INTRODUCTION

INTRODUCTION
Metal toxicity is a major medical concern. Of particular concern are Heavy metals which occur naturally in the earths crust and are defined in physiochemical terms as metal with density at least five times as great as water. The definition translates into an approximate heavy metal minimum density of 5, and in addition to cadmium, lead and mercury the metals zinc, copper, iron, cobalt, nickel, tin, manganese and molybdenum also qualify. Metal toxicity affects all organ systems and can result in wide-ranging and nonspecific symptoms; however, the central nervous system (CNS) is especially susceptible to damage from metals (Neustadt et al., 2007). Heavy metal pollution of water is a major environmental problem facing the modern world. The global heavy metal pollution is increasing in the environment due to increase in number of industries. Many industrial wastewaters contain heavy metals like cadmium, lead, zinc, cobalt and chromium. Among heavy metals, chromium plays a major role in polluting our water environment. Chromium can co-exist in the environment in two oxidation states viz., trivalent chromium and hexavalent chromium. The hexavalent chromium is released from various industries such as electroplating, leather tanning, textile printing, textile preservation and metal finishing. The compounds of chromium have been known to be strong carcinogens and mutagens that can reach the target organs of human through drinking water (Chidambaram et al., 2009). Metals generate many of their deleterious effects through the formation of free radicals, resulting in DNA damage, lipid peroxidation, depletion of protein sulfhydryls (e.g. glutathione), and other effects. These reactive radicals include a wide range of chemical species, including oxygen, carbon, and sulfur radicals originating from the superoxide radical, hydrogen peroxide, and lipid peroxides, and also from chelates of amino acids, peptides, and proteins complexes with the toxic metals. One of the major mechanisms of metal toxicity is damaging of mitochondria via depletion of glutathione, an endogenous thiol containing antioxidant which result in the generation of free radical and mitochondrial damage (John, et al., 2007). Chromium (Cr) is a metallic element belonging to the first transitional series of the periodic table has atomic number 24 and atomic mass 51.996 amu. The three more stable forms in which chromium occurs in the environment are the 0 (metal and alloys), +3 (trivalent chromium), and +6 (hexavalent chromium), valence states. In the +3 valance state, the chemistry of chromium is dominant by the formation of stable complexes with the both
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organic and inorganic ligands (Hartford, 1979). In the +6 valance state, chromium exist s as oxo species such as CrO3 and CrO42- that are strongly oxidizing (Peter et al., 1998). Chromium in the ambient air occurs from natural sources, industrial and product uses, and burning of fossil fuels and wood. The most important industrial sources of chromium in the atmosphere originate from ferrochrome production. Ore refining, chemical and refractory processing, cement-producing plants, automobile brake lining and catalytic converters for automobiles, leather tanneries, and chrome pigments also contribute to the atmospheric burden of chromium. Chromate chemicals used as mist inhibitors in cooling towers and the mist formed during chrome plating are probably the primary sources of Cr (VI) emitted as mists in the atmosphere (Towill et al., 1978). Chromium (Cr) is considered as an essential nutrient and health hazard. It is because Cr exists in more than one oxidation states. Specifically, Cr in oxidation state +6 written as Cr(IV)considered as harmful even in small intake quantity whereas Cr in oxidation state +3 written as Cr(III) considered as an essential for good health (Jacques Guertin., 2004). Cr (VI) is unstable in the body, and is rapidly reduced to Cr (V), Cr (IV) and ultimately to stable Cr (III) by endogenous reducing agents (Assem et al., 2007). Cr toxicity depends on its concentration. The federal maximum concentration level (MCL) for total Cr drinking water is 100g/l (Jacques Guertin., 2004). The concentration of Cr occurring naturally in the earth normal mineral soil ranges from about with a mean of 200mg/kg worldwide. Human activities further contribute to Cr in the environment. The greatest anthropogenic source of Cr (VI) emissions are (1) Chromium plating, (2) chemical manufacturing of Cr and (3) evaporative cooling towers. While combustion of coal and oil also release large quantities of chromium only approximately 0.2% of this Cr (VI). For any substance to have an adverse health effect there must first be an exposure to that substance and then it must enter the body. The common exposure routes or intake modes are, (1) Ingestion (eating and drinking), (2) Dermal contact (3) Inhalation (breathing). Chromium ingestion pathways are usually drinking water and contaminated soil. It is only 2% to 3% and extra quantity excreted through urine. The gastric juice rapidly reduced the Cr (VI) to Cr (III) which is an essential nutrient and no any hazardous effect. But Cr causes cancer and some other disorders through inhalation and dermal contact cause cancer and some other disorders (Jacques., 2004). Studies on mice exposed that Cr (VI) is carcinogenic for animals. Chronic inhalation in mice causes lung tumors for exposure to 4.3mg/m3 of Cr (VI). However, a number of
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chronic animal studies showed no carcinogenic effects in rats, rabbits, or guinea pigs exposed to 1.6 mg/m3 of Cr (VI). Thus, cancer effects on animals seem to depend on the type of animal. A number of studies carried out to evaluate the carcinogenicity of chromium compounds in various solubility and oxidation states on rodents (mainly mice, rabbits and hamsters). The intramuscular injection of lead chromate in rates resulted in development of renal carcinomas. (Silvio., 2000) Taxonomy Picrorhiza karroo ROYLE ex BENTH (family Scrophulariaceae) Synonyms Picrorhiza scrophulariiflora PENNELL (part); Neopicrorhiza scrophulariiflora (PENNELL)HONG (part)The genus Picrorhiza was originally considered monotypic,

comprising the single widespread species P. kurrooa, until PENNELL (1943) distinguished a second species, Picrorhiza scrophulariiflora, which was subsequently placed in a separate genus, Neopicrorhiza, by H ONG (1984), although the original generic name is still widely used for the latter species. The two species are apparently largely or entirely allopathic, with P. kurrooa occurring in the Western Himalaya and N. (P.) scrophulariiflora found further east, although a sketch map in SMIT indicates a small area of apparent sympatry in northeast Uttar Pradesh (the Himalayan sections of which are now Uttaranchal), India. MILL has subsequently described a second species of Neopicrorhiza (N. minima) from northern Bhutan. Trade names Gorki (Gurung), Hodling (she), Honglen (she), Honglen (tib), Hugling (she), Kadu (Himachal Pradesh), Karroo (Pakistan), Katuka (san), Kaur Kutki (Pakistan), Kuraki (Tamang), Kutaki (Gurung, Lhotshampkha), Kutki (Lhotshampkha, nep), NgoHonglen, Picrorhiza rhizome (chi), Putishing (Dzongkha), Xuanhulian (chi) (Somesh et al., 2012). Picrorhiza kurrooa is recorded from India and Pakistan. In India, SMIT in 2000 lists localities in Jammu & Kashmir, Himachal Pradesh and Uttar Pradesh (the Himalayan sections of which are now Uttaranchal). The main altitudinal range is 3000-4300 m, although there are records from as low as 2500 m and high as 5300 m. It occurs from 2700-4500 m in Himachal Pradesh, with its distribution in the Great Himalayan National Park complex fairly well known both through scientific surveys and the native knowledge of collectors. In Pakistan the species is reported to be declining because of habitat disturbance related to changes in land use brought about by increased tourism, human settlement and road building. Unsustainable harvest and natural disasters, e.g. floods and landslides, are also considered a threat, though less severe than habitat disturbance. Pollution is considered a
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lesser threat. Climate change is causing an upward shift in the permanent snow cover and therefore population declines in the lower elevation range of distribution. Animal studies have shown that picrorhiza kurroa has a powerful antioxidant and antiinflammatory effect. It has also shown that the active constituents of picrorhiza kurroa may prevent liver toxicity and the ensuing biochemical changes caused by numerous toxic agents. In other animal studies picrorhiza raised depleted glutathione levels in rats infected with malaria, boosting detoxification and antioxidation (Murphy et al., 2000). Silymarin is chemical extract of milk thistle. The terms milk thistle, flavonoids, silymarin, and silybin are generally used interchangeably; however, each of these compounds has specific characteristics and actions, with an intrinsic beneficial or toxic effect. In the last 10 years, about 12,000 papers have been published on these substances, used as antioxidants or chemopreventives and anticancer agents, and especially as hepatoprotectants. Other names: Marian thistle, St. Mary's thistle, Our Lady's thistle, Family: Asteraceae, Distribution: Native to the Mediterranean region of Europe, but naturalized in California and the eastern US. The plant has following features, tall herb with prickly leaves and a milky sap. Small, hard fruits (achenes), a feathery tuft or pappus is removed Milk white veins in the leaves (originated in the milk of the Virgin Mary which once fell upon the plant).The parts of plants which are used for medicine are ripe fruit (not seeds), root, leaves, and hull. The chemically silymarin is chemical mixture of antihepatotoxic principles; 1-4% conc. in fruit. Shown to consist of a large number of flavonolignans, including principally silybin accompanied by isosilybin, dehydrosilybin, silydianin, silychristin, etc. Oxidative stress may be a key factor in the onset of certain diseases, including cancer. Oxy-radicals play important roles in the initiation, promotion, and progression of carcinogenesis. It is considered that a significant event in oxy radical mediated carcinogenesis is the extensive oxidative damage to the nuclear membrane, which leads to deoxyribonucleic (DNA) damage such as DNA single strand breaks and possibly facilitation of carcinogenesis. To prevent cellular damage leading to cancer caused by oxy-radicals, the level of tissue antioxidants is critical. Interest in natural sources of antioxidant molecules for use in the food, beverage and cosmetic industries has resulted in a large body of research in recent years. It is well known that natural antioxidants extracted from herbs and spices have high antioxidant activity and are used in many foods applications. Of these substances, the phenolic compounds, which are widely distributed, have the ability to scavenge free radicals by single-electron transfer. Silymarin is isolated from the fruits and seeds of the milk thistle

(Silybum marianum) and in reality area mixture of three structural components: silibinin, silydianine, and silychristine (Sharker et al., 2010). Milk thistle is a member of the Asteraceae family. It has been reported as havingmultiple pharmacological activities includingantioxidant, hepatoprotectant and antiinflammatory agent, antibacterial, antiallergic, antimutagenic,antiviral, antineoplastic, antithrombotic agents, andvasodilatory actions.Asghar et al. 2008 suggested that silymarin may beused in preventing free radical-related diseases as adietary natural antioxidant supplement.The carob tree is widelycultivated in the Mediterranean countries. The fruit of the carob tree is abrown pod 10-25 cm in length. The two principalcomponents of the carob fruit are the pulp and seed.The important ingredient of the seeds is galactomannan which is known for its thickening effectsand is widely used in the food industry. The main application of carob pods is animalfeed production, but in a few countries the pods arealso used as a cocoa substitute. Carob pods contain lots of polyphenols, especiallyhighly condensed tannins. A phenolic analysisrevealed high contents of different forms of Gallicacid (freegallic acid, gallotannins, and methyl gallate) and large amounts of quercetin andmyricetin derivatives.Thus, carob fiber combinestwo positive nutritional ingredients,

namelypolyphenols and dietary fiber. Recent studiesdiscovered that carob fiber has cholesterol loweringactivities in persons suffering from hypercholesterolemia. There are otherreported antioxidants properties in different in vitro test systems (Akkaya and Yilmaz., 2012). The aim of study was to demonstrate the hepatoprotective activity of silymarin and Picrorhiza against the heavy metal chromium induced hepatotoxicity in mice. For this the antioxidant status of liver was assessed by measuring the activities of the intracellular antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and MDA and also measure the creatinine, urea and chromium metal level in mice blood serum.

