Chapter 1 INTRODUCTION

Introduction

Since time immemorial, human has made use of plants in the treatment of various ailments and has been documented in history of all civilizations to rescue humanity from the clutches of disease. As the man began to acquire knowledge of his environment, he began associate curative characteristics with certain plants classifying them as pain killer, febrifuge, antiflogistics, soporic and so on (Rangari, 2002). Medicinal herbs are very much like foods, and in many cases they are indistinguishable from them. They contain a variety of different nutritious and therapeutic constituents such as vitamins, minerals, trace elements as well as active ingredients with a variety of medicinal actions that are considered to play a beneficial role in health care. These include volatile oils, tannins, mucilage, alkaloids, bitters and flavonoids. Herbs are compatible with the chemistry of the human body, which has adapted over thousands of years to assimilate them. Herbal drugs have gained importance and popularity in recent years because of their safety, efficacy and cost effectiveness. The World Health Organization (WHO) has listed 21,000 plants, which are used for medicinal purposes around the world. Among these 2500 species are in India, out of which 150 species are used commercially on a fairly large scale. According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs. Therefore, such plants should be investigated to better understand their properties, safety and efficacy (Nascimento et al., 2000; Cheng et al., 2003; Kumar et al., 2009). India represented by rich culture, traditions and natural biodiversity, offers a unique opportunity for drug discovery researchers (Jachak et al., 2007). India is the largest producer of medicinal herbs and is called as botanical garden of the world. The Indian traditional medicine system like ayurveda, siddha and unani are predominantly based on the use of plant materials. The herbal products symbolize safety in contrast to the synthetics that are regarded as unsafe to human and environment.Today there is an enormous resurgence of interest in all herbal products and a rediscovery of the traditional use of medicinal herbs. Treatment with herbs can often provide a gentle and safe alternative or complement to modern drugs and other orthodox medical treatments. Over the centuries we have collected a great number of data about the use and efficacy of herbal medicine. Traditional medicines derived from medicinal plants are used by about 60% of the worlds population. The World Health Organization estimates that 80% of the people in developing countries of the world rely on traditional medicine for their primary health care needs, and about 85% of traditional medicine involves the use of plant extracts. This means 1

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that about 3.5 to 4 billion people in the world rely on plants as sources of drugs (Farnsworth et al., 1985). Today, pharmacognosy is a highly specialized science that represents one of the major disciplines of pharmaceutical education. It embraces knowledge of the history, distribution, collection, selection, preparation, commerce, isolation, identification, evaluation, preservation and use of drugs and economic substances that affects the health of human being (Tyler, 1977). Pharmacognosy deals with the standardization, authentication and study of natural drugs. It is closely involved with allied fields, viz. phytochemistry and toxicological screening of natural products. Much of the research in pharmacognosy has been done in identifying controversial species of plants, authentication of commonly used traditional medicinal plants through morphological, histological, physicochemical and toxicological parameters, especially heavy-metal estimation and radiobiological contamination in plants, prescribed by an authoritative source. The importance of pharmacognosy has been widely felt in recent times (Kumar, 2007). Undoubtedly, the plant kingdom still holds large number of species with medicinal value which have yet to be discovered. Lots of plants were screened for their possible pharmacological values like hypoglycemic, hepatoprotective, hypertensive, anti-inflammatory, anti-fertility, anti-cancer, anti-diarrhoeal etc. Pharmacognosists with a multidisciplinary background are able to make valuable contributions in the field of phytomedicine (Evans, 2006). With the onset of research in medicine, it was concluded that plants contain active principles, which are responsible, for curative action of the herbs. Around 170 phytoconstituents has been isolated from 110 plants belonging to 55 families and reported to possess hepatoprotective activity. An estimate of 600 commercial herbal formulations with claimed hepatoprotective activity is being sold all over the world. In India, about 93 medicinal plants are used in different combinations in the preparations of 40 patented herbal formulations (Girish et al., 2009). Some of the patented Indian herbal preparations sold for liver ailments are Acilvan, Adliv, Biligen, Hepa- 10, Hepex, Hipex, Kalmegh Compound, Liv- 52, Liv- 77, Liva-16, Livarin, Livatone, Livergen, Livodin, Livomyn, Neoliv- 100, Stimuliv, Syliv, Tefroli, Vimiliv etc ( Reddy et al.,2010). The world is rich in medicinal plants growing wildly or cultivated, forming a huge natural and economical health which must be protected, increased and industrialized for the sake of humanity, development of economy, wealth of nation and health of people. Developing countries have rich sources of new biologically active substances. There are thousands of medicinal plants, which are medicinally important. However the researchers of
Pharmacognostical, Phytochemical and Pharmacological Evaluation of the Bark of Plant Holoptelea integrifolia (Roxb.) Planchon.

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Phytochemistry have exploited very less plants species remained unravelled for their phytochemical composition and pharmacological properties (Kamboj et al., 2000).

1.1 HOLOPTELEA INTEGRIFOLIA (ROXB) PLANCH.

1.1.1 HABITAT AND CULTIVATION Holoptelea integrifolia is wildly grown in deciduous forests and cultivated in gardens. The tree is commonly found in India up to an altitude of 600 m; Lower ranges of Himalaya from Jammu to Oudh, Rohilkhand, forests of Dehra Dun, Saharanpur, Orissa, including Chota Nagpur ,Central India, Ajmer, Bihar, Assam, W. Bengal, Hills of Deccan, easteran slopes of W. Ghats and North Circars. It is also found in Burma, W. Peninsula, Ceylon, Cochin, China and tropical and sub-tropical parts of Asia and Africa. It thrives in deep porous soil with good drainage but becomes stunted and crooked on poor shallow soil. It requires moderate conditions for optimum growth. The tree sheds its seeds during the hot season and they germinate at the commencement of the rains. Protection from sun in early stages is beneficial. The rate of growth is fast and the mean annual girth increment is 1.05 inch (Kirtikar et al., 1988; The wealth of India, 1959; Sharma et al., 2001).

