risk factors that leads to HCC

(a). Hepatitis B Virus (HBV)

HBV is a DNA virus and is recognized as a major etiological factor in the

development of diseases such as fatty liver (steatosis), cirrhosis, hepatocellular

adenoma and HCC. The risk of HCC in chronic HBV carriers is more than 100

times greater than in uninfected individuals (Beasley et al., 1981; Ito et al., 2010).

(b). Hepatitis C Virus (HCV)

HCV is an enveloped positive stranded RNA virus belongs to the genus

Hepacivirus. It is a completely cytoplasmic-replicating virus that induces oncogenic

transformation (Tellinghuisen and Rice, 2002). Chronic HCV infection mostly leads

to hepatic cirrhosis before developing HCC (Donato et al., 1997). Generally, the

prevalence of HCV-infection is accepted to be a major morbidity factor in hepatic

carcinogenesis. However, it also now established that many viral proteins are

implicated in malignant transformation and HCC development. Of these proteins,


58

core proteins, NS3 an dNS4, were shown to have transformation potential in tissue

culture (Sakamuro et al., 1995; Gale et al., 1999; Park et al., 2000). These viral

proteins, in addition to the viral RNA, interact with many host-cell factors, while

still regulating the viral life cycle. They modulate host-cell activities such as cell

signaling, transcription, transformation, apoptosis, membrane rearrangement,

vesicular trafficking and protein translation. This ultimately misleads the host

transcription factors, disturbing cell mitosis and protein synthesis, leading to

carcinogenesis (Levrero, 2006).

Fig. 1.10. Progression of HCV infection to HCC

(c). Diabetes mellitus

Many studies around the world have found a significant relationship between

diabetes and the development of HCC (Lagiou et al., 2000). Between 10 and 20% of

cirrhosis patients have overt diabetes and a higher percentage present impaired

glucose tolerance.


59

(d). Exposure to environmental pollutants

An environmental pollutant such as aflatoxins is a type of mycotoxin, toxic

chemicals made by some types of fungi. Aflatoxin is produced by Aspergillus fungi

when the fungus grows on improperly stored food products. Aflatoxins are capable

of causing DNA mutations, including the tumor suppressor, TP53 (p53). Aflatoxins

may be found in peanuts, tree nuts, corn, wheat and other grains, and oil seeds.

Other known chemical carcinogens are chlorination byproducts in drinking water.

Uncontrolled water chlorination converts many organic traces in water into

dangerous intermediates, such as di-and tri-chloroacetic acids, which are

experimentally known to induce HCC. Many other chemical contaminants, such as

solvents, food additives, drugs and hormones are also thought to contribute to HCC

(Abdel-Hamid, 2009).

(e). Alcoholism

Alcohol is the second most common risk factor for HCC after infection with

hepatits virus, steatohepatitis (fatty liver) and cirrhosis (Donato et al., 2002). In

developed countries, alcohol drinking seems to be the most common source for

HCC. Alcohol either directly initiates HCC after its oxidation into acetaldehyde,

which is genotoxic, or indirectly through the development of cirrhosis (London

et al., 1996).


60

(f). Obesity

Obesity showed a 5 fold increase in cancer mortality in people with great

body mass index in contrast to those who had a normal body mass index. Liver

cancer is frequently found in patients with metabolic disarrangements.

(g). Hereditary hemochromatosis

Hereditary hemochromatosis is an autosomal recessive condition

characterized by excessive iron deposition in hepatocytes due to an increased

intestinal absorption. Among hemochromatotic patients, 6% of men and 1.5% of

women are at absolute risk of liver cancer (Elmberg et al., 2003).

1.2.13.2. Developmental stages of hepatocellular carcinoma

Fig. 1.11. Histopathological progression and molecular features of HCC

After hepatic injury incurred by any one of several factors (hepatitis B virus

(HBV), hepatitis C virus (HCV), alcohol and aflatoxin B1), there is necrosis

followed by hepatocyte proliferation. Continuous cycles of this destructive–

regenerative process foster a chronic liver disease condition that culminates in liver


61

cirrhosis. Chronic hepatitis leads to cirrhosis within 15-40 years. Mostly, HCC

develops among 70%-90% of cirrhotic patients, while only 10% of HCC patients

have a non-cirrhotic liver, or even have inflammatory lesions. Cirrhosis is

characterized by abnormal liver nodule formation surrounded by collagen deposition

and scarring of the liver. Subsequently, hyperplastic nodules are observed, followed

by dysplastic nodules and ultimately hepatocellular carcinoma (HCC), which can be

further classified into well differentiated, moderately differentiated and poorly

differentiated tumours - the last of which represents the most malignant form of

primary HCC. (Levrero, 2006; Farazi and DePinho, 2006).