LITERATURE REVIEW

LITERATURE REVIEW
Exposure to toxic metals has become an increasingly recognized source of illness worldwide. Both cadmium and arsenic are ubiquitous in the environment, and exposure through food and water as well as occupational sources can contribute to a well-defined spectrum of disease. The symptom picture of arsenic toxicity is characterized by dermal lesions, anemia, and an increased risk for cardiovascular disease, diabetes, and liver damage. Cadmium has a significant effect on renal function, and as a result alters bone metabolism, leading to osteoporosis and osteomalacia. Cadmium-induced genotoxicity also increases risk for several cancers. The mechanisms of arsenic- and cadmium-induced damage include the production of free radicals that alter mitochondrial activity and genetic information. The metabolism and excretion of these heavy metals depend on the presence of antioxidants and thiols that aid arsenic methylation and both arsenic and cadmium metallothionein-binding. Sadenosyl-methionine, lipoic acid, glutathione, selenium, zinc, N-acetylcysteine (NAC), methionine, cysteine, alpha-tocopherol, and ascorbic acid have specific roles in the mitigation of heavy metal toxicity. Several antioxidants including NAC, zinc, methionine, and cysteine, when used in conjunction with standard chelating agents, can improve the mobilization and excretion of arsenic and cadmium (Lily et al., 2008). Continued human population growth and industrialization result in increased contamination of wildlife habitats. Effects of such habitat deterioration on the well-being of natural populations are unclear. Exposure to contaminants may impair immune competence, thereby in-creasing disease susceptibility. The mammalian immune system is important in maintaining health and in its sensitivity to toxins. In our study conducted from May 1999 through May 2001, we examined assays of immune competence in the white-footed mouse (Peromyscusleucopus ) that inhabited reference sites and sites significantly contaminated with mixtures of heavy metals. We estimated potential exposure and uptake of heavy metals by measuring the level of each contaminant in representative soil and tissue samples. Intra individual variation across mice, but not sex, explained a large portion of the overall variance in immune response, and spleen weight was significantly affected by mouse age. We found no evidence that residence on contaminated sites had any effect on immune pathology and humoral immunity as measured in our study. We suggest that field and laboratory studies in eco toxicology provide estimates of exposure to contaminants (i.e., tissue analyses) to establish a database suitable to clarify the dose-response relationship between contaminants and target systems (Jannifer et al., 2004).
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The liver is the largest organ inside the body. In an adult, it is about the size of a football and weighs close to three pounds. It is located behind the ribs in the upper right-hand portion of the abdomen. Shaped like a triangle, the liver is dark reddish-brown and consists of two main lobes. There are over 300 billion specialized cells in the liver that are connected by a well organized system of bile ducts and blood vessels called the biliary system. (Hepatitis B Foundation) The liver lies almost entirely within the rib cage, caudal to the diaphragm. The gallbladder is attached to the liver via the hepatic, cystic, and common bile ducts. The hepatic ducts convey bile from the liver lobes and may join the cystic duct by one or more stems. The major pancreatic duct joins the common bile duct before its entry into the duodenum (Waltham., 1999). The liver receives blood from the intestinal tract via the portal vein, which is then delivered to the vena cava through the hepatic vein. The liver therefore receives all materials absorbed from the gastrointestinal tract except for some lipids that passes through the mesenteric lymphatic as chyle. The histologic unit of the liver is the lobule. In cross section, the lobule appears as a hexagon with the central vein (a branch of the hepatic vein) at the center and the portal triad at the corners. The portal triad consists of branches of the portal vein, the hepatic artery, and the bile duct. This lobular pattern is a result of the hydrodynamics of the blood flow through the liver (Shivaraj et al., 2009). Human are exposed to a great number of xenobiotics during the course of our lifetime, including a variety of pharmaceuticals and food components. Many of these compounds show little relationship to previously encountered compounds or metabolites, and yet our bodies are capable of managing environmental exposure by detoxifying them. To accomplish this task, our bodies have evolved complex systems of detoxification enzymes. These enzyme systems generally function adequately to minimize the potential of damage from xenobiotics. However, much literature suggests an association between impaired detoxification and disease, such as cancer, Parkinsons disease, fibromyalgia, and chronic fatigue/immune dysfunction syndrome. Therefore, accumulated data suggests an individuals ability to remove toxins from the body may play a role in etiology or exacerbation of a range of chronic conditions and diseases. The detoxification systems are highly complex, show a great amount of individual variability, and are extremely responsive to an individuals environment, lifestyle, and genetic uniqueness.

When food is eaten, the nutrients travel down the throat, into the stomach and then on to the intestines. These organs break up and dissolve the food into small pieces that can be absorbed into the bloodstream. Most of these small particles travel from the intestines to the liver, which filters and converts the food into nourishment that the bloodstream delivers to cells that need it. The liver stores this nourishment and releases it throughout the day, as the body needs it. The liver is such an important organ that we can survive only one or two days if it shuts downif the liver fails, your body will fail, too. Fortunately, the liver can function even when up to 75% of it is diseased or removed. This is because it has the amazing ability to create new liver tissue (i.e. it can regenerate itself) from healthy liver cells that still exist. The liver has a number of important functions, some of the main ones being: Detoxification of potentially toxic chemicals from both inside and

outside of the body including drugs, alcohol and toxins from intestinal microbes. Accomplished with antioxidant nutrients and enzymes such as glutathione. The liver detoxifies these harmful substances by a complex series of chemical reactions. The role of these various enzyme activities in the liver is to convert fat soluble toxins into water soluble substances that can be excreted in the urine or the bile depending on the particular characteristics of the end product. Storage of sugar as 'glycogen' and regulation of blood sugar levels. Production and storage of proteins as well as the regulation of many

substances involved in protein metabolism. Production of bile which aids in the digestion of fats. Production of blood proteins, clotting factors and substances important

to the production of red blood cells (erythrocytes) Regulation of a number of hormones. Neutralization of 'free-radicals' by antioxidants. Free radicals are

highly reactive oxygen molecules that can damage tissues. Storage of vitamins, mainly iron, copper, B12, vitamins A, D, E and K It plays an important role in digestion (breaking nutrients down) Involved with assimilation (building up body tissues). Red blood cells, which are responsible for carrying oxygen around the

body, are also produced in the liver The liver is the organ in the body that breaks down poisons present in the blood, such as alcohol, and removes toxic compounds such as bio toxins and heavy metals.
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The gallbladder then secretes into the digestive tract with its bile, for removal from the body in the faces. Bile serves a number of functions, but helps to lubricate the digestive tract and acts as a medium to eliminate toxins from the liver. Detoxification Mechanisms, R.T. Williams defined the field of detoxification. Williams proposed that these non-reactive compounds could be bio transformed in two phases: functionalization, which uses oxygen to form a reactive site, and conjugation, which results in addition of a water-soluble group to the reactive site these two steps, functionalization and conjugation, are termed Phase I and Phase II detoxification, respectively. The result is the biotransformation of a lipophilic compound, not able to be excreted in urine, to a water-soluble compound able to be removed in urine (Gramenzi et al., 2006). Laboratory liver tests are broadly defined as tests useful in the evaluation and treatment of patients with hepatic dysfunction. The liver carries out metabolism of carbohydrate, protein and fats. Some of the enzymes and the end products of the metabolic pathway which are very sensitive for the abnormality occurred may be considered as biochemical marker of liver dysfunction. Some of the biochemical markers such as serum bilirubin, alanine amino transferase, aspartate amino transferase, ratio of amino transferases, alkaline phosphatase, gamma glutamyl transferase, 5 nucleotidase,ceruloplasmin, fetoprotein are considered here. An isolated or conjugated alteration of biochemical marker of liver damage in patients can challenge the clinicians during the diagnosis of disease related to liver directly or with some other organs. The term liver chemistry tests is a frequently used but poorly defined phrase that encompasses the numerous serum chemistries that can be assayed to assess hepatic function and/or injury. Bilirubin is the catabolic product of hemoglobin produced within the