1.1.2 TAXONOMIC CLASSIFICATION Domain Kingdom Sub Kingdom Phyllum Subphyllum Class Subclass Order Family Genus Species Subspecies : : : : : : : : : : : : Eukaryota Plantae Viridaeplantae Tracheophyta Euphllophytina Magnoliopsida Dilleniidae Urticales Ulmaceae Holoptelea. integrifolia Planch

Pharmacognostical, Phytochemical and Pharmacological Evaluation of the Bark of Plant Holoptelea integrifolia (Roxb.) Planchon.

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1.1.3 PLANT DESCRIPTION

Introduction

A medium to large sized spreading glabrous deciduous tree grows up to 15-18 m tall in height (Kirtikar et al., 1988; The wealth of India, 1959).

Bark:

whitish and yellowish grey, pustular exfoliate with regular intervals with offensive smell when cut, covered with blisters, peeling in corky scales on old trees.

Leaves:

Alternately arranged leaves are elliptic-ovate, glabrous, 8-13cm long and 3.2-6.3 cm wide, glabrous, margins entire (those of the seedlings and shoots often serrate), apex acute or acuminate, crushed leaves emit an unpleasant odour, base rounded or cordate, main nerves 5 to 7 pairs, petioles 6-13 mm, long, stipules lateral lanceolate and scarious.

Flowers:

In clusters, appearing before the leaves. Usually male without a rudimentary ovary and Polygamous or hermaphrodite mixed in short racemes or fascicles near leaf axils, small, greenish-yellow to brownish, pubescent, borne in short racemes or fascicles at the scars of fallen leaves (at scars of previous years shoots which are scaly but leafless), Sepals are velvety, often four in number, pubescent 1.5-2.5 mm long; Stamens 4-8 (often 6 or 7); filaments glabrous; anthers pubescent; Ovary compressed, pubescent, single celled and stalked, The stalk lengthens as the seed ripens; styles 2.5-4 mm long, stigmatose inside through their whole length.

Fruit:

Fruits are circular/sub orbicular (winged) samara compressed with membranous wings, 2.5 cm in diameter on a slender stalk, indehiscent, pubescent, reticulately veined wings tip bifid and lobes incurved, edible, seed flat.

Pharmacognostical, Phytochemical and Pharmacological Evaluation of the Bark of Plant Holoptelea integrifolia (Roxb.) Planchon.

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Figure-1.1: Fruits of Holoptelea integrifolia

Figure-1.2: Flower of Holoptelea integrifolia

Figure-1.3: Leaves of plant H. integrifolia 5

Pharmacognostical, Phytochemical and Pharmacological Evaluation of the Bark of Plant Holoptelea integrifolia (Roxb.) Planchon.

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1.1.4 COMMON VERNACULAR NAMES (Kirtikar et al., 1988; Sharma et al., 2001) Hindi Sanskrit English Nepali Bengali Madrasi Gujrati Tamil Malayalam Kannada Marathi Oriya Tamil Telgu Papri, Kanju, Banchilla, Dhamna, Begana Chirivilva, Pootikaranja Indian Elm, Jungle cork tree Sano pangro Nata Karanja Perunaruvuli Charal,charel,kanjo, Waola Aya, Ayil, Kanci, Vellaya Aavil, Njettaval, Aaval Thavasai, Rasbija, Kaladri, Nilavahi Ainasadada, Vavala, Vavli, Papara Dhauranjan, Turuda Aya, Ayil, Kanci, Vellaya Nali, Ayil, Kanci, Vellaya

1.1.5 TRADITIONAL USES The plant has been used for long time in various ailments and conditions as: Table 1.1: Traditional use of plant Holoptelea integrifolia
S. No. 1 Used in/or as Fish poison Part of Plant The crushed stem bark Reference Katewa et al., 2008; Murthy et al., 2010 2 3 4 Eczema treatment Scabes treatment Ring worm treatment Leaf past Leaf past Leaf past Jain Anita et al., 2008 Khare, 2007 Shukla et al., 2010; Jain, 2004; Khare, 2007 5 6 7 Chronic wound healing Toothache Swelling or torsion on right side of stomach 8 9 Cancer treatment (applied externally) Inflammation affected part) (applied on the Stem bark paste Seed oil added with sulphur and kept for 5 days 10 Herpes treatment Bark Parinitha et al., 2004 Jain et al., 2010 Meena et al., 2010 Bark Powder Leaves pieces Warmed stem bark Pawar et al., 2007 and 2004 Patil and Patil, 2005 Patil and Patil, 2005

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11 Hair loss (applied externally) leaf bud mixed with lime juice 12 13 Hydrocoel treatment For edible purpose Stem bark tied on arm Oil/ seed

Introduction
Parinitha et al., 2004

Kumar et al., 2006 Das et al., 2002; Ganesan et al., 2009

14

Rheumatism treatment

Mucilaginous bark is boiled, juice squeezed out and applied to

Wealth of India, Vol. V: H-K, 1959

rheumatic swellings 15 Spiders elimination Leaf juice sprayed Ganesan et al., 2006; Khare, 2007 Patil et al., 2006 Sharma et al., 2001

over spider 16 17 Pile (applied externally) Diabetes, vitiligo, leprosy, colic Crushed leaf Bark and leaves

1.2 LIVER
1.2.1 ANATOMY AND HISTOLOGY The liver is the largest gland in the human body, weighing approximately 3 pounds and occupying a large region mostly on the right side of the body, below the diaphragm and behind ribs 5 through 10. The liver is divided into 4 lobes: right, left, caudate, and quadrate. The right and left lobes are the largest, while the caudate and quadrate are smaller and located posteriorly. The liver plays an astonishing array of vital functions in the maintenance, performance and regulating homeostasis of the body. The basic functional unit of the liver is the lobule and a single lobule is about the size of a sesame seed, roughly hexagonal in shape. The basic structures in a lobule include irregular, branched epithelial cells called hepatocytes arranging in interconnected plates around a central vein forming the bulk of the lobule; bile canaliculi formed between walls of adjacent hepatocytes; portal triads (hepatic artery, portal vein branch and bile duct) at each corner of hexagon; liver sinusoids that run from the central vein to the portal triads; hepatic macrophages (Kupffer cells) and a small space between the sinusoids and the hepatocytes known as Space of Disse (Ross and Wilson, 2001; Guyton 2000; Harsh 2000; Maher, 1997).