1.2.13.3. Experimental model – NDEA induced hepatocellular carcinoma

N-nitrosodiethylamine (NDEA) is a potent carcinogenic dialkylnitrosoamine

frequently used to induce liver cancer in animal models. NDEA belongs to the group

of N-nitrosamines, causing a wide range of tumors in all animal species and

suspected to be health hazards to man (Loeppky, 1999; Pandi perumal et al., 2006).

It is found in a wide variety of foods such as cheese, soybeans, smoked, salted and

dried fish, cured meat and alcoholic beverages and producing reproducible

hepatocellular carcinoma after repeated administration (Singh et al., 2009).

NDEA becomes metabolically active in the liver by the action of cytochrome

P450 enzymes to produce reactive electrophiles, which increase oxidative stress

level leading to cytotoxicity, mutagenecity and carcinogenicity (Archer, 1989).

It has been shown that the mechanism of action is due to metabolism of NDEA to

alkylating agents and reactive oxygen species and further interaction with DNA

molecule, forming various DNA adducts that can lead to mutations (Jeena et al,

1999). ROS are continuously generated in vivo as a result of NDEA administration


62

causing oxidative stress that seriously damaged the biological systems by injuring

tissues, altering biochemical compounds, causing chromosomal instability, eroding

cell membranes and mutation, which are involved in all steps of carcinogenesis, i.e.

initiation, promotion and progression (Karbownik et al., 2001).

The conventional therapy of hepatocarcinoma including chemotherapy,

radiation, surgical resection and ablation gives little hope for restoration of health

because of poor diagnosis and serious side effects. Liver transplantation is

considered to be the most effective treatment for patients with hepatocarcinoma.

However, low availability of organs limits the offer of this option to all candidates,

and the high risk of tumor recurrence after transplantation further com-promises its

efficiency.

Numerous components of plants, collectively termed “phytochemicals” have

been reported to possess substantial chemopreventive properties. For many years

cancer chemotherapy has been dominated by potent drugs that either interrupt the

synthesis of DNA or destroy its structure once it has formed. Unfortunately, their

toxicity is not limited to cancer cells and normal cells are also harmed. So efforts to

develop less toxic drugs that affect only malignant cells and mechanism based

approach are necessary in cancer therapy (Sivalokanathan et al., 2006). Several

herbal drugs have been investigated for their chemopreventive potential against

hepatocellular carcinoma.


63

Table 1.5. Chemopreventive effects of certain plants against hepatocellular carcinoma

SI. No Plant /Compound Experimental model Reference

1. Abrus precatorius Hep G2 cell lines and NDEA induced hepatocellular carcinoma in rats

Kartik et al., 2010

2. Acacia nilotica (polyphenolics)

NDEA induced hepatocellular carcinoma in rats

Singh et al., 2009

3. Achyranthes aspera NDEA and CCl4 induced hepatocellular carcinoma in rats

Kartik et al., 2010b

4. Alocasia macrorrhiza Hepatocellular carcinoma cell lines SMMC-7721 and Murine hepatoma H22 cell lines

Fang et al., 2012

5. Callilepis laureola Human hepatoma Hep G2 cells

Popat et al., 2002

6. Cuphea hyssopifolia (Cuphiin D1, cuphiin D2, woodfordin C, oenothein B)

Hepatoma Hep 3B cells Wang et al., 1999

7. Emblica officinalis NDEA induced hepatocellular carcinoma in rats

Jeena et al., 1999

8. Feronia limmonia Human liver hepatoma cells Hep G2

Jain et al., 2011

9. Genistein DEN induced hepatocellular carcinoma in rats

Chodon et al., 2007

10. Glycyrrhizin NDEA induced hepatocellular carcinoma in rats

Shiota et al., 1999

11. Gynandropsis gynandra Aflatoxin B1 induced hepatocellular carcinoma in rats

Sivanesan and Begum, 2007


64

Table 1.5. (Cont.) Chemopreventive effect of certain plants against hepatocellular carcinoma