reticuloendothelial system, released in unconjugated form which enters into the liver, converted to conjugated forms bilirubin mono and diglucuronides by the enzyme UDPglucuronyl transferase. Normal serum total bilirubin varies from 2 to 21mol/L. The indirect (unconjugated) bilirubin level is less than 12mol/L and direct (conjugated) bilirubin less than 8mol/L. The serum bilirubin levels more than 17mol/L suggest liver diseases and levels above 24mol/L indicate abnormal laboratory liver tests. Jaundice occurs when bilirubin becomes visible within the sclera, skin, and mucous membranes at a blood concentration of around 40 mol/L. The occurrence of unconjugated hyperbilirubinemia due to over production of bilirubin, decreased hepatic uptake or conjugation or both. It is observed in genetic defect of UDP-glucuronyl transferase causing Gilbert\'s syndrome,
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Crigler-Najjar syndrome and reabsorption of large hematomas and ineffective erythropoiesis. In viral hepatitis, hepatocellular damage, toxic or ischemic liver injury higher levels of serum conjugated bilirubin is seen. Hyperbilirubinemia in acute viral hepatitis is directly proportional to the degree of histological injury of hepatocytes and the longer course of the disease. It has been observed that the decrease of conjugated serum bilirubin is a bimodal fashion when the biliary obstruction is resolved. Parenchymal liver diseases or incomplete extrahepatic obstruction due to biliary canaliculi give lower serum bilirubin value than those occur with malignant obstruction of common bile duct but the level remains normal in infiltrative diseases like tumours and granuloma. Raised Serum bilirubin from 20.52 mol/L to 143.64mol/L in acute inflammation of appendix has been observed. In normal asymptomatic pregnant women total and free bilirubin concentrations were significantly lower during all three trimesters and a decreased conjugated bilirubin was observed in the second and third trimesters. The recent study has shown that a high serum total bilirubin level may protect neurologic damage due to stroke (Shivaraj et al., 2009). Alanine amino transferase (ALT) ALT is found in kidney, heart, muscle and greater concentration in liver compared with other tissues of the body. ALT is purely cytoplasmic catalysing the transamination reaction. Normal serum ALT is 7-56 U/ L. Any type of liver cell injury can reasonably increases ALT levels. Elevated values up to 300 U/L are considered nonspecific. Marked elevations of ALT levels greater than 500 U/L observed most often in persons with diseases that affect primarily hepatocytes such as viral hepatitis, ischemic liver injury (shock liver) and toxin-induced liver damage. Despite the association between greatly elevated ALT levels and its specificity to hepatocellular diseases, the absolute peak of the ALT elevation does not correlate with the extent of liver cell damage. Viral hepatitis likes A, B, C, D and E may be responsible for a marked increase in amino transferase levels. The increase in ALT associated with hepatitis C infection tends to be more than that associated with hepatitis A or B. Moreover in patients with acute hepatitis C serum ALT is measured periodically for about 1 to 2 years. Persistence of elevated ALT for more than six months after an occurrence of acute hepatitis is used in the diagnosis of chronic hepatitis. Elevation in ALT levels is greater in persons with nonalcoholic steato hepatitis than in those with uncomplicated hepatic steatosis. In a recent study the hepatic fat accumulation in childhood obesity and nonalcoholic fatty liver disease causes serum ALT elevation. Moreover increased ALT level was associated with reduced insulin sensitivity, adiponectin and glucose tolerance as well as increased free fatty acids and triglycerides. Presence of Bright liver and elevated plasma ALT level was independently associated with increased risk
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of the metabolic syndrome in adults. ALT level is normally elevated during 2nd trimester in asymptomatic normal pregnancy. In one of the study, serum ALT levels in symptomatic pregnant patients such as in hyperemesis gravidarum was 103.5U/L, in preeclampsia patients was 115U/L and in haemolysis with low platelet count patients showed 149U/L. However, in the same study ALT rapidly drops more than 50% of the elevated values within 3 days indicating the improvement during postpartum. One of the recent studies has shown that coffee and caffeine consumption reduces the risk of elevated serum ALT activity in excessive alcohol consumption, viral hepatitis, iron overload, overweight, and impaired glucose metabolism. AST catalyse transamination reaction. AST exist two different isoenzyme forms which are genetically distinct, the mitochondrial and cytoplasmic form. AST is found in highest concentration in heart compared with other tissues of the body such as liver, skeletal muscle and kidney. Normal serum AST is 0 to 35U/L. Elevated mitochondrial AST seen in extensive tissue necrosis during myocardial infarction and also in chronic liver diseases like liver tissue degeneration and necrosis. About 80%of AST activity of the liver is contributed by the mitochondrial isoenzyme, whereas most of the circulating AST activity in normal people is derived from the cytosolic isoenzyme. However the ratio of mitochondrial AST to total AST activity has diagnostic importance in identifying the liver cell necrotic type condition and alcoholic hepatitis. AST elevations often predominate in patients with cirrhosis and even in liver diseases that typically have an increased ALT. AST levels in symptomatic pregnant patient in hyperemesis gravidarum were 73U/L, in preeclampsia 66U/L, and 81U/L was observed in hemolysis with low platelet count and elevated liver enzymes. The ratio of AST to ALT has more clinical utility than assessing individual elevated levels. A coenzymepyridoxal-5\'-phosphate deficiency may depress serum ALT activity and consequently increases the AST/ALT ratio. The ratio increases in progressive liver functional impairment and found81.3% sensitivity and 55.3% specificity in identifying cirrhotic patients. Whereas mean ratio of1.45 and 1.3 was found in alcoholic liver disease and post necrotic cirrhosis respectively. The ratio greater than 1.17 was found in one year survival among patients with cirrhosis of viral cause with 87% sensitivity and 52% specificity. An elevated ratio greater than 1 shows advanced liver fibrosis and chronic hepatitis C infection. However, an AST/ALT ratio greater than 2characteristically is present in alcoholic hepatitis. A recent study differentiated nonalcoholic steato hepatitis (NASH) from alcoholic liver disease showing AST/ALT ratio of 0.9 in NASH and 2.6 inpatients with alcoholic liver disease. A mean ratio of 1.4 was found in patients with cirrhosis related to NASH. Wilson\'s
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disease can cause the ratio to exceed 4.5 and similar such altered ratio is found even in Hyperthyroidism (Shivaraj et al., 2009). ALP is present in mucosal epithelia of small intestine, proximal convoluted tubule of kidney, bone, liver and placenta. It performs lipid transportation in the intestine and calcification in bone. The serum ALP activity is mainly from the liver with 50% contributed by bone. Normal serum ALP is 41 to 133U/L. In acute viral hepatitis, ALP usually remains normal or moderately increased. Elevation of ALP with prolonged itching is related with Hepatitis A presenting cholestasis. Tumours secrete ALP into plasma and there are tumour specific isoenzymes such as Regan, Nagao and Kasahara. Hepatic and bony metastasis can also cause elevated levels of ALP. Other diseases like infiltrative liver diseases, abscesses, granulomatous liver disease and amyloidosis may cause a rise in ALP. Mildly elevated levels of ALP may be seen in cirrhosis, hepatitis and congestive cardiac failure. Low levels of ALP occur in hypothyroidism, pernicious anaemia, zinc deficiency and congenital

hypophosphatasia. ALP activity was significantly higher in the third trimester of asymptomatic normal pregnancy showing extra production from placental tissue. ALP levels in hyperemesis gravidarum were 21.5U/L, in preeclampsia 14U/L, and 15U/L in haemolysis with low platelet count was seen during symptomatic pregnancy. Transient hyper phosphataemia in infancy is a benign condition characterized by elevated ALP levels of several folds without evidence of liver or bone disease and it returns to normal level by 4 months. ALP has been found elevated in peripheral arterial disease, independent of other traditional cardiovascular risk factors. Often clinicians are more confused in differentiating liver diseases and bony disorders when they see elevated ALP levels and in such situations measurement of gamma glutamyl transferase assists as it is raised only in cholestatic disorders and not in bone diseases. GGT is a microsomal enzyme present in hepatocytes and biliary epithelial cells, renal tubules, pancreas and intestine. It is also present in cell membrane performing transport of peptides into the cell across the cell membrane and involved in glutathione metabolism. Serum GGT activity mainly attributed to hepatobiliary system even though it is found in more concentration in renal tissue. The normal level of GGT is 9 to 85 U/L. In acute viral hepatitis the levels of GGT will reach the peak in the second or third week of illness and in some patients remain elevated for 6 weeks. Increased level is seen in about 30% of patients with chronic hepatitis C infection. Other conditions like uncomplicated diabetes mellitus, acute pancreatitis, myocardial infarction, anorexia nervosa, Gullianbarre syndrome, and hyperthyroidism, obesity and dystrophicamyotonica caused elevated levels of GGT. Elevated
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serum GGT levels of more than 10 times is observed in alcoholism. It is partly related to structural liver damage, hepatic microsomal enzyme induction or alcoholic pancreatic damage. GGT can also be an early marker of oxidative stress since serum antioxidant carotenoids namely lycopene, -carotene, -carotene, and -cryptoxanthin are inversely associated with alcohol-induced increase of serum GGT found in moderate and heavy drinkers. GGT levels may be 23 times greater than the upper reference value in more than 50% of the patients with nonalcoholic fatty liver disease. There is a significant positive correlation between serum GGT and triglyceride levels in diabetes and the level decreases with treatment especially when treated with insulin. Whereas serum GGT does not correlate with hepatomegaly in diabetes mellitus. Serum GGT activity was significantly lowers in the second and third trimesters of normal asymptomatic pregnancy. The levels of GGT in hyperemesis gravidarum were 45U/L, in preeclampsia 17U/L, and 35U/L in hemolysis with low platelet count and elevated liver enzymes was found during symptomatic pregnancy. The primary usefulness of GGT is limited in ruling out bone disease as GGT is not found in bone. NTP is a glycoprotein generally disseminated throughout the tissues of the body localised in cytoplasmic membrane catalyzing release of inorganic phosphate from nucleoside-5-phosphates. The normal range established is 0 to 15U/L. Raised levels of NTP activity were found in patients with obstructive jaundice, parenchymal liver disease, hepatic metastases and bone disease. NTP is precise marker of early hepatic primary or secondary tumors. ALP levels also increased in conjugation with NTP showing intra or extra hepatic obstruction due to malignancy. Elevation of NTP is found in acute infective hepatitis and also in chronic hepatitis. In acute hepatitis elevation of NTP activity is more when compared with chronic hepatitis and it is attributed to shedding of plasma membrane with ecto NTP activity due to cell damage, or leakage of bile containing high NTP activity. Serum NTP activity was slightly but significantly higher in the second and third trimesters of pregnancy. Ceruloplasmin is synthesized in the liver and is an acute phase protein. It binds with the copper and serves as a major carrier for copper in the blood. Normal plasma level of ceruloplasmin is 200 to 600mg/L. The level is elevated in infections, rheumatoid arthritis, pregnancy, non Wilson liver disease and obstructive jaundice. Low levels may also be seen in neonates, menkes disease, kwashiorkor, marasmus, protein losing enteropathy, copper deficiency and aceruloplasminemia. In Wilson\'s disease ceruloplasmin level is depressed. Decreased rate of synthesis of the ceruloplasmin I responsible for copper accumulation in liver because of copper transport defect in golgi apparatus, since ATP7B is affected. Serum ceruloplasmin levels were elevated in the chronic active liver disease (CALD) but lowered in
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the Wilsons disease (WD). Hence it is the most reliable routine chemical screening test to differentiate between CALD and WD (David et al., 2005). The AFP gene is highly activated in fetal liver but is significantly repressed shortly after birth. The mechanisms that trigger AFP transcriptional repression in postpartum liver are not properly understood. AFP is the major serum protein in the developing mammalian fetus produced at high levels by the fetal liver and visceral endoderm of the yolk sac and at low levels by fetal gut and kidney. AFP is required for female fertility during embryonic development by protecting the developing female brain from prenatal exposure to estrogen. In response to liver injury and during the early stages of chemical hepato-carcinogenesis led to the conclusion that maturation arrests of liver-determined tissue stem cells give rise to hepato-cellular carcinomas. The normal level of AFP is 0 to 15g/L. An AFP value above 400 - 500g/L has been considered to be diagnostic for hepato-cellular carcinoma (HCC) in patients with cirrhosis. A high AFP concentration 400g/L in HCC patients is associated with greater tumors size, bilobar involvement, portal vein invasion and a lower median survival rate. Higher serum AFP levels independently predict a lower sustained virological response (SVR) rate among patients with chronic hepatitis C. There are three different AFP variants, differing in their sugar chains (AFP-L1, AFP-L2, and AFP-L3). AFP-L1, the nonLens culinaris agglutinin (LCA) -bound fraction, is the main glycol form of AFP in the serum of patients with nonmalignant chronic liver disease. In contrast, Lens culinaris-reactive AFP, also known as AFP-L3, is the main glycol form of AFP in the serum of HCC patients and it can be detected in approximately one third of patients with small HCC (< 3 cm), when cut-off values of 10% to 15% are used. AFP-L3 acts as a marker for clearance of HCC after treatment. It is reported that an AFP-L3 level of 15% or more is correlated with HCCassociated portal vein invasion. Estimating the AFP-L3 / AFP ratio is helpful in diagnosis and prognosis of HCC. There is a direct association between second trimester maternal serum alpha-fetoprotein levels and the risk of sudden infant death syndrome (SIDS), which may be mediated in part through impaired fetal growth and preterm birth (Shivaraj et al., 2009). Chromium has atomic number 24 and atomic mass 51.996 g.mol-1. It has 6 isotopes. It is a lustrous, brittle, hard metal. Its color is silver-gray and it can be highly polished. It does not tarnish in air, when heated it borns and forms the green chromic oxide. Chromium is unstable in oxygen, it immediately produces a thin oxide layer that is impermeable to oxygen and protects the metal below (Jacques., 2004). Chromium main uses are in alloys such as stainless steel, in chrome plating and in metal ceramics. Chromium plating was once widely used to give steel a polished silvery
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mirror coating. Chromium is used in metallurgy to impart corrosion resistance and a shiny finish; as dyes and paints, its salts color glass an emerald green and it is used to produce synthetic rubies; as a catalyst in dyeing and in the tanning of leather; to make molds for the firing of bricks. Chromium (IV) oxide (CrO2) is used to manufacture magnetic tape. (Assem et al., 2007) Chromium is mined as chromite (FeCr2O4) ore. Chromium ores are mined today in South Africa, Zimbabwe, Finland, India, Kazakihstan and the Philippines. A total of 14 million tonnes of chromite ore is extracted. Reserves are hestimated to be of the order of 1 billion tonnes with unexploited deposits in Greenland, Canada e USA (Russel et al., 2001) Chromium and nickel individually considered potential health hazards. These are components of various steels and their salts are used extensively in plating. Thus both these metals are important materials in many industries and hence it is not possible to stop exposure to them. Oral exposure to human to level much greater than background has resulted in death, gastrointestinal, hematological, and hepatic renal and neurological effects (Mandava et al., 2006). Chromium (VI) and Chromium (0) are generally produced by industrial processes. Chromium (VI) compounds are oxidizing agents capable of directly inducing tissue damage. Accidently or international swallowing of large amount of Chromium (VI) causes stomach upset and ulcer, convulsions, liver and kidney damage and even death. It has also been reported to cause severe liver effect in workers exposed to CrO3 in chrome platinge industry. Hexavalent chromium result in enhanced formation of reactive oxygen species (ROS), including superoxide anion, hydroxyl radical and nitric oxide, decreased cell vaibility, increase cellular and genomic hepatic DNA fragmentation, enhanced intracellular oxidized states, membrane damage apoptic and necrotic death. People can be exposed to chromium through breathing, eating or drinking and through skin contact with chromium or chromium compounds. The level of chromium in air and water is generally low. In drinking water the level of chromium is usually low as well, but contaminated well water may contain the dangerous chromium(IV); hexavalent chromium. For most people eating food that contains chromium (III) is the main route of chromium uptake, as chromium (III) occurs naturally in many vegetables, fruits, meats, yeasts and grains. Various ways of food preparation and storage may alter the chromium contents of food. When food in stores in steel tanks or cans chromium concentrations may rise (Peter et al., 1898).