Pharmacognostical, Phytochemical and Pharmacological Evaluation of the Bark of Plant Holoptelea integrifolia (Roxb.) Planchon.

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Figure-1.4: Architecture of liver lobule

Figure-1.5: Architecture of liver sinusoids

Pharmacognostical, Phytochemical and Pharmacological Evaluation of the Bark of Plant Holoptelea integrifolia (Roxb.) Planchon.

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Hepatocyte

Introduction
Pit cell Kupffer cell

Endothelial cell
Figure-1.6: Inner structure of a lobule

Stellate cell

1.2.2 FUNCTIONS AND PHYSIOLOGY Liver is involved with almost all the biochemical pathways to growth, immunity, nutrient supply, energy provision and reproduction. Its primary function is to control the flow and safety of substances absorbed from the digestive system before distribution of these substances to the systemic circulatory system. Liver has very complex physiology (Gines et. al., 2010). Metabolism: Hepatocytes synthesize proteins of importance for the whole body. These proteins include cargo proteins (e.g., albumin, transferrin, ceruloplasmin, haptoglobin, lipoproteins), immune-related proteins (proteins of the complement system, acute-phase proteins), and coagulation factors. C-reactive protein is an acute-phase protein, whose hepatocellular production is massively stimulated by cytokines such as IL-6 and IL-1. Albumin is the most abundant plasma protein maintaining intravascular oncotic pressure. Aminotransferases transfer an amino group from a donor molecule to a recipient molecule. ALT (alanine transferase) is strictly present in cytosol whereas AST (aspartate transferase) can be cytosolic and mitochondrial. Increased serum aminotransferase levels are nonspecific markers for hepatocellular damage. Destruction of proteins is facilitated by two major 9

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pathways i.e. autophagiclysosomal pathway in which a part of the cytosol is engulfed in vacuole and digested by lysosomal enzymes and ubiquitinproteasome-related pathway in which proteins are enzymatically linkaged with ubiquitin residues for degradation. About 90% of the intestinally absorbed glucose is taken up by the liver to maintain blood glucose levels within physiologic range. Glucose cross membranes via glucose transporters (Glucose transporter (GLUT); GLUT-2, 9, and 10 are expressed in the liver). Once in the cytoplasm, glucose is phosphorylated by hexokinase or glucokinase to access cellular metabolism. Glucokinase is expressed only in the liver and phosphorylates only glucose. Glucose- 6-phosphate is sequentially transformed into glucose-1-phosphate by phosphoglucomutase and into uridine-diphosphate-glucose by glucose-1-

phosphouridyltransferase to be finally stored as glycogen. Glucose-6- phosphate is not solely the initial compound for glycolysis, it can also enter the pentose phosphate pathways via glucose-6-phosphate dehydrogenase to produce NADPH and precursors for nucleotides. Other carbohydrates such as fructose and galactose are enzymatically transformed to join the glycolysis pathway. When glucose blood levels drop, glucagon and adrenaline stimulate phosphorylase reverting glycogen to glucose- 1-phosphate (-glycanphosphorylase) and to glucose-6-phosphate (phosphoglucomutase). Glucose-6-phosphate is converted to glucose by glucose-6- phosphatase. Glucose may be synthesized from other sources. Two third of the glucose derived from gluconeogenesisis synthesized from lactate, which results from anaerobic metabolism and can be supplied to the liver by the muscles. Glucose can also be produced from amino acids, mostly alanine, and from glycerine which is a degradation product of triglycerides. Gluconeogenesis is triggered by hormonal signals. Glucagon increases gluconeogenesis in the short term, while glucosteroids enhance gluconeogenesis in the long term. Insulin inhibits gluconeogenesis. A hallmark of hepatic insulin resistance is the failure of insulin to inhibit hepatic glucose output. Within each liver lobule, the periportal zone is where oxidative energy metabolism, amino acid catabolism, cholesterol metabolism, and fatty acid -oxidation take place, whereas the perivenous zone is where de novo lipid synthesis, ketogenesis and xenobiotic metabolism occur. Liposynthesis occurs by esterification of free fatty acids via acetyl-CoA and glycerol and is driven by glycerophosphate acyltransferase (GPAT). GPAT is activated by nutritional status and insulin and inhibited by glucagon. De novo lipogenesis is regulated by insulin via activation of sterol regulatory element binding protein-1c (SREBP-1c), which controls the transcription of lipogenic enzymes such as fatty acid synthase. Insulin stimulates the conversion of carboxyl-CoA to malonyl-CoA, acting as regulator for the distribution of
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free fatty acids toward esterification or oxidation. Low levels of malonyl-CoA direct free fatty acids to the mitochondriae and -oxidation via carnitine palmitoyltransferase-1 (CPT-1), an outer mitochondrial membrane enzyme. High levels of malonyl-CoA inhibit CPT-1, thus enhancing esterification of free fatty acid into triglycerides. Fatty acids can also be oxidized in peroxisomes (-oxidation) and microsomes (-oxidation). Triglycerides trigger apolipoprotein B (Apo-B) synthesis and are secreted as VLDL-Apo-B while Insulin inhibits Apo-B synthesis and impair secretion of triglycerides as very low density lipoprotein (VLDL). Liver also regulates copper and iron homeostasis. Biliary Circulation: The enterohepatic circulation retains over 95% of the bile acids. Bile acids are derived from cholesterol and facilitate cholesterol homeostasis. About 600-ml bile is produced daily. Bile acid that is present in the terminal ileum return to the liver where they are taken up by hepatocytes and excreted in to bile. Hepatocytes and cholangiocytes have a basolateral sides and an apical side. On their apical side hepatocytes have ATP-dependent pumps- Bile salt export pump (BSEP) that accumulate bile acids, phospholipids, and organic anions in the canalicular bile. A Na+-dependent carrier (apical sodium bile acid transporter (ASBT)) situated on the apical side of enterocytes, hepatocytes and cholangiocytes recycle the bile acids. Also an organic solute and steroid transporters (Ost, Ost) transporters on the basolateral side of enterocytes and cholangiocytes have been implicates in the transport. Another Na+-dependent transporter, Na+-Taurocholate cotransporting polypeptide (NTCP) take up the bile in to hepatocytes and retain the essential cholesterol metabolite. Bile acids activate the TGR5 (also called GPBAR1, membrane bile-acid receptor or BG37) as well as an intracellular receptor, FXR (farnesoid X receptor). Bile acids affect the energy