SI. No Plant /Compound Experimental model Reference

12. Lygodium flexuosum

NDEA induced hepatocellular carcinoma in rats

Wills et al., 2006

13. Morin NDEA induced hepatocellular carcinoma in rats

Sivaramakrishnan et al., 2008

14. Phyllanthus amarus NDEA induced hepatocellular carcinoma in rats

Rajeshkumar and Kuttan, 2000

15. Picrorrhiza kurroa NDEA induced hepatocellular carcinoma in rats

Jeena et al., 1999

16. Scutia myrtina NDEA induced hepatocellular carcinoma in rats

Ramanathan et al., 2011

17. Silymarin NDEA induced hepatocellular carcinoma in rats

Ramakrishnan et al., 2008; Ramakrishnan et al., 2009

18. Strychnos nux-vomica (Brucine, Strychnine, Brucine N-oxide, isostrychnine)

Human hepatoma HepG2 cell lines

Deng et al., 2006

19. Terminalia arjuna NDEA induced hepatocellular carcinoma in rats

Sivalokanathan et al., 2006

1.2.14. Selection of the plant for present study

When selecting a plant for pharmacological activities, four basic methods are

usually followed (Suffness and Douros, 1979):


65

a. Random choice of plant species

b. Choice based on ethnomedical use

c. Follow up of existing literature on the use of the species

d. Chemotaxonomic approaches

Based on the ethnopharmacological relevance and reported activities

Woodfordia fruticosa Kurz flowers were selected for the study.

1.2.14.1. Plant profile

Botanical name : Woodfordia fruticosa (Linn.) Kurz

: Woodfordia floribunda (Salib.)

Family : Lythraceae

Vernacular names : Eng. : Fire-flame bush, Shiranjitea, Woodfordia

Hin. : Davi, Tavi

Kan : Bela, Tamrapurpi

Mal : Tatri, Tatirippu

San : Dhataki, Madaniyahetu

Tam : Dhattari, Jargi, Velakkai

Tel : Dhataki, Jargi, Serinji

Distribution : Throughout India, but more abundant in north India, ascending up to

an altitude of about 1600 m, and also in a majority of the countries of South East and

Far East Asia like Malaysia, Indonesia, Sri Lanka, China, Japan and Pakistan as well

as Tropical Africa (Kirtikar and Basu, 1935).


66

The plant: A much branched beautiful deciduous shrub attaining a height of 3-7m

with much long arching branches and bark of the plant, characteristically cinnamon-

brown coloured and smooth, peels off in fibres and the young shoots are terete, often

clothed with fine white pubescence. Leaves simple, opposite or sub- opposite, entire,

ovate - lanceolate, acute, subcoriaceous with black granular dots on the under

surface. Flowers numerious, brilliant, red in dense, axillary, paniculate - cymose

clusters. Fruits ellipsoid, irregularly dehiscent capsule about 1cm long.

Parts used : Flowers

Properties : The flowers are astringent, acrid, refrigerant, stimulant, depurative,

styptic, uterine sedative, anthelmintic, constipating, antibacterial, vulnerary,

corrective of urinary pigments, alexeteric and febrifuge. They are used in vitiated

condition of kapha and pitta, leprosy, skin disease, burning sensation, haemorrhages,

menorrhagia, leucorrhoea, haemoptysis, erysipelas, diarrhea, dysentery, foul ulcers,

diabetes, bilious fever, hepatopathy and verminosis. They are an important

ingredient of Aristam and Asavam as they aid in fermentation; they are also highly

valued as a stimulant in pregnancy.

1.2.14.2. Reported activities of Woodfordia fruticosa

In India, Woodfordia fruticosa Kurz is a much used medicinal plant in

Ayurvedic and Unani systems of medicines (Chopra et al., 1956; Watt, 1972;

Dymock et al., 1995). Although all parts of this plant possess valuable medicinal

properties, there is a heavy demand for the flowers, both in domestic and

international markets specialized in the preparation of herbal medicines

(Oudhia, 2003).