17

Chromium (Cr) is considered an essential nutrient and a health hazard. How is this possible? The answer is that Cr exists in more than one oxidation state. Specifically, Cr in oxidation state +6, written as Cr (VI), is considered harmful even in small intake quantity (dose) whereas Cr in oxidation state +3, written as Cr (III), is considered essential for good health in moderate intake. The health effects or nutritional benefits of Cr in other oxidation states are unknown although there are regulatory limits for the metal, Cr (0) or Cr 0, and Cr (II) along with those for Cr (III) and Cr (VI). For example, the federal maximum concentration level (MCL) for total Cr in drinking water is 100 mg/l, the California MCL is 50 mg/l (Calder, 1988), and the National Institute for Occupational Health and Safety (NIOSH) recommends an exposure limit for Cr(VI) of 1 mg/m3 and an exposure limit for Cr(0), Cr(II), and Cr(III) of 500m g/m3 for a 10-hour workday, 40-hour week. The concentration of chromium occurring naturally in the Earths normal mineral soil ranges from about 1 to 2000 mg/kg in the United States with a mean of 200 mg/kg worldwide. In conterminous United State soils, Cr concentration ranges from 1 to 2000 mg/kg with a mean of 37 mg/kg and most of this Cr is Cr(III) (Shacklette and Boerngen., 1984). Human activity further contributes to Cr in the environment (air, surface water, groundwater, soil). The greatest anthropogenic sources of Cr (VI) emissions are: (1) chromium plating, (2) chemical manufacturing of chromium, and (3) evaporative cooling towers (ATSDR, 2000). While combustion of coal and oil also release large quantities of chromium (1700 metric tons per year), only approximately 0.2% of this is Cr (VI). Approximately 35% of Cr released from all anthropogenic sources is Cr (VI). However, the ratio of Cr (III)/Cr(VI) in the natural environment varies considerably, from perhaps 0.3 to 1.5, depending on oxidation/reduction and acid/base conditions. Chromium metal or elemental chromium, Cr (0), rarely occurs naturally and Cr (II) is unstable in the environment, readily oxidizing to Cr(III). Only small quantities of Cr (II) are used in industry. Thus, most exposures to Cr in the environment will be to C r(III) and not to Cr(VI), the toxic constituent of total Cr. Occupational exposure to Cr(VI) is the most likely potential for adverse health effects (Gorshkov et al., 1996) This hazard identification document pertains to chromium (hexavalent compounds). Hexavalent chromium, or (Cr (VI)), compounds are those that contain the metallic element chromium (Cr) in its +6 valence (hexavalent) state. In this document these compounds are denoted as chromium (hexavalent compounds), compounds. Chromium has six oxidation states. The hexavalent state is one of the three most stable forms in which chromium is found in the environment. The other two of these forms are the 0 (metal and alloys), and the +3
18

(trivalent chromium, valence states. In nature, chromium generally occurs in small quantities associated with other metals, particularly iron. Its atomic weight is 51.996. Hexavalent chromium, in contrast to the trivalent form, exists as highly-oxidizing species. As noted by NTP (2008), Cr (VI) is usually present in complexes with halide (chromyl chloride) and oxygen ligands (chromium trioxide, chromate, and dichromate). There are numerous Cr (VI) compounds. Some examples are potassium chromate, dichromate, sodium chromate, chromium trioxide, and lead chromate. Hexavalent chromium compounds can vary considerably in their water solubility and other physical properties. Most chromate (Cr(VI)) results from man-made production, as the form is rare in nature. Hexavalent chromium reduces readily to Cr (III); the rate increases with decreasing pH.The NTP (2008) notes that Cr(VI) is easily reduced to Cr(III) in acidic solutions containing organic molecules such as proteins, DNA, or glutathione. Glutathione is also capable of reducing Cr(VI) at neutral pH at a slower rate than under acidic conditions (Silvio., 2000). Chromium metal is usually produced by reducing the chromite (FeCr2O4) ore with aluminum. Chromium is used to harden steel, in the manufacture of stainless steel, and in the production of a number of industrially important alloys. Chromium is used in making of pigments, in leather tanning and for welding. Chromium plating produces a hard mirrorlike surface on metal parts that resists corrosion and enhances appearance. The general public and communities have been exposed via air to Cr(VI) through manufacturing emissions, its use as an anticorrosive agent in cooling systems, chrome plating, and combustion releases; for example, in fly ash from power plants and cigarette smoke. The California Air Resources Board, consequent to the identification of hexavalent chromium as a toxic air contaminant, has taken a number of steps to reduce the publics exposure to Cr(IV) in air, including a prohibition on its use in cooling towers and development and enforcement of standards on chrome plating operations. In the environment, Cr (VI) may be reduced to the trivalent form Cr (III), although hexavalent forms may also persist: Cr(VI) compounds occur as contaminants in ambient air, drinking water, soil, house dust and food. Trivalent chromium and Cr (VI) are inter-convertible in the environment. Oze et al. (2006) note the occurrence of naturally occurring Cr (VI) in ground and surface waters, and its generation through natural processes. Mechanisms for its generation from Cr (III) from serpentinederived soils and sediments and migration into water sources have been described by these authors. Serpentine, the California State Rock, is prevalent in central and northern California. It is unclear how much exposure in the State to Cr (VI) in drinking water results from such processes. Contamination of drinking water with Cr (VI) also has resulted from industrial
19

uses, such as in plating operations. In water that is rich in organic content, Cr (VI) is most likely to react quickly with reducing agents to form Cr (III). However, Cr (VI) may persist in water as water-soluble complex anions. Legacy contamination of drinking water sources from previous uses in cooling towers and in manufacturing continues to result in site clean-up orders in the State. Virtually all foods contain some chromium, ranging from 20 to 590 g/kg. The foods with the highest levels of chromium are meats, mollusks, crustaceans, vegetables, and unrefined sugar. Trivalent chromium tends to form stable complexes with organic and inorganic legends, and is presumed to be the form found in foodstuffs due to the presence of reducing agents in food (U.S. EPA, 1988; NAS and FNB, 2000). There are debates over the essentiality of Cr (III) (Sterns, 2000), and its use as a nutritional supplement in sports medicine and to treat insulin resistance. The National Academy of Sciences (NAS) did not find sufficient evidence to set an Estimated Average Requirement (EAR) for chromium, but did set an Adequate Intake (AI) for chromium of 35 g/day for young men and 25 g/day for young women. Most recently the Institute of Medicine (IOM, 2004) reviewed the safety of chromium picolinate, the nutritional supplement form of Cr(III) and found there is neither consistent evidence of reasonable expectation of harm from chromium picolinate nor sufficient evidence to raise concern regarding the safety or toxicity of chromium picolinate when used in the intended manner for a length of time consistent with the published clinical data. Workers experience the highest exposures to Cr (VI) through chrome plating, chromate production and stainless steel welding. Usually the route of occupational exposure is inhalation or dermal contact (Jacques Guertin., 2004). Chromium is one of the metallic elements for which maximum concentrations in the environment are limited by the law due to its toxic properties. In nature it may exist in two oxidation states: (III) and (VI). The effects of chromium on health have been widely studied; Cr (VI) is about 300 times more toxic than Cr (III). Its impact on the environment also depends on the oxidation degree. Chromium compounds are used in many industries such as leather tanning, metal plating, and other metallurgical procedures. The inadequate disposal of their wastes may give rise to concentrations above the natural values. Human activity further contributes to Cr in the environment (air, surface water, groundwater, soil). The greatest anthropogenic sources of Cr (VI) emissions are: (1) chromium plating, (2) chemical manufacturing of chromium, and (3) evaporative cooling towers. While combustion of coal and oil also release large quantities of chromium (1700 metric tons per year), only approximately 0.2% of this is Cr (VI). Approximately 35% of Cr released from all anthropogenic sources is Cr(VI). However, the ratio of Cr (III)/Cr(VI) in the
20

natural environment varies considerably, from perhaps 0.3 to 1.5, depending on oxidation/reduction and acid/base conditions (Russel et al., 2001). Chromium occurs naturally in the earths crust, predominantly in the trivalent, chromium (III), form, and it is ubiquitous in air, water, soil and biological materials. Chromium (VI) compounds are especially anthropogenically produced and do not occur naturally in environment. Large amount are produced through a range of activities, including the production of chromates and bio chromates, stainless steel, welding, chromium plating, ferrochrome alloys and chrome pigment production, material tanning, the combustion of coal and oil, cement works, and waste incineration with the global production of the major chromium (VI) compounds estimated to be about 17.5 T/year, will be released into various environmental media. The releases of chromium (VI) from any sources are excepted to be reduced via abiotic and biotic processes to chromium (III) in most situations in the environment , and the impact of the chromium (VI) from is therefore likely to be limited to the area around an exposure source. In biological system, the oxidation of chromium (III) to chromium (VI) never occurs. In food stuffs, chromium is generally considered to be present as chromium (III). (Assem et al., 2007). It was reported the effects of small doses of chromium, lead, cadmium, nickel and titanium in drinking water on the growth and survival of mice up to 21 months of age in experiments attempting to duplicate human concentrations. Chromium and titanium increased growth rates in both sexes; in males chromium lessened early mortality, whereas cadmium and lead increased mortality at older ages. This report is concerned with our total experience for the lifetime of these animals, regarding mortality, gross causes of death, effects on incidence of tumors and organ accumulations of metals. Chromium (VI) reducing capacity of metabolic system was derived from human and different species i.e. mice, rats, hamsters, woodchuck and some avian species (Chicken, Pekin duck,) and different analytical techniques give the result the Cr(VI) and colonized the human body parts i.e. epithelial-lining fluid, saliva, gastric juice etc. Cr (VI) in extra concentration act as carcinogenicity and genotoxicity. The respiratory tracts have successful defence against Cr. But other parts of body cells act can affected. Cr (VI) can enter in the cell and attack like as a sort of Trojan horse and can penetrate in the cell. It enters in the different compartments of cells. Cr enters in endoplasmic reticulum, mitochondria and nucleus and act as reducing agent and destroying the cell. Cr (VI) also attack on DNA and damage the structure of DNA act as mutagenic (Silvio, 2000).