homeostasis via the TGR5 pathway and their cellular uptake exert direct signalling functions in cholangiocytes and hepatocytes via calcium, PKC, MEK, ERK, and PI3K pathways, altering gene expression, cell proliferation, apoptosis, and secretion. FXR, a nuclear hormone receptors functions as a transcription factor, hinders the accumulation of bile acids and thereby prevents toxic damage. FXR influence the bile acid, cholesterol, triglyceride, and glucose metabolism. As a Filter: The liver contain cells of first line defence against pathogens including Kupffer cells (KCs, mobile), dendritic cells (DCs), and natural killer (NK) cells that function as antigen-presenting cells (APCs). KCs are crucial for the development of hepatic antigen tolerance. Depletion of KCs leads to upregulation of T cells. CD4 T cells transform into different T-helper (Th) cells or regulatory T (Treg) cells expressing different chemokines (Th1: IFN-, Th2: IL-4, IL-10, Th17: IL-17) that plays a key role in liver immunotolerance.
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T-cell stimulation is inhibited short term by CTLA-4 and long-term inhibition by PD-1. NK and NKT cells, which are identified by expression of CD56 together with KCs and LSECs to provide an effective first-line innate immune defense against invading pathogens, toxins, food antigens, and circulating tumor cells. Liver sinusoidal endothelial cells (LSECs), KCs, and DCs function as antigen-presenting cells (APCs) and support immune pathways by expressing CD 40, CD 80, and CD86, similar to mature DCs. LSECs express mannose and scavenger receptors and antigen uptake molecules. As a detoxifier: Kidney remove water-soluble substances while lipophilic substances biotransform as detoxification and sometime activation (prodrugs) product within the liver. Liver detoxify exo and endogenous substances. Detoxification is carried out in three steps. Polarity of molecule is increased in first step by adding such moieties as NH2, COOH, OH, or SH groups. This is accomplished by cytochrome P-450 of microsomal mixed functional monooxygenases. In the second phase the molecule is made more hydrophilic by conjugation with glucuronic acid, amino acids, activated sulfuric acid or mercapturic acid. In the third phase of transmembrane transporters noxious compounds conjugated with charged moieties such as glucuronide, glutathione, and sulfate are subsequently pumped into bile across the canalicular membrane by different ATP-binding cassette (ABC) transporters. Pumps such as ABCC3, ABCC4, and ABCC5 transport the conjugated compounds back into the blood. ABCC2 perform on organic anions while many charged and uncharged compounds are pumped by ABCG2 transporter.

1.2.3 LIVER DISEASES Liver disease is any condition that causes liver inflammation or tissue damage and affects liver function. Characteristics of liver diseases are following: (a) Fibrosis or scarring: Liver fibrosis refers to the accumulation of tough, fibrous scar tissue in the liver. Formation of scar tissue is a normal bodily response to injury, but in fibrosis this healing process goes wrong. When hepatocytes (functional liver cells) are injured due to infection with a virus, heavy alcohol consumption, toxins, trauma, or other factors, the immune system is activated to repair the damage. The injury or death (necrosis) of hepatocytes stimulates inflammatory immune cells to release cytokines, growth factors, and other chemicals. These chemical messengers direct support cells in the liver called hepatic stellate cells to activate and produce collagen, glycoproteins (such as fibronectin), proteoglycans, and other substances. These substances are deposited in the liver, causing the

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build-up of extracellular matrix (nonfunctional connective tissue). At the same time, the process of breaking down or degrading collagen is impaired. In a healthy liver, the synthesis (fibrogenesis) and breakdown (fibrolysis) of matrix tissue are in balance. Fibrosis occurs when excessive scar tissue builds up faster than it can be broken down and removed from the liver. (Ramachandren, 2009) (b) Cirrhosis: In this condition scarring and inflammation spread through the liver and irreversibly disrupt its shape or function causing permanent cell damage and ultimately liver failure and leading to liver cancer. Cirrhosis is usually caused by toxins (including alcohol) in the blood or by hepatitis. Scarring and inflammation causes hypoxia, ischaemic cell injury or death followed by collagen formation and finally cirrhosis (Georgios et al., 2005). In cirrhosis, dead and damaged liver cells are replaced by fibrous tissue, which can form masses of scar tissue and dramatically change the structure of the liver. These fibrous areas can slow the flow of blood through the liver. (c) Hepatitis or inflammation: It is the inflammation of the liver tissue and occurs as a result of injury, infections, and other immunophysiological response. The condition leads to chronic liver diseases. (d) Cholengitis: The liver performs many functions. One of them is manufacturing bile. Bile is a watery liquid made by the cells of the liver that is important for digesting food in the intestine, particularly fat, and ridding the body of toxins. Liver cells secrete the bile they make into small canals within the liver. The bile flows through the canals and into larger collecting canals (ducts) within the liver (the intrahepatic bile ducts). The bile then flows within the intrahepatic bile ducts out of the liver and into the extrahepatic bile ducts. From the extrahepatic bile ducts, the bile flows into the intestine where the bile mixes with food. In primary sclerosing cholangitis, the intrahepatic and extrahepatic bile ducts become inflamed, scarred and thickened (sclerotic), narrowed, and finally obstructed. Obstruction of the ducts can lead to abdominal pain, itching, jaundice, infection in the bile ducts (cholangitis), and liver scarring that leads to liver cirrhosis and liver failure. (e) Fatty Liver Disease: Fatty liver disease refers to a reversible condition in which large vacuoles of triglyceride fat accumulate in liver cells via the process of steatosis. NONalcoholic steatohepatitis (NASH) is a distinct hepatic disorder observed in patients without a history of significant alcohol consumption, which histologically resembles alcoholinduced liver damage. It was first described in obese and diabetic women. NASH is considered to be part of the spectrum of non-alcoholic fatty liver disorders (NAFLD), ranging from bland steatosis to steatohepatitis and cirrhosis. NAFLD has four histological stages: (i) Fatty
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infiltration of the liver; (ii) Fatty infiltration plus inflammation; (iii) Fatty infiltration with ballooning degeneration; (iv) Fatty infiltration with lesions similar to alcoholic hepatitis and sinusoidal fibrosis, polymorphonuclear infiltration with or without Mallory hyaline. NASH is the name given to the third and fourth stages. NASH has been associated with insulin resistance, which includes obesity, diabetes, hypertriglyceridemia and hypertension. In addition, NASH has also been associated with hyperlipoproteinemia, jejunal bypass, parenteral nutrition, drugs (tamoxifen, steroid, massive estrogen, amiodarone, antiviral drugs nucleoside analogues aspirin/NSAIDS, methotrexate, nifedipine, perhexiline maleate, tetracycline, valproic acid), -1-antitrypsin, cryptogenic cirrhosis, requiring liver transplantation could have been the result of NASH (Das et al., 2006). (f) Ascites: Ascites is excess fluid in the space between the tissues lining the abdomen and abdominal organs (the peritoneal cavity). Increase in hepatic sinusoidal pressure is common and essential condition in ascites. Pathophysiological cause of ascites is systemic arteriolar vasodilation, activation of sodium and water retention, and sinusoidal portal hypertention (Arun et al., 2008). (g) Jaundice: There is yellowish discoloration of the skin, the conjunctival membranes over the sclerae and other mucous membranes caused by hyperbilirubinemia. The underlying causes of jaundice may be pre-hepatic, hepatic or post-hepatic. (h) Portal hypertension: The normal pressure gradients between the portal vein and the