67

The flower is pungent, acrid, cooling, toxic, sedative and anthelmintic, and is

useful in thirst, dysentery, leprosy, erysipelas, blood diseases, leucorrhoea,

menorrhagia and toothache. It is considered as ‘Kapha’ (mucilage type body

secretion) and ‘Pitta’ (energy-dependent metabolic activity) suppressant in the

Ayurvedic concepts of medicine. Many marketed drugs comprise flowers, fruits,

leaves and buds mixed with pedicels and thinner twigs of the plant (Chopra et al.,

1956).

The flowers are used in the preparation of Ayurvedic fermented drugs called

“Aristhas” (hot extraction followed by month-long slow fermentation) and “Asavas”

(cold percolation followed by month-long slow fermentation) (Atal et al., 1982).

Aristhas are believed to be general health tonics in nature, having overall health

stimulating properties via ameliorating and/or delaying one or other systemic

disorders. Of the 18 aristhas mentioned in the Indian Ministry of Health & Family

Welfare’s monograph (CCRIMH, 1978), 17 have been found to contain Woodfordia

fruticosa. Tribal people in the Chhattisgarh district of central India uses fresh

flowers to stop bleeding in emergency cuts, but they prefer to employ dried flower

powder to heal wounds more efficiently. It is also one of the ingredients of a

preparation used to increase fertility in women (Burkill, 1966; Dey, 1984).

Flowers used for the ayurvedic preperation “Kutajarista” for Sprue,

dysentery, diarrhoea (Shenoy and Yoganarasimhan, 2008), “Lukol” for the

leucorrhoea DUB (dysfunctional uterine bleeding) and symptoms of pelvic

inflammatory disease. Oil based flower extract has always been recommended for

open wounds (Tewari et al., 2001; Das et al., 2007). The dried flowers powder

sprinkled over ulcers and wounds to diminish discharge and promote granulation


68

(Khorya and Katrak, 1984). They are also used as tonic in disorders of mucous

membranes, hemorrhoids and in derangement of the liver (Chopra et al., 1956; The

Wealth of India, 1988; Mhaskar et al., 2000). An Ayurvedic medicine called

“Balarishta”, a drug of ‘Asava’ and ‘Aristha’ group, contains Woodfordia fruticosa

flowers as one of the major constituents and is used for burning sensation in stomach

(Agnimandya), weakness (Daurbalya) and rheumatic diseases (Vataja roga)

(Anonymous, 1978). A popular crude drug (Sidowaya or Sidawayah) of Indonesia

and Malaysia mainly contains dried flowers of Woodfordia fruticosa (Burkill, 1966).

It has been used as an astringent to treat dysentery and sprue and also for the

treatment of bowel complaints, rheumatism, dysuria and hematuria in many

Southeast Asian countries.

Table 1.6. Reported activities of Woodfordia fruticosa Kurz with part and solvent used

SI.No Activity Part used Solvent used Reference

1. Antibacterial Flowers Petroleum ether, Chloroform, Methanol Ethanol, Water

Kumaraswamy et al., 2008

2. Antibacterial Flowers Methanol Parekh and Chanda, 2007

3. Antibacterial Leaves Ethanol Bajracharya et al., 2008

4. Antibacterial Leaves - Chougale et al., 2009

5. Antibacterial Kutajarista- An ayurvedic

preparation

- Shenoy and Yoganarasimhan, 2009


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Table 1.6. (Cont.) Reported activities of Woodfordia fruticosa Kurz with part and solvent used


6. Antidiarrhoeal Kutajarista- An ayurvedic

preparation

- Shenoy and

Yoganarasimhan,

2008

7. Antifertility Flowers Alcohol Khushalani et al.,

2006

8. Antimicrobial Flowers Hexane, Chloroform,

Acetone, Methanol,

Water

Dabur et al., 2007

9. Antimicrobial Leaves Essential oil and Hexane, Methanol, Acetone

Kaur and Kaur,

2010

10. Antioxidant Flowers Petroleum ether, Chloroform, Methanol, Water

Kumaraswamy and Satish, 2008

11. Antioxidant Flowers (gallic acid) Petroleum ether, Chloroform, Methanol, Water

Lok Ranjam Bhatt, 2005

12. Antitumor Flowers(WoodfordinA, B and C dimeric hydrolysable tannins)

- Yoshida et al., 1989

14. Antitumor Isolated compound - Kuramochi-Motegi et al., 1992

15. Antiulcer Roots Ethanol Mihira et al., 2011


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Table 1.6 (Cont.) Reported activities of Woodfordia fruticosa Kurz with part and solvent used