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Milk thistle is widely used in Europe for hepatic and biliary disorders, and is beginning to be used to protect against nephro toxicity as well. It protects the liver from several heap totoxins, including Amanita mushrooms, acetaminophen and alcohol. Its primary active ingredient is silymarin, which is a potent antioxidant composed of several flavonoid compounds. Further studies are needed to evaluate milk thistle's renal protectant effects, such as prevention of cisplatin toxicity, its use in treating alcoholic liver disease, and its use to prevent cancer or as a complementary treatment for cancer. There are no known long-term risks to adults associated with milk thistle use. Its safety in pediatrics, pregnancy, and during lactation is unknown (Murphy et al., 2000). Oxidative stress may be a key factor in the onset of certain diseases, including cancer. Oxy-radicals play important roles in the initiation, promotion, and progression of carcinogenesis. It is considered that a significant event in oxy radical mediated carcinogenesis is the extensive oxidative damage to the nuclear membrane, which leads to deoxyribonucleic (DNA) damage such as DNA single-strand breaks and possibly facilitation of carcinogenesis. To prevent cellular damage leading to cancer caused by oxy radicals, the level of tissue antioxidants is critical. Interest in natural sources of antioxidant molecules for use in the food, beverage and cosmetic industries has resulted in a large body of research in recent years. It is well known that natural antioxidants extracted from herbs and spices have high antioxidant activity and are used in many food applications. Of these substances, the phenolic compounds, which are widely distributed, have the ability to scavenge free radicals by single electron transfer. Silymarin is isolated from the fruits and seeds of the milk thistle (Silybum marianum) and in reality are a mixture of three structural components: silibinin, silydianine, and silychristine. Milk thistle is a member of the Asteraceaefamily. It has been reported as having multiple pharmacological activities including antioxidant, hepatoprotectant and antiinflammatory agent, antibacterial, antiallergic, antimutagenic, antiviral, antineoplastic, antithrombotic agents, and vasodilatory actions. Asghar et al. (2008) suggested that silymarin may be used in preventing free radical-related diseases as a dietary natural antioxidant supplement. Milk thistle has been used medicinally in Europe since the first century. Pliny the Elderclaimed that it was helpful in improving bile flow. It was also mentioned in the writings of Dioscorides, Jacobus Theodorus and Culpepper1. Its leaves, flowers and roots have historically been considered a vegetable in European diets, and its fruits (achenes), which resemble seeds, have been roasted for use as a coffee substitute. The leaves of the plant are
22

eaten in fresh salads and as spinach substitute, the stalks eaten like asparagus, and the flower heads served as one would anartichoke (Wahsha and Jassabi., 2009). In Traditional Chinese Medicine, milk thistle seeds are known as ShuiFeiJi; in China milk thistle is used to protect the liver, increase bile secretion and protect against oxidative injuries suchas radiation. Ripe milk thistle seeds are used in Europe in the treatment of various hepatobiliaryproblems, such as hepatitis, cirrhosis, gallstones, and jaundice, as well as for kidney ailments.Milk thistle are used as an antidote for Amanita mushroom poisoning and to protect the liver and kidneys from toxic medications. It is used to treat hepatitis and biliary disease, lower cholesterol, and even improve psoriasis. Some herbalists also recommend it to treat insufficient lactation (Murphy et al., 2000). The German Commission E recommends it for the treatment of dyspeptic complaints, toxin-induced liver damage, and hepatic cirrhosis and as a supportive therapy for chronic inflammatory liver conditions; sales there exceeded $180 million in 1997. Medicinal species: Silybum marianum L. Gaertn., Cardusmarianus L. Common names: Holy thistle, marian thistle, Mary thistle, milk thistle, Our Ladys thistle, St. Mary thistle, wild artichoke, Mariendistel (Ger), Chardon-Marie (Fr). Milk thistle should not be confused with blessed thistle, Cnicusbenedictus. Milk thistle is sold as Legalon in Germany. Botanical family: Compositae/Asteraceae Plant description: Milk thistle is a tall, biennial herb, five to ten feet high, with hard, green, shiny leaves that have spiny edges and are streaked with white along the veins. The solitary flower heads are reddish purple with bracts ending in sharp spines. The small hard fruits in the flowers, known technically as achenes, resemble seeds and are the part of the plant used medicinally. Where its grown: Southern and western Europe, South America and North America in the eastern United States and California. Milk Thistle: Potentially active chemical constituents are Flavonoids/flavonolignans: silymarin (which includes silybin [silibinin], silidianin, silychristin [silichristin] and isosylibin), apigenin, dehydrosilybin, deoxysilycristin, deoxysildianin, siliandrin, silybinome, silyhermin, neosilyherminother: silybonol; myristic, oleic, palmitic and stearic acids; betaine hydrochloride. (Fraschini et al., 2002) The dried seeds contain 1-4% silymarin flavonoids. Silymarin is a mixture of at least threflavonolignans, including silybin (silibinin), silidianin, and silychristin. It is the primary active ingredient in milk thistle, and is also found in related species such as artichokes. The bioavailability of enterally administered silymarin is limited; the compound is poorly soluble in water, and only 20-50% is absorbed from the gastrointestinal tract after ingestion.
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Absorption is significantly enhanced if silybin is administered in a complex with phosphatidlycholine. There is rapid absorption after an oral dose with the peak plasma concentration reached after two to four hours and an elimination half-life of six hours; it undergoes extensive entero hepatic circulation. Three to eight percent is excreted in the urine, and 80% is excreted in the bile as glucuronide and sulfate conjugate. Bioavailability can vary up to three-fold depending on the formulation; the brand used in most European studies, Legalon, contains approximately twice as much available silybin as other preparations. Silybin is the most biologically active component with regard to antioxidant and hepato protective properties; it is concentrated in the bile, achieving concentrations 60 times highe than that found in the serum. Other flavonolignans identified in S. marianum include dehydro silybin, deoxysilycistin, deoxy silydianin, silandrin, silybinome, silyhermin and neo silyhermin. In addition, milk thistle contains apigenin; silybonol; myristic, oliec, palmitic and stearic acids; and betaine hydrochloride, which may have a hepato protective effect. Potential Clinical Benefits of Milk Thistle are as following. 1. Cardiovascular: none 2. Pulmonary: none 3. Renal and electrolyte balance: Renal protestant 4. Gastrointestinal/hepatic: Hepatoprotectant; treatment of hepatitis, antilipidemic 5. Neuro-psychiatric: none 6. Endocrine: Antidiabetic and pancreatic protectant 7. Hematologic: none 8. Rheumatologic: none 9. Reproductive: none 10. Immune modulation: Anti-inflammatory 11. Antimicrobial: none 12. Anti neoplastic: Chemoprevention 13. Antioxidant: Antioxidant 14. Skin and mucus membranes: Psoriasis: Traditional use, no data. 15. Other/miscellaneous: none Flavonoids usually possess good antioxidant activity. The water-soluble dehydrosuccinate sodium salt of silibinin is a powerful inhibitor of the oxidation of linoleic acidwater emulsion catalyzed by Fe2+ salts. It also inhibits in a concentration-dependent way the microsomal peroxidation produced by NADPH-Fe2+-ADP, a well known experimental system for the formation of hydroxy radicals.In studies performed in rat hepatic microsomes,
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it has been demonstrated that lipid peroxidation produced by Fe(III)/ascorbate is inhibited by silibinin dihemi succinate; the inhibition is concentration-dependent. It has been shown that silymarin is as active as quercetin and dihydroquercetin, and more active than quercitrin, in terms of antiperoxidant activity, independent of the experimental model used to produce peroxidation. It has recently been reported that in rat hepatocytes treated with tert-butyl hydroperoxide (TBH), silymarin reduces the loss of lactate dehydrogenase (LDH), increases oxygen consumption, reduces the formation of lipid peroxides, and increases the synthesis of urea in the perfusion medium. Furthermore, silymarin is able to antagonise the increase in Ca2+produced by TBH, reducing ion levels down to below 300 nmol/L. The protective effect of silymarin is mediated by the inhibition of lipidperoxidation, and the modulation of hepatocyte Ca2+content seems to play a crucial role (Fraschini et al., 2002). Protective Effects in Models of Oxidative Stress Oxidative stress is defined as structural and/or functional injury produced in tissues by the uncontrolled formation of prooxidant free radicals. Oxidative stress usually develops when the pro-oxidant action of an inducer exceeds the anti-oxidant capacity of the cell defense system, altering its homeostatic capacity. Numerous substances induce oxidative stress, including carbontetrachloride, TBH, ethanol, paracetamol (acetaminophen) and phenylhydrazine. It has been shown in rats that silibinin protectsneonatal hepatocytes from cell damage produced by erythromycin, amitriptyline, nortriptyline and TBH. Erythrocytes obtained from rats treated with silymarin exhibited high resistance against the haemolysis produced byphenylhydrazine and the lysis induced by osmotic shock. This suggests that silymarin may act by increasing the stability of the erythrocyte membrane. The cytoprotective activity of silymarin has also been shown in hepatocytes of rats subjected to osmotic stress produced by hypotonic saccharose solutions. The perfused liver is a valid experimental model for the evaluation of the effect of substances that induce oxidative stress and of the protection provided by scavengers. Using this experimental model, it has been shown that phenylhydrazine produces anincrease in oxygen consumption in rat liver in vitroand in the release of thiobarbituric acid reactive substances (TBARS) in the perfusate. This stress is associated with a reduction in the amount of reduced glutathione (GSH) in the liver; GSH exerts important protective activity against chemically induced oxidative stress. Using liver from rats pretreated in vivo with silibinin50 mg/kg intravenously, a significant reduction in the oxygen consumption stimulated by phenyl hydralazine and in the release of TBARS was observed, without any changes in GSH levels. The antioxidant effect of silibinin was observed in rats with acute intoxication caused by ethanolor paracetamol which are peroxidation inducers that produce marked GSH depletion
25

in the liver. Treatment with silymarin or silibinin was able to protect animals from oxidative stress produced in the liver by ethanol or paracetamol. Furthermore, it has been reported that treatment with silibinin attenuates the increase in plasma levels of AST, ALT and gamma glutamyl transpeptidase (GGT) observed after intoxication by paracetamol. The hepato protective activity of silibinin has also been studied in rats with liver cirrhosis induced by the long term administration of carbon tetrachloride. Muriel & Mourelle have shown that silibinin preserves the functional and structural integrity of hepatocyte membranes by preventing alterations of their phospholipid structure produced by carbon tetrachloride and byrestoring alkaline phosphatase and GGT activities. Another interesting property of silibinin and silymarin is their role as regulators of the content of GSH in various organs. In ratstreated with silibinin intravenously or silymarin in traperitoneally, a significant increase in the amount of the GSH contained in the liver, intestine and stomach was found, whereas there were no changes in the lungs, spleen and kidneys (Boigk et al., 1997). Activity against Lipid Peroxidation Lipid peroxidation is the result of an interaction between free radicals of diverse origin and unsaturated fatty acids in lipids. Lipid peroxidation involves a broad spectrum of alterations, and the consequent degeneration of cell membranes may contribute towards the development of other disorders of lipoprotein metabolism, both in the liver and in peripheral tissues. Silymarin appears to act as an antioxidant not only because it acts as a scavenger of the free radicals that induce lipid peroxidation, but also because it influences enzyme systems associated with glutathione and superoxide dismutase. It has been shown that all the components of silymarin inhibit linoleic acid peroxidation catalysed by lipoxygenase and that silymarin protects rat liver mitochondria and microsomes in vitro against the formation of lipid peroxides induced by variousagents. Effects on Liver Lipids. The influence of silymarin on cellular permeability are closely associated with qualitative and quantitative alterations of membrane lipids (both cholesterol and phospholipids). This suggests that silymarin may also act on other lipid compartments in the liver; this may influence lipoprotein secretion and uptake. It has been shown that silymarin and silibinin reduce the synthesis and turnover of phospholipids in the liver of rats. Furthermore, silibinin is able to neutralize two effects of ethanol in rats: the inhibition of phospholipid synthesis and the reduction in labelled glycerol incorporation into lipids of isolated hepatocytes. In addition, silibinin stimulates phosphatidylcholine synthesis and increases the activity of cholinephosphat ecytidyl transferase in rat liver both in normal conditions and after intoxication by galactosamine. Data on the influence of silymarin on triglyceride metabolism in the liver are scanty. It is known that in rats silibinin is able to
26