inferior vena cava are 3 to 6 mm Hg. A pressure gradient higher than 10 mm Hg confronts a life threatening condition known as portal hypertension. It has been demonstrated that an increase in the resistance to portal blood flow caused by certain hepatic conditions elevates the portal vein pressure (Moan, 2009). (i) Focal liver disease: Focal liver disease (i.e., a lesion detected by imaging) may be

cystic, solid, vascular (or combinations thereof) or considerable overlap in the appearance between benign and malignant lesions. These are formed in response to underlying liver diseases e.g. HCC, cirrhosis. Various types of hepatic mass lesions are as follow (Rockey et al., 2009): BenignCysts, Hemangioma, Adenoma, Liver abscess (amebic or pyogenic), Focal nodular hyperplasia, Fatty infiltration, Rare primary liver neoplasms. MalignantHepatocellular cancer, Cholangiocarcinoma, Metastatic, Rare primary liver, neoplasms, Rare primary bile duct neoplasms.

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(j) Hepatic Failure and Hepatic Encephalopathy: Hepatic failure is the impairment of normal liver function. The biochemical effects of hepatic failure include significant alterations in glucose metabolism, buildup of tissue and serum ammonia, failure to remove metabolites and toxins from the circulation, and changes in the bioavailability of drugs normally cleared by the liver. Acute hepatic failure is most commonly caused by infection, inflammation, acute hypotension, toxin exposure, or fulminant primary liver disease. This combination of events can cause progressive encephalopathy, a spectrum of neurological dysfunction classically divided into four stages. Stage I is euphoria or depression, with mild confusion, slurred speech, and disordered sleep; stage II is lethargy and moderate confusion; stage III is marked confusion, incoherent speech, and somnolence; stage IV is coma with cerebral edema formation and elevated intracranial pressure. Special cases of liver failure with associated neurological dysfunction include Wilsons disease, Reyes syndrome, and the hepatic porphyrias (Panayiotis, 2005).

1.2.4 TYPES OF LIVER DISEASE The codes defined by the 10th revision of the International Classification of Diseases (ICD10) for all diseases of the liver are K70-K76. Chronic liver disease, which includes alcoholic liver disease, is specified by the codes K70 and K73-K74. ICD-10 codes were published by the World Health Organization in 1992 and have been used for coding and classifying causes of death since 1999 (Russo et al., 2004). Liver disorders can be grouped as:

(i) AUTOIMMUNE DISORDER

(a) Primary Biliary Cirrhosis: Primary biliary cirrhosis is a chronic cholestatic liver disease. Formation of antimitochondrial antibody against the E2 subunit of the pyruvate dehydrogenase complex is reported in more than 95% of patients. Cell mediated-immune system is also implicated in the pathology of disease. Progressive bile-duct injury from portal and periportal inflammation could result in progressive fibrosis and eventual cirrhosis (Talwalkar and Lindor, 2003). (b) Primary Schlerosing Cholagitis: PSC is a chronic cholestatic liver disease associated with inflammatory bowel disease (IBD) characterized by progressive destruction of bile ducts, which may result in the development of biliary cirrhosis. Immunologic as well as nonimmunologic factors (e.g. infection, toxins, and ischemia) could be responsible in

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genetically susceptible individuals. Antineutrophil cytoplasmic antibodies (ANCA) have been reported in approximately 85% of PSC patients (Mendes et al., 2004). (c) Autoimmune Hepatitis: Autoimmune hepatitis is a non-resolving inflammation of the liver having interface hepatitis on histologic examination with hyper-gammaglobulinemia, and serum auto-antibodies. On the basis of pattern of auto-antibodies detected there are two types of autoimmune hepatitis (Czaja et. al., 2002). Type 1 Signature auto-antibodies = (ANA) antinuclear antibodies and (SMA) = smooth

muscle antibodies Ancillary auto-antibodies = (pANCA) perinuclear antineutrophil cytoplasmic antibodies Auto-antigen = Unknown Type 2 Signature auto-antibodies = (LKM1) antibodies to liver/kidney microsome type 1 Ancillary auto-antibodies = (LC1) liver cytosol type 1 Auto-antigen = CYP2D6

(ii) INHERITED LIVER DISEASE

(A) Familial unconjugated hyperbillirubinemia In these disorders bilirubin cannot be broken down. One normal UGT1A1 allele is adequate to maintain normal plasma bilirubin concentration. Gilbert s syndrome (bilirubin level rarely exceeding 4mg/ dl and normal liver functions) is reported as an autosomal dominant disorder while Crigler-Najjar syndrome type 1(bilirubin level 20-45 mg/dl) an autosomal recessive disorder (Eugene et al., 2007). (a) Crigler-Najjar syndrome: Mutations in exons 2-5 of UGT1 gene or less often in exon 1 affecting the glucuronidation of bilirubin. In Crigler-Najjar syndrome type II phenobarbital induced enzyme induction is sufficient to prevent the development of kernicterus. (b) Gilberts syndrome: Bilirubin clearance reduced to one third of the normal. The molecular mechanism responsible for the disease is the addition of an extra dineucleotide sequence TA, to the transcription initiation sequence (TATAA box) of the promoter for A1 exon. Normal sequence i.e. A (TA)6 TAA is replaced by mutated sequence A(TA)7TAA.