16. Hepatoprotective

Flowers 50 % Alcohol Brindha and

Geetha, 2009


Flowers Water Chandan et al.,

2008


Flowers Methanol Baravalia et al., 2011

19. Immunomodulatory

Fermented product from flowers

- Kroes et al., 1993

20. Immunostimulatory

Flowers Ethanol Shah and Juvekar,

2010

1.2.14.3. Chemical constituents of Woodfordia fruticosa

The extracts of Woodfordia fruticosa flowers showed the presence of

carbohydrates, gums, flavonoids, sterols and phenolic compounds/tannins

(Khushalani et al., 2006). A series of publications have appeared on the structural

characterization of the secondary metabolites of the plant. The compounds identified

are predominantly phenolics, particularly hydrolysable tannins and flavonoids. The

following chemical constituents are found in different part of the Woodfordia

fruticosa.

Octacosanol and β-sitosterol (Chauhan et al., 1979a), steroid sapogenin

hecogenin and meso-inositol from the flowers (Chauhan et al., 1979b), lupeol,

betulin, betulinic acid, oleanolic acid and ursolic acid from the leaves (Dan and Dan,

1984) .


71

The phenolic constituents include gallic acid in leaves and stems (Kalidhar

et al., 1981; Kadota et al., 1990), ellagic acid in leaves and flowers (Nair et al.,

1976), bergenin (a C-glycoside of gallic acid) and the norbergenin in stems

(Kalidhar et al., 1981), chrysophanol-8-O-β-D-glucopyranoside in flowers

(Chauhan et al., 1979a), and the naphthaquinone pigment lawsone in leaves (Saoji

et al., 1972).

The flavonoid constituents: six quercetin glycosides; 3-rhamnoside from

flowers (Chauhan et al., 1979b), 3-β-L-arabinoside (polystachoside) from flowers

and leaves (Nair et al., 1976), and 3-O-α-L-arabinopyranoside, 3-O-β-D-

xylopyranoside, 3-O- (6″-galloyl)-β-D-glucopyranoside from leaves (Kadota et

al., 1990). Three myricetin glycosides; 3-O-β-D-galactoside in flowers and leaves

(Nair et al., 1976), and 3-O-α- L-arabinopyranoside, 3-O-(6″-galloyl)-β-D-

galactopyranoside in leaves (Kadota et al., 1990), as also naringenin 7-glucoside and

kaempferol 3-O-glucoside in flowers (Chauhan et al., 1979b).

A large number of new and known hydrolysable tannins have been isolated

from the flowers. The known tannins reported are: 1,2,3,6-tetra-O-galloyl-β-D-

glucose, 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-

glucose, tellimagrandin, gemin D, heterophyllin A and oenothein B (Yoshida et al.,

1989, Yoshida et al., 1990), woodfordins A- C (Yoshida et al., 1989, Yoshida et al.,

1990), woodfordin D, oenothein A (Yoshida et al., 1991), as also isoschimawalin A

and woodfordins E-I (Yoshida et al., 1992; Kuramochi-Motegi et al., 1992)


72

The traditional system of medicine in India recommends the hepatoprotective

potential of this plant. Yet there is paucity of information regarding the liver

protective efficacy of Woodfordia fruticosa. Hence this study was undertaken to fill

the lacuna in this regard.

So the thesis embodies the study regarding the antioxidative, antifibrotic and

anticancer activities of Woodfordia fruticosa in experimental animals. The study

pertaining to the isolation and identification of the active phytochemical constituents

responsible for these effects too forms a portion of this thesis.

Fig. 1.12. Woodfordia fruticosa Habit


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Fig. 1.13. Woodfordia fruticosa flowers

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CHAPTER 1shodhganga.inflibnet.ac.in/bitstream/10603/35283/11/11_chapter1.pdf · Introduction and Review of LiteratureIntroduction and Review of Literature 11 Fig. 1.3 Organs affected