partly antagonise the increase in total lipids and triglycerides produced in the liver by carbon tetra chloride and, probably, to activate fatty acid -oxidation. It has also been suggested that silymarin may diminish triglyceride synthesis in the liver. Letter on et al. studied the mechanisms of action of silymarin that provide protection against lipid peroxidation and the hepato toxicity of carbon tetrachloride in mice, and came to the conclusion that silymarin works by reducing metabolic activation by carbon tetrachloride and by acting as an antioxidant that prevents chain rupture. Other authors have shown that silymarin affords hepatoprotection against specific injury induced by microcystin (a hepatotoxin), paracetamol, halothane and alloxan in several experimental models (Fraschini et al., 2002). Effects on Plasma Lipids and Lipoproteins the administration of silymarin reduces plasma levels of cholesterol and low-density lipoprotein (LDL) cholesterol inhyperlipidaemic rats, whereas silibinin does not reduce plasma levels of cholesterol in normal rats; however, it does reduce phospholipid levels, especially those transported in LDL. Data obtained in experimental models of hepatic injury have shown that silymarin is able to normalize the increase in plasma lipids observed after administration of carbon tetrachloride and to antagonize the reduction in serum free fatty acids induced bythioacetamide. In the experimental model of hepatic injury produced by thio acetamide, silymarin did not appear to be able to normalize the reduction in triglycerides in serum. In the experimental model of hepatic injury produced by paracetamol in rats, it was evident that silymarin improves LDL binding to hepatocytes, an important factor for the reduction of LDL in plasma. Stimulation of liver regeneration one of the mechanisms that can explain the capacity of silymarin to stimulate liver tissue regeneration is the increase in protein synthesis in the injured liver. In vivo and in vitro experiments performed in the liver of rats from which part of the organ had been removed, silibinin produced a significant increase in the formation of ribosomes and in DNA synthesis, as well as an increase in protein synthesis. Interestingly, the increase in protein synthesis was induced by silibinin only in injured livers, not in healthy controls. The mechanism whereby silibinin stimulates protein synthesis in the liver has not been defined; it may be the physiological regulation of RNA polymerase I at specific binding sites, which thus stimulates the formation of ribosomes. In rats with experimental hepatitis caused by galactosamine, treatment with intraperitoneal silymarin 140 mg/kg for 4 days completely abolished the inhibitory effect of galactosamine on the biosynthesis of liver proteins and glycoproteins. These data support the results of previous experiments in a similar model of acute hepatitis in the rat, in which silymarin protected hepatic structures, liver glucose stores and enzyme activity in vivo from injury produced by galactosamine. The
27

capacity of silymarin to stimulate protein synthesis has also been studied in neo plastic cell lines, in which no increase in protein synthesis, ribosome formation or DNA synthesis has been found after treatment with silymarin (Srinivasan et al., 2012). Effects during Experimental Intoxication with Amanita phalloides the therapeutic activity of silymarin against mushroom poisoning is worthy of particular attention. The hepato protective properties of silymarin have been tested in dogs, rabbits, rats and mice. A dose of 15 mg/kg of silymarin was administered intravenously 60 minutes before intra peritoneal administration of a lethal dose of phalloidin, and was able to protect all animal species tested (100% survival) from the action of the toxin. When it is injected 10 minutes after phalloidin, silymarin affords similar protection only at doses of 100 mg/kg. The longer the time that has elapsed after administration of the toxin, the less effective the drug becomes, and after 30 minutes it is no longer effective even at high doses. Histochemical and histo enzymological studies have shown that silymarin, administered 60 minutes before or no longer than 10 minutes after induction of acute intoxication with phalloidin, is able to neutralise the effects of the toxin and to modulate hepatocyte function. Similar results were obtained in dogs treated with sublethal oral doses of A. phalloides, in which hepatic injury was monitored by measuring enzymes and coagulation factors. Amongst the numerous substances tested (prednisolone, cytochromec, benzylpenicillin, silymarin), only benzylpenicillin (1000 mg/kg intravenous infusion after 5 hours) and silymarin (50 mg/kg intravenous infusion after 5 hours and 30 mg/kg after 24 hours) were able to prevent the increase in hepatic enzymes and the fall in coagulation factors induced by experimental intoxication (table II). The cyclopeptides of fungi of the genus Amanita, including amatoxins and fallo toxins, are captured by hepatocytes through the sinusoidal system, which is also involved in the mediation of liver uptake of biliary salts. It has been demonstrated that silibinin is able to inhibit uptake of amanitin in isolated preparations of hepatocyte membranes, and the same effect has been shown for taurocholate, antamanide, prednisolone and phalloidin. The effect of silibinin appears to be competitive. Recently, the role of tumour necrosis factor- (TNF) in hepatic injury produced by -amanitin has been investigated in primary cultures of rat hepatocytes. At a concentration of 0.1 mol/L, the toxin inhibits RNA and protein synthesis within 12 hours, but cyto toxicity appears only much later (36 hours). TNF is not indispensable for the development of cyto toxicity, but exacerbatesitand markedly increases lipid peroxidation. The addition of silibinin at a concentration of 25mol/L to the culture medium prevented the effects of TNF (50g/L).

28

Anti Inflammatory and Anti carcinogenic properties a significant anti-inflammatory effect of silymarin has been described in liver tissue. Studies have shown that silymarin exerts a number of effects, including inhibition of neutronphil migration, inhibition of Kupffer cells, marked inhibition of leukotriene synthesis and formation of prostaglandins. The protection afforded by silymarin against carcinogenic agents has been studied in various experimental animal models. A series of experiments have been performed in nude mice with non melanoma skin cancer produced by UVB radiation, studying its initiation, promotion and complete carcinogenesis. In all the stages studied, silymarin applied onto the skin at different doses appeared to reduce significantly the incidence, multiplicity and volume of tumors per animal. Furthermore, in a short term experiment (using the same experimental model), the application of silymarin significantly reduced apoptosis, skin oedema, depletion of catalase activity and induction of cyclooxygenase and ornithine decarboxylase activity. This effect provides protection against photo carcinogenesis. Similar results were also obtained in the model of skin carcinogenesis produced by chemical carcinogenic agents in carcinogenesissensitive mice. The molecular bases of the anti-inflammatory and anti-carcinogenic effects of silymarin are not yet known; they might be relatedto the inhibition of the transcription factor NF-B, which regulates the expression of various genes involved in the inflammatory process, in cytoprotection and carcinogenesis. It has also been hypothesized that silymarin may act by modulating the activation of regulating substances of the cellular cycle and of mitogen activated protein kinase. Antifibrotic effects are stellate hepatocytes have a crucial role in liver fibrogenesis. In response to fibrogenic influences (for example protracted exposure to ethanol or carbon tetrachloride), they proliferate and transform into myofibroblasts responsible for the deposition of collagen fibres in the liver. Recently, the effects of silibinin on the transformation of stellate cells into myofibroblasts have been investigated. The results have shown that silibinin, at a concentration of 100mol/L reduce the proliferation of stellate cells isolated from fresh liver of rats by about 75%, reduce the conversion of such cells into myofibroblasts, and down regulate gene expression of extracellular matrix components indispensable for fibrosis. Furthermore, it has been demonstrated that silymarin improves hepatic fibrosis in vivo in rats subjected to complete occlusion ofthe biliary duct, a manoeuvre that causes progressive hepatic fibrosis without inflammation. Silymarin, administered at a dosage of 50 mg/kg/day for 6 weeks, is able to reduce fibrosis by 30 to 35% as compared with controls. A dose of 25 mg/kg/day is not effective. Colchicine and silymarin, administered at a dose of 50 mg/kg orally for 55 days, were able to prevent
29

completely all the alterations induced by carbon tetrachloride in rats (peroxidation of lipids, Na+ , K+ and Ca2+ -ATPase), except for the hepatic content of collagen, which was reduced only by 55% as compared with controls; moreover, alkaline phosphatase and ALT were unchanged as compared with controls. In the group of rats treated with silymarin, the loss of glycogen was inhibited completely (Fraschini et al., 2002). Silymarin can inhibit the hepatic cytochrome P450 (CYP) detoxification system (phase I metabolism). It has been shown recently in mice that silibinin is able to inhibit numerous hepatic CYP enzyme activities, whereas other researchers have not detected any effect of silymarin on the CYP system. This effect could explain some of the hepato protective properties of silymarin, especially against the intoxication due to A. phalloides. The Amanita toxin becomes lethal for hepatocytes only after having been activated by the CYP system. Inhibition of toxin bio activation may contribute to the limitation of its toxic effects. Additionally, silymarin, together with other antioxidant substances, could contribute towards protection against free radicals generated by enzymes of the CYP system (Hatice et al., 2012). The hepato protection provided by silymarin appears to rest on four properties: Activity against lipid peroxidation as a result of free radical scavenging and the ability to increase the cellular content of GSH; Ability to regulate membrane permeability and to increase membrane stability in the presence of xenobiotic damage; Capacity to regulate nuclear expression by means of a steroid-like effect; and Inhibition of the transformation of stellate hepatocytes into myofibroblasts, which are responsible for the deposition of collagen fibres leading to cirrhosis. Silymarin and silibinin inhibit the absorption of toxins, such as phalloidin or -amanitin, preventing them from binding to the cell surface and inhibiting membrane transport systems. Furthermore, silymarin and silibinin, by interacting with the lipid component of cell membranes, can influence their chemical and physical properties. Studies in erythrocytes, mast cells, leucocytes, macrophages and hepatocytes have shown that silymarin renders cell membranes more resistant to lesions. Furthermore, the well documented scavenging activity of silymarin and silibinin can explain the protection afforded by these substances against hepatotoxic agents. Silymarin and silibinin may exert their action by acting as free radical scavengers and interrupting the lipid peroxidation processes involved in the hepatic injury produced by toxic agents. Silymarin and silibinin are probably able to antagonise the depletion of the two main detoxifying mechanisms, GSH and superoxide dismutase (SOD), by reducing the free radical
30

load, increasing GSH levels and stimulating SOD activity. Furthermore, silibinin probably acts not only on the cell membrane, but also on the nucleus, where it appeared to increase ribosomal protein synthesis by stimulating RNA polymerase I and the transcription of rRNA. The stimulation of protein synthesis is an important step in the repair of hepatic injury and is essential for restoring structural proteins and enzymes damaged by hepatotoxins (Kannampalli et al., 2007) Silymarin has been reported to protect liver cells from a wide variety of toxins, including acetaminophen, ethanol, carbon tetra-chloride, and D-galactosamine. Silymarinhas also had been found to protect liver cells from ischemic injury, radiation, iron toxicity, and viral hepatitis. The mechanisms which provide silymarins hepato protective effects are many and varied, and include antioxidation, anti-lipid peroxidation, enhanced detoxification, and protection against glutathionedepletion. Silymarin has been found to inhibit the formation of leukotrienes from poly-unsaturated fatty acids in the liver, via its inhibition of the enzyme lipoxygenase. These leukotrienes are known to be some of the most damaging chemicals found in man.Studies also demonstrated that silymarin increased hepatocyte protein synthesis, decreased the activity of tumor promo-ters,stabilized mast cells,modulated immune functions,and was anti-inflam-matoryand antifibrotic.Stimulation of Liver