(B) Familial conjugated hyperbillirubinemia Familial conjugated hyperbillirubinemia have following disorders (Eugene et al., 2007): Without cholestasis: include Dubin-Johnson syndrome, Rotor Syndrome, hepatic storage disease. With cholestasis: include (BRIC) benign recurrent intrahepatic cholestasis and (PFIC) progressive familial intrahepatic cholestasis.
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(a) Dubin-Johnson syndrome: The disease (person has mild jaundice throughout life) is caused by MRP2 gene encodeing an ATP-dependant canalicular plasma membrane transporter for bilirubin conjugate. (b) Rotor Syndrome: It is characterized by chronic mainly conjugated hyperbillirubinemia and phenotypically resembles the Dubin-johnson syndrome but hepatosplenomegaly and hepatic pigmentation has not been reported. (c) BRIC disease: This is the rare autosomal recessive disorder characterize by recurrent attacks of pruritis and jaundice with an episode of mild malaise, elevated transaminases level followed by increase in alkaline phospatase and conjugated bilirubin. In this disease FIC 1 gene that encode FIC1 protein is mutated. The protein belong to P-type ATPase family implicate in the transport of aminophospolipid from outer to inside of various type of cell membranes. Another mutation associated with BRIC type 2 of is bile salt export pump BSEP located on the bile canaliculli. (d) PFIC disease: Three phynotypically identical syndrome of PFIC are PFIC type 1or Byler disease (FIC1 mutation), type 2(Mutation of BSEP) and type 3(Mutation of MDR3 protein). Symptoms of all types are nearly same phenotypically such as malnutrition, growth retardation and end stage liver disease. High serum level of gamma glutamyle transferase activity has been observed in type 3 PFIC. (C) Carolis syndrome It is an autosomal recessive disorder associated with the family of fibropolycystic diseases and consists of Carolis disease (bouts of cholangitis, hepatolithiasis, and gallbladder stones) and congenital hepatic fibrosis (portal hypertension and oesophageal varices). Portal hypertension may result in hematemesis or melena.The disease is characterized by multiple segmental cystic dilatations of the intrahepatic bile ducts (Bayraktar, 2007). Other inherited liver disorders are Cystic fiberosis, Hemochromatosis, Alpha-1 antitrypsin deficiency, or Wilson's disease (Gines et al., 2010).

(iii) INFECTIOUS LIVER DISEASE

These include parasitic infections and infestations. (a) Viral Hepatitis: This include chronic hepatitis infections from viruses such as hepatitis A (enterovirus), hepatitis B (DNA virus), hepatitis C (single stranded RNA flavivirus), hepatitis D delta agent (RNA virus), hepatitis E (a non enveloped, single stranded RNA virus) and

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hepatitis G (two isolated, almost identical single-stranded, positive-sense RNA viruses) (Golla et al., 2004). (b) Pyogenic Liver Abscesses: Pyogenic liver abscesses have a polymicrobial infection usually with Gram negative aerobic and anaerobic organisms such as Escherichia coli, Klebsiella pneumoniae, bacteroides, enterococci, anaerobic streptococci, and microaerophilic streptococci that results in pus-filled area in the liver (Krige et al., 2001). (c) Amebic Liver Abscess: ALA is a protozoal (Entamoeba histolytica) infestation and form abscess by necrotic lysis of the liver tissue, which varies in size from a few centimeters to a large lesion. The abscess consists of the chocolate brown material of necrotic liver tissue, bile, fat and other products. The parasite is transmitted through contaminated water or vegetables by swallowing the cysts (Perez, 2006). (d) Hydatid Disease Of The Liver: Hydatid disease is a zoonotic infection caused by adult or larval stages of the cestode Echinococcus granulosus. Humans become infected via ingestion of eggs as a result of direct contact with infected dogs. (e) Schistosomiasis Of Liver: The infection is caused by trematodes (flukes)Schistosoma mansoni, Schistosoma japonicum. Adults S. mansoni and S. japonicum reside in the venules of the intestine. The liver disease manifestations are due to the deposition of eggs in the liver and the consequent liver fibrosis (Adonis et al., 2008).

(iv) ALCOHOL INDUCED LIVER DISEASE

Alcohol related liver damage can be categorised in to fatty liver, alcoholic hepatitis and alcoholic cirrhosis. Alcohol is metabolized in liver cells by CYP2E1 producing free radicals. Alcohol decreases the concentrations of vitamins A and E and one of the important antioxidant glutathione. Researchers have demonstrated that chronic alcohol consumption induces lipid peroxidation due to free radicals. Other mechanism in alcoholic liver damage are hypoxia, Inflammatory Agents (eicosanoids or cytokines (TGF by kuffer cells and TGF by various cells)), fibrosis and adduct formation. Free radicals and aldehyde, can attach chemically to proteins forming adduct that initiate the immune response by forming antibodies. Long term alcohol consumption stimulates the livers fat-storing (i.e., stellate) cells to produce collagen, the protein that forms scar tissue and finally cirrhosis (Maher, 1997).

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(v) NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) AND NONALCOHOLIC STEATOHEPATITIS (NASH)

The term Non-Alcoholic Fatty Liver Disease (NAFLD) was proposed by Matteoni et al., According to two hits hypothesis fat is accumulated in liver in the first hit, while the second hit of oxidative damage causes progression to non-alcoholic hepatitis. The initial fatty metamorphosis is accompanied by inflammation, cell ballooning, and Mallory bodies and the process is called non-alcoholic steatohepatitis. Free fatty acids are substrates that induce microsomal lipoxygenase of cytochrome p-450 and produce hepatotoxic free radicals. Formation of free radicals increases by mitochondrial beta oxidation (Santos et al., 2010).

(vi) DRUG-INDUCED LIVER DISEASE

Liver metabolizes the drug and other exogenous substances. Drug induced effects may be predictable (dose dependent response) or unpredictable (time dependant). Idiosyncratic druginduced liver injury (IDILI) is caused by drugs and has been categorized as being due to either immune idiosyncrasy or metabolic idiosyncrasy. The hepatic damage produced by drugs include interference with bilirubin uptake, excretion &conjugation cytotoxic injury, chorlestatic injury, steatosis, cirrhosis, phospholipidosis, liver tumours, hepatic necrosis, vascular lesions.

(vii) LIVER CANCER

Liver cancer can either arise in the hepatobiliary system itself (primary liver cancer) or metastasize from a tumour elsewhere in the body (secondary liver cancer).Primary liver cancer consists of either Hepatocellular carcinoma (HCC) which arises in liver cells (hepatocytes) or Cholangiocarcinoma (CCA) which arises in ductular epithelium of the biliary either within (intrahepatic) or outside of the liver (extra-hepatic). Hepatocellular carcinoma is not the same as metastatic liver cancer while it is usually caused by scarring of the liver. Patients with hepatitis B or C are at risk for liver cancer, even if they do not have cirrhosis (Reuben, 2005).

(viii) SPECIFIC LIVER DISEASES

There are some obstructive liver diseases that cause many complications as follows:

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(a) The BuddChiari Syndrome: It is a heterogeneous group of disorders characterized by hepatic venous outflow obstruction at the level of the hepatic venules, the large hepatic veins, the inferior vena cava, or the right atrium. This results in increased hepatic sinusoidal pressure and portal hypertension. The various cause of disease includes hypercoagulable states (Antithrombin III deficiency Prothrombin mutation, Myeloproliferative disorders, Protein C deficiency, Protein S deficiency etc.); tumoral invasion and idiopathic conditions etc. (b) Hepatic veno-occlusive Disease: There is obstruction of hepatic venous outflow at the level of the central or sublobular hepatic veins, or both (Menon, 2004).
Hepatic venous outflow tract Obstruction
Portal venous perfusion Portal venous thrombosis

Venous stasis and congestion

Cirrhosis

Hypoxic damage to adjacent hepatic parenchymal cells

Nodular regenerative hyperplasia

Ischemic injury to the sinusoidal lining cells

Centrilobular fibrosis

Hepatocyte necrosis in the centrilobular regions

Free radicals, and oxidative injury to the hepatocytes

Figure- 1.7: Consequences of obstructive liver disease

(ix) LIVER DISEASE IN PREGNANCY

Very few alterations in standard liver tests occur as a result of pregnancy. Table shows that increase in aminotransferases and GGTP signify pathology, and should prompt a search for disease. Table 1.2: Liver enzymes during pregnancy
Standard liver tests Aminotransferases (AST and ALT) g-glutamyl transpeptidase (GGTP) Serum bile acid Albumin Cholesterol Affects of pregnancy on enzymes Normal range Normal range Normal range Lower Higher

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(a) Cholestasis of pregnancy: The pathogenesis may involve inheritance, progesterone or cholestatic effects of estrogen. (b) HELLP (hemolysis, elevated liver tests, low platelets) syndrome: In HELLP syndrome, patients have symptoms of pre-eclampsia as well as thrombocytopenia. (c) AFLP (acute fatty liver of pregnancy): Patients with acute fatty liver of pregnancy have true hepatic dysfunction, and may, or may not, have signs of pre-eclampsia and HELLP syndrome. The hepatic histology is different from that of HELLP syndrome, and shows a microvesicular fatty infiltrative disorder similar to that in Reyes syndrome. Affected infants are deficient in one of the enzymes of mitochondrial beta oxidation of fatty acids, long chain 3-hydroxyl-acyl CoA dehydrogenase (LCHAD) and may develop nonketotic hypoglycaemic coma. The pre-existing liver disorders of pregnant women may be exacerbated by underlying pregnancy state. Such diseases are viral hepatitis, drug-induced hepatitis, gallstone disease and malignancy. Cholestasis of pregnancy is common, and should be considered in the differential diagnosis of abnormal liver tests presenting initially in the second trimester. HELLP and AFLP are considered in the differential diagnosis of abnormal liver tests in the second half of pregnancy, usually in the third trimester (Riely, 1999).

(x) PAEDIATRIC LIVER DISEASE

Common liver paediatric problems are as follow: (a) Biliary Atresia (BA): It is a cholangiodestructive disease of both intra- and extrahepatic parts of the biliary system and remains the commonest indication for pediatric liver transplantation throughout the Western country. BA leads to cirrhosis and ultimately liver failure (Davenport, 2006). (b) Cystic Fibrosis: CF transmembrane conductance regulator (CFTR) is located at the apical membrane of the cells lining the intra-hepatic bile ducts. The abnormalities of chloride transport inhibit the hydration of the canalicular-produced bile, thereby increased viscosity. In addition, excessive mucus composed of proteoglycans produced by intra-hepatic biliary epithelial cells increases the viscosity of CF bile. It has been proposed that other factors such as environmental, nutritional or non-CFTR genetic influences may be significant. Mutations in the alpha-1-antitrypsin, mannose binding lectin (and glutathione-S-transferase genes may impose independent risk factors for CF liver disease (Bush et al., 2006).