Regeneration, one of the mechanisms to explain the ability of silymarin to stimulate the regeneration of hepatic tissue is the increase in protein synthesis in damaged livers. In both in vivo and in vitro experiments, significant increases in the formation of ribosomes and DNA synthesis were measured in addition to the increase in protein synthesis. Interestingly, the increased protein synthesis was only measured in damaged livers (partial hepectomy), not in controls. The mechanism of increased protein synthesis is currently not known but some authors speculate silymarin imitates a physiologic regulator, so the silybin fits into a specific binding site on the polymerase, thus stimulating ribosome formation. The potential for stimulation of protein synthesis by silymarin was investigated in malignant liver tissue, and no increases in protein synthesis, ribosome formation, or DNA synthesis were found in malignant cell lines. Anti-inflammatory Effects: The main-stays of the current medical management of non viral chronic hepatitis are immune suppressive/anti-inflammatory medications (e.g., prednisone, azathioprine). While use of these drugs may be lifesaving, long-term use may result in debilitating, life-threatening side effects. Doctors and patients need safe and effective alternative anti-inflammatory medications. Botanical antiinflammatories may constitute such a group. Silymarin has been shown to have significant anti inflammatory effects on hepatic tissue. Several studies have demonstrated a variety of
31

anti inflammatory effects, including mast cell stabilization, inhibition of neutrophilmigration, Kuppfer cell inhibition, strong inhibition of leukotriene synthesis, and prostagland in formation. Antifibrotic effects hepatic stellate cells play a central pathogenic role in liverfibrogenesis. In response to some fibrotic influences (e.g., chronic ethanol exposure, carbon tetrachloride, etc.), they proliferate and transform into myofibroblasts, which are responsible for the deposition of collagen fibers in the liver. One recent study investigated the effect of silybin on the transformation of hepatic stellate cells into myofibroblasts. Silybin (10-4mol/l concentration) was found to reduce the proliferation of freshly isolated rat hepaticstellate cells by about 75 percent. It also reduced the conversion of stellate cells into myofibroblasts and down-regulated the gene expression of extracellular matrix components necessary for fibrosis Silymarin has been shown to slow or reverse liver fibrosis in animals. Rats were subjected to a complete bile duct occlusion which consistently causes progressive liver fibrosis without inflammation. Silymarin was able to reduce the fibrosis by 30-35 percent in comparison with controls (50 mg/kg/day, human dose = 3500 mg/day). Silymarin worked equally if used continuously for six weeks after the bile duct occlusion or only for the final two weeks. Dosage at 25 mg/kg/day (human dose = 1750 mg/day) was not found to be effective.Colchicine is currently used to inhibit fibrosis of the liver. It functions as an anti fibrotic and anti-inflammatory by inhibiting macrophage stimulation of fibrosis. Unfortunately colchicine has a narrow, unpredictable therapeutic window, and serious, lifethreatening side-effects, including liver failure, renal failure, myocardial injury, severe gastrointestinal damage, shock, and death. Ina rat study using carbon tetrachloride induced liver fibrosis, silymarin was found to be very similar to colchicines for the prevention of chronic liver fibrosis, but without any side effects. Inhibition of P450: Paradoxically, silymarin may have an inhibitory effect on the cytochrome P450 (Phase I) detoxification system. In recently published animals silybin was found to inhibit several speinduced P450 enzymes in mice. Researchers have noted the lack of stimul effect on the P450 detoxification system this effect may explain some of hepato protective effects of silym especially against Amanita poison Amanitin toxin becomes deadly to hepatoonly after it becomes bio activated by the system. The inhibition of the bio active tiamanit in could reduce its toxic effect addition; silymarin and other antioxid afford some protection against the free regenerated by P450 enzymes. Enhanced glucuronidation Glucuronidation is an important Phase II liver detoxification pathway. More toxins are removed from the body via glucuronidation than any other single detoxification pathway. Glucuronic acid is conjugated with toxins to facilitate
32

their elimination from the body via the bile. In addition, many other substances, including estrogen, are removed from the body via glucuronidation. Unfortunately, some in testinal bacteria (mostly pathogenic) possess an enzyme, beta-glucuronidase that enables them to remove glucuronic acid from the conjugated substance and use it as an energy source. This allows the original molecule to be reabsorbed through the GI mucosa, thus reexposing the person to the removed substance (Boigk et al., 1997) Silymarin was found to inhibit the activity of beta-glucuronidase 53 percent in healthy humans and in one patient with colon cancer. Immunomodulation researchers have investigated the immunomodulatory effects of silymarin on the diseased liver. A pair of Hungarian studies demonstrated a positive effect of silymarin on immune function. The first study looked at patients with histologically proven chronic alcoholic liver disease. These patients originally had low T cell percentage, high CD8+ cell percentage, and an enhanced antibody-dependent increase in lymphocytecy to toxicity. All of these abnormal immune findings were normalized by a six-month course of silymarin. No significant changes were found after six months in the control group. The second study looked at the hepato protective effect of silymarin in addition to its effects on normalizing immune function. Forty patients with alcoholic cirrhosis of the liver were given either silymarin, amino-imidazole carboxamide phosphate, or placebo in a one-month, double-blind clinical trial. In the treated groups, silymarin normalize delevated levels of AST, ALT, and total bilirubin, markedly reduced the high level of GGT, decreased the percentage of OKT8+cells, and suppressed lymphocy totoxicity. Dosage/Toxicity Silybum marianum is not water soluble and is typically administered as an encapsulated standardized extract (70-80%silymarin). In animals, silymarin has proven to be non-toxic when administered at high doses for short periods of time and long term dosage in rats has also failed to demonstrate any toxicity. Human studies have shown silymarin to be generally without side-effects. The typical adult dosage for silymarin is 240-900 mg/day in two or three divided doses. At higher doses (>1500 mg/day) silymarin may produce a laxative effect due to increased bile flow and secretion. Mild allergic reaction shave also been noted, but neither of these side effects was severe enough to discontinuous treatment discontinue treatment (Muriel et al., 1990).

The bitter tasting roots of picrorhiza kurroa are hard, about 6-10 inches in length, and creeping. The leaves are 2-4 inches long, oval in shape with a sharp apex, flat, and serrate. The flowers are white or pale purple on a long spike, blooming in June through August. The fruit is inch long and oval in shape. The rhizome of picrorhiza kurroa is manually harvested in October through December. Like many species of medicinal plants, picrorhiza is
33

threatened to near extinction due to over-harvesting. Its common names are Indian Name: Kutki and Kuru, Botanical Name: Picrorhiza kurroa, Other Names: Katuka, Kuru & Kadu (Somesh et al., 2012). Parts of plant which are used are leaf, bark, root and rhizomes.Picorhizakurrorais also known as kutki. It belongs to the family Scrophulariaceae. The plant is widely distributed north-western Himalayas at an altitude 2,700 to 4,500m from Kashmir to Kumaun and Garhwal regions in India and Nepal. The root is bitter, cooling, stomachic, cardiotonic, antipyretic, an thelmintic, laxative, promotes appetite, useful in biliousness, bilious fevers, urinary discharges asthma hiccough blood troubles burning sensations,

leucoderma and jaundice. In China and Malaya, the rhizome is a favorite remedy for bilious dyspepsia accompanied by fever. It is a good stomachic and very useful in almost all forms of dyspepsia and in nervous pain of the stomach and bowels. Drug is reported to exhibit protective effect against CCl4 induced liver damage in rats. In combination with and rographolidepicrolivreported to exhibit anti cholestatic effect. It is also reported that the plant is a potent immune stimulant of both cell mediated and humoral immunity. The plant contains Iridoidglycosides peroxide I and kutko side as major constituents. Other minor constituents are as picroside-III, veronicoside, minecoside, phenol glycoside picein and and rosin, cucurbitacin glycosides and 4-hydroxy-3-methoxy acetophenone.

Because of its widespread use in various geographic regions, and to detect its adulteration, it is important to standardize the root of picrorhiza kurroa and its formulation. Therefore we have developed a HPTLC method for standardization of its extract and formulations using picroside I and kutkoside as marker compound (Murelle et al., 1989). Nature has been a source of medicinal agents for thousands of years and an impressie number of modern drugs have been isolated from natural sources. India is a

land of rich biodiversity. The total number of lower and higher plants in India is about 45,000 species. Many plants have been sources of medicines since ancient times. According to World Health Organization, 80% of the population of the world depends on traditional medical practitioners for their medicinal needs. Yet a scientific study of plants to determine their antimicrobial active compounds is a comparatively new field. Numerous surveys on biological important medicinal plants had been made in United States and in many countries through out the world. Such study had demonstrated the wide occurrence of active compounds in higher plants. Picrorhiza kurroa (Scrophulariaceae) is a small

perennial herb that grows in northwest India on the slopes of the Himalayas between 3000 and 5000 meters. It is an important herb in the traditional Ayurvedic system of medicine
34

and has been usedto treat liver and bronchial problems. Other tradition uses treatment of dyspepsia (Similar

include

to gentian in its bitter quality), bilious fever, chronic

dysentery and scorpion sting. The most important active constituents of Picrorhiza kurroa are the cucurbitacin glycosides, apocynin, drosin, kutkin. iridoid glycoside picrosides and

Picorrhizakurroa has hepatoprotective effect against Amanita poisoning Carbon

tetrachloride,and Aflotoxin B1.Bioactivity studies on Picrorhiza kurroa established its anti infamammatory, immunomodulatory and hydrocholeretic effects in rats and dogs and antiviral activity on vaccina virus. The present study was carried out to test the antibacterial efficacy of the rhizome extract of Picrorhiza kurroa with reference to bacteria spp. (Kumar et al., 2010). Kutkin, a bitter glycosidal principle, is reported. Also isolated D-mannitol, vanillic acid and some steroids are present. Kutkin was later shown to be a stable mixed crystal of two C-9 iridoid glycosides-Picroside I and Kutakosid. Apocynin has been isolated from the plant. Picroside II has been isolated and shown to have hepatoprotective activity. With the help of preparative HPLC, larger Quantities of picrosides have been isolated, permitting precise structure identification and biological experiments (Anjali et al., 2011). Therapeutic Uses, Benefits and Claims of Picrorhizakurroa are following. The root contains a number of very bitter glucosides including kitkin and picrorhizin, nine cucurbitacin glycosides, D-mannitol, benetic acid, kutkisterol, vanillic acid and some steroids. Picrorhiza kurroa also contains apocynin, a powerful anti-inflammatory agent, which also reduces platelet aggregation. The actions of Picrorhiza kurroa are antibacterial, antiperiodic, cathartic (in large doses), laxative (in smaller doses) stomachic and bitter tonic, hepatoprotective, anticholestatic (relieves obstruction of bile salts), anti-inflammatory, anti-allergy, antioxidant; modulates the immune system and liver enzyme levels. Picrorhiza kurroa is an important herb in the traditional Chinese and Ayurvedic systems of medicine, used to treat liver and upper respiratory conditions. Its traditional uses include treatment of a wide range of conditions, including fevers, chronic diarrhea, constipation, dyspepsia and jaundice. Picrorhiza kurroa is traditionally used to treat disorders of the upper respiratory tract, and is thought to be beneficial as an herbal treatment for bronchial asthma. Animal studies have shown that picrorhiza kurroa has a powerful antioxidant and antiinflammatory effect. It has also shown that the active constituents of picrorhiza kurroa may prevent liver toxicity and the ensuing biochemical changes caused by numerous toxic agents.
35

In other animal studies picrorhiza raised depleted glutathione levels in rats infected with malaria, boosting detoxification and antioxidation (Shaker et al., 2010) Picrorhiza kurroa is thought to be helpful as a remedy for a number of auto-immune diseases such as vitiligo and psoriasis. Research also indicates that picrorhiza kurroa may be of therapeutic value in treating viral hepatitis and that some constituents of picrorhiza kurroa may protect against liver damage due to Amanita mushroom poisoning. Studies have shown that the curcubitacins in picrorhiza kurroa are highly cytotoxic and have antitumor actions and that it may reduce blood cholesterol levels and reduce coagulation time. Furthermore studies of the rhizome, was shown to boost the immune system and to have a specific action against the parasite Leishmaniadonovani, which causes the tropical parasitic disease called leishmaniasis (Sood and Chauhan, 2009). Picrorhiza is a traditional herbal treatment for scorpion stings and snake bites. Alcohloic extract of the plant and kutkin possess hepatoprotective activity. Plant is a potent immunostimulant of both cell mediated and humoral immunity and exhibits choleretic activity in dogs. Picrorhizakurroa is also benefical in the management of bronchial asthma. Picrorhiza remedies for Protects the liver against hepatotoxins, hepatoprotective properties, potent antioxidant activity, Modulates liver enzyme levels, anti-inflammatory action anti-allergy action. Oxidative stress is one of the mechanisms with a central role involved in the pathogenesis of antitubercular drugs (isoniazid and rifampicin) induced hepatitis. In the present study the antihepatotoxic effect of the ethanol extract of Picrorhiza kurroa rhizomes and roots (PK) on liver mitochondrial antioxidant defense system in antitubercular drugs (isoniazid and rifampicin) induced hepatitis in rats has been investigated. In liver mitochondria of anti tubercular drugs administered rats, asignificant elevation in the level of lipid peroxidation with concomitant decline in the level of reduced glutathione and the activities of antioxidant enzymes was observed. Coadministration of PK (50 mg/kg/day for 45 days) significantly prevented these anti tubercular drugs induced alterations and maintained the rats at near normal status. The results of the present investigation indicated that the hepatoprotective effect of the ethanol extract of P. kurroa rhizomes and roots (PK) might be ascribable to its membrane-stabilizing action and/or antioxidant property (Friso Smit, 1968). Mechanisms of Action of Picrorhiza in the liver have following steps. Antioxidant: The mechanism by which Picrorhiza affords protection to the liver is not completely understood, but several possibilities have come to light. Like silymarin, Picrorhiza does possess significant antioxidant methosulphate NADH system, inhibited oxidative malonaldehyde
36

generation by both the ascorbate-Fe2+ and NADPH-ADP-Fe2+ systems, and scavenged superoxide (O2) anions generated in a xanthine-xanthine oxidase system. In other words, Picrorhiza demonstrated antioxidant activity similar to that of superoxide dismutase, metalion chelators, and xanthine oxidase inhibitors. Glutathione is vital to maintaining a variety of intracellular functions, including detoxification, antioxidation, tertiary protein configuration, and redox balance. Picrorhiza was found to restore depleted glutathione levels in African desert rats infected with activity in vitro which may contribute to the hepatoprotective effect by reducing lipid peroxidation and free radical damage. Chander et al found that Picrorhiza and its main constituents, picroside-I and kutkoside, inhibited the non-enzymatic generation of O2- anions in a phenazine methosulphate NADH system, inhibited oxidative malonaldehyde generation by both the ascorbate-Fe2+ and NADPH-ADP-Fe2+ systems, and scavenged superoxide (O2) anions generated in a xanthine-xanthine oxidase system. In other words, Picrorhiza demonstrated antioxidant activity similar to that of superoxide dismutase, metal-ion chelators, and xanthine oxidase inhibitors. Glutathione is vital to maintaining a variety of intracellular functions, including detoxification, antioxidation, tertiary protein configuration, and redox balance. Picrorhiza was found to restore depleted glutathione levels in African desert rats infected with Plasmodium berghei (malaria). Several enzymes

associated with glutathione function were also restored, including glutathione-S-transferase, glutathione reductase, and glutathione peroxidase. Generation of lipid peroxides in African desert rats infected with Plasmodium berghei was significantly reduced by Picrorhiza at the oral dose of 6 mg/kg for two weeks, revealing Picrorhiza also possesses anti-lipid peroxidative effects. Stimulation of Liver Regeneration: Like silymarin, Picrorhiza may have an effect on liver regeneration. A 1992 study demon-strated stimulation of nucleic acid and protein synthesis in rat liver with oral administration of Picrorhiza. The authors stated the results were comparable to silymarin. Anti-inflammatory: Another factor in the

hepatoprotection of Picrorhiza may be its anti-inflammatory effects. Picrorhiza extracts were found to have an inhibitory effect on such Pro-inflammatory cells as neutrophils, macrophages, and mast cells. The authorssug-gested Picrorhiza extract inhibited membranemediated activation of these cells (inhibited 8-adrenergic receptors). The researchers found no effect of the Picrorhiza extract on prostaglandin production. Picrorhiza contains apocynin, a catechol, as one of its minor constituents. Apocynin has been found to exhibit powerful anti-inflammatory effects on a variety of inflammatory models. Apocynin was found to inhibit neutrophil oxidative burst in vitro without affecting beneficial activities such as chemotaxis, phagocytosis, and intracellular killing of bacteria. In vivo animal models,
37

apocynin inhibited lipopoly saccharide-induced emphysema in hamsters. Apocynin prevented the formation of ulcerative lesions in rats injected intracutaneously with Freunds complete adjuvant, and reduced swelling in collagen-immunized rats. No effects on humeral and cellular immunity were observed after treatment with apocynin. What is remarkable about the last study is the effective daily dose of apocynin was only 0.024 mg/kg. Such a dose is readily achieved from normal use of Picrorhiza root instead of the concentrated apocynin extract. Choleretic: Several hepatotoxins, in-cludingparacetamol and ethynylestradiol, have a cholestatic effect on the production of bile. Picrorhiza has been shown to reverse acetaminophen and ethynylestradiol-induced cholestasis, maintaining both bile volume and flow. Silymarin was tested simultaneously for comparison. Picrorhiza was found to be a more potent choleretic and anticholestatic agent than silymarin (Scott Luper, 1998) Dosage/Toxicity of Picrorhiza is poorly soluble in water and so is usually not taken as a tea. It is soluble in ethanol and so can be taken in tincture form (very bitter), but is usually administered as an encapsulated standardized extract (4% kutkin). The usual adult dosage is 400 to 1500mg/day, although daily doses as high as 3.5 g/day have been recommended for fevers. Picrorhiza use is widespread in India and no major adverse reactions have been reported. The oral LD50 of kutkin is greater than 2600 mg/kg in rats. The LD50 of picrocide and kutkoside is greater than 1000 mg/kg in rats. By comparison, the maximum dose achievable with oral ingestion of Picrorhiza root is about 3-6 mg/kg (Rajaprabhuet al., 2007) Side Effects of Picrorhiza are rarely. It has some side effect on liver which are loose stools and colic have been reported when unprepared picrorhiza rhizomes are used as medicine. However, extracts in alcohol have shown much less tendency to cause such effects. No other adverse effects have been reported with picrorhiza. Although the use of the herb is not discouraged in India during pregnancy and breast-feeding, there is little information to determine the safety of the herb during these times (Teresa et al., 2008).

38

MATERIAL AND METHOD

39

METHODOLOGY
The purpose of the study was to investigate, The Combined Impact of Picrorhiza and Silymarin on Chromium Induced Hepatotoxicity in mice. The plane of work and methodology adopted are presented in this chapter. The study was carried out in three phases. In phase I, Albino mice were induced hepatotoxicity by chromium and then treated with extract of picrorhiza kurroa and silymarin. In the phase II, all the mice were slaughtered in order to get blood samples. Following six different parameters are assessed through following biochemical tests. Estimation of GLUTATHIONE S-TRANSFERASE (GST) Estimation of SUPEROXIDE DISMUTASE (SOD) Estimation of CATALASE (CAT) Estimation of MALONDIALDEHYDE (MDA) Estimation of CREATININE concentration Estimation of UREA concentration

In the final phase (phase III) of the study the analysis of confined impact of picrorhiza and silymarin on chromium induced toxicity was carried out. The study named as The combined impact of picrorhiza and silymarin was experimental in nature.

Experimental design:
Healthy Albino mice aged 6-8 weeks were purchased from Tolintan market Lahore. The animals were randomly divided in 4 groups of 3 animals in each group. They were fed standard pallet diet and drinking water. The protocol was approved by Institute of Molecular Biology and Biotechnology of The University of Lahore.

Treatment:
The group I was control group i.e. it was never given any treatment. Group II was treated with1ml of 100mg/L Cr solution dissolved in 250ml of water for 12 days. After 12 days they were treated with 200mg of silymarin extract mixed with their feed. Group III was treated with 1ml of 200mg/L Cr solution dissolved in 250ml of water for 12 days. After 12 days they were treated with 200mg of picrorohiza kurroa extract mixed with their feed. Group IV was treated with 1ml of 300mg/L Cr solution dissolved in 250ml of water for 12 days. After 12 days they were treated with 200mg of picrorohiza kurroa extract combined with 200mg of silymarin extract mixed with their feed.

40

Summary of grouping:
Group I Group II Group III Group IV : : : : Control group Cr solution + Silymarin Cr solution + Picrorhiza Cr solution + Picrorhiza +Silymarin

After 24 days the animals were slaughtered, blood was collected, and serum was separated by centrifugation of blood at 400 rpm. The serum sample were analyzed for liver marker enzymes, urea and creatinine concentration in blood for their antioxidant status.

ASSAY OF SUPEROXIDE DISMUTASE (SOD):

SOD was assayed according to the method of Kakkar et al. (1984).

PRINCIPLE:
The assay of SOD is based on the inhibition of the formation of NADH-phenazine methosulphate nitro blue tetrazolium formazon. The colour formed at the end of the reaction can be extracted into butanol and measured at 560nm.

REAGENTS:
1. Sodium pyrophosphate buffer (0.025M, pH 8.3) 2. Phenazine methosulphate (PMS) (186M) 3. Nitroblue tetrazolium (NBT) (300M) 4. NADH (780M) 5. Glacial acetic acid 6. n-butanol 7. Potassium phosphate buffer (50mM, pH 6.4)

PROCEDURE: PREPARATION OF ENZYME EXTRACT:

The different samples, namely leaves, stolon and roots (0.5g), were ground with 3.0ml of potassium phosphate buffer, centrifuged at 2000g for 10 minutes and the supernatants were used for the assay.

ASSAY:
The assay mixture contained 1.2ml of sodium pyrophosphate buffer, 0.1ml of PMS, 0.3ml of NBT, 0.2ml of the enzyme preparation and water in a total volume of 2.8ml. The reaction was initiated by