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1.3 EPIDEMOLOGY OF LIVER DISEASE

Introduction

It has been estimated that 170 million persons has been chronically with the virus with 3 to 4 million persons newly infected each year having the global prevalence of Hepatitis C virus (HCV) infection is around 2%. In India, studies on voluntary or mixed donors have reported a prevalence of hepatitis C below 2% while professional donors have noted a prevalence of 55.3% and 87.3% (Mukhopadhya , 2008). It is commonly seen in daily clinical practice that diabetes mellitus (DM) coexist with chronic liver disease. DM may have a metabolic pathway leading to CLD via the development of NAFLD. Epidemiology of NAFLD suggests that it affects a large proportion of the general population of several countries and recognized as one of the most common liver diseases worldwide (Arab et al., 2010). NAFLD has the potential for major economic impact on healthcare costs because of liver-related morbidity and mortality. The overall prevalence of NAFLD is 15 to 40% in western countries and 9-40% in Asian countries. In India NAFLD prevalence is 9% with 19% in adult population (Amarapurkar et al., 2007). Nonalcoholic fatty liver disease (NAFLD) affects 10%-30% of the general U.S. population and can progress to significant fibrosis and cirrhosis (Neuschwander et al., 2010). NAFLD/NASH will become one of the primary reasons for liver transplants throughout the world by the year 2020. NAFLD has been reported North America, Australia , Japan , India , the Middle East , South America , North Europe , South Europe , New Zealand , and Southeast Asia (Santos et al., 2010). About 2 billion people acutely and 350 million people chronically affected with Hepatitis B virus infection (HBV) and become a global public health problem. In 2005, 6% of liver-related deaths and 7% of orthotopic liver transplants (OLT) was attributed to ALF (Acute Liver Failure) in the United States (Khashab et al., 2007).

1.4 HEPATOTOXINS
Hepatotoxins are classified as: 1.4.1 INORGANIC AGENTS Inorganic agents such as iodides, hydrazine derivatives and trace Elements (Metal and Metalloids) i.e., antimony, arsenic, beryllium, bismuth, boron, cadmium, chromium, cobalt, copper, iron, lead, manganese, mercury, gold, phosphorous, selenium, tellurium, thallium zinc causes hepatotoxicity (Zimmerman, 1999).

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1.4.2 ORGANIC AGENTS This can be categorized in to natural and synthetic
(i) NATURALS HEPATOTOXINS

Introduction

(a) Fungal (Mycotoxins) The fungi flourish on the food material in various climatic and living conditions and produce hepatotoxins such as amanitin, phalloidin (Amanita phalloides); aflatoxin B1; G1(Aspergillus flavus); luteoskyrin, chlorine containing polypeptide(Penicillium isiandicum) (Schoental R, 1963). Other examples are ochratoxins, rubratoxins, sterigmatocystin, griseofulvin, sporidesmin, tetracycline and other antibiotics (Zimmerman, 1999). (b) Plant Hepatotoxins Pyrrolizidine alkaloid represent a large group of substances present in the various family of genera Senecio, Echium, Erechtites, Crotalaria, Cytisus, Heliotopium, Macrotomia etc. Examples of these include Retrorsine, retronecine (Senecio retrorsus L.) etc. Another important class of pant hepatotoxins is N-oxide alkaloids differ from other alkaloids by having an oxygen on nitrogen atom e.g. N-oxide of Lasiocarpine. Other plant hepatotoxins are Cycasin, Methylazoxymethanol (Cycas circinalis L.) (Schoental, 1963). (c) Bacterial Toxins These agents have exotoxins such as Cyanobacteria (mycrocystins), Bacillus cereus (cereulide) and some stains of Escherichia coli (ethionine). Streptococcus hemolyticus (i.e., S. pyogenes), Corynebacterium diphtheriae, Clostridium botulinus are also included in this group. Some endotoxins also produce hepatotoxicity (Zimmerman, 1999).

(ii) SYNTHETIC HEPATOTOXINS These are categorized in to non medicinal and medicinal agent. Non medicinal agents are azo compounds, chloroaromatic compounds, haloalkanes and haloolephins, nitroalkanes, nitroaromatic compounds, organic amines, phenol and derivatives etc. It has been estimated that more than 900 medicinal drugs reported hepatotoxicity (Zimmerman, 1999).

1.5 OXIDATIVE STRESS

Liver, the aerobic organ generates reactive oxygen species that cause oxidative tissue damage. These radicals react with cell membranes and induce lipid peroxidation or inflammation that have been implicated as important pathological mediators in many clinical disorders such as heart disease, diabetes, gout and cancer. A major defense mechanism is the

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antioxidant enzymes, which convert active oxygen molecules into non-toxic compounds. In recent years oxidative stress has been implicated in a variety of degenerative processes, diseases and syndromes (Dash et al., 2007).

1.5.1 Free Radicals A radical is defined as any atom or biomolecule that contains unpaired electrons. These unpaired electrons render the free radical chemically reactive. The important free radicals in biological systems are the superoxide anion (O 2), the hydroxyl radical (HO) and nitric oxide (NO.). The oxygen is metabolized in the mitochondria and converted to the radical O 2. Some other species are intermediate in the metabolism of O2 or NO but are not radicals. These intermediate species along with the radical species are called reactive oxygen species (ROS) and reactive nitrogen species (RNS) respectively. Examples of non radical ROS are hydrogen peroxide (H2O2), hypochlorous acid and that of nonradical RNS is peroxynitrite ONOO (Georgios et al., 2005).

1.5.2 Antioxidants An antioxidant is any substance that when present at low concentrations, compared with those of an oxidizable substrate significantly delays or prevents oxidation of the substrate. In the biological system, small molecular weight substances known as endogenous antioxidants are able to prevent initiation of oxidative damage or limit its propagation and enzymatically convert and detoxify ROS and RNS. When produce at levels that cannot be counteracted by endogenous antioxidant systems, an imbalance takes place, called oxidative stress and leads to the damage of lipids, proteins, carbohydrates, and nucleic acids. Hepatocytes tend to be resistant to injury by ROS and RNS, since they contain high intracellular concentrations of glutathione (GSH), superoxide dismutase (SOD), catalase, and lipid soluble antioxidants. The plants contain carotenoids, a group of colored pigments that is the precursor of vitamin A and exert their antioxidant action as free-radical scavengers. Another group of antioxidants are phenols. Plants contain a large range of phenols, including tocopherols, tocotrienols, flavonoids, anthocyanidins, and phenylpropanoids. They inhibit peroxidation by acting as chain-breaking peroxyl-radical scavengers. Besides, they scavenge ROS and RNS such as HO, ONOO, and hypochlorous (Georgios et al., 2005).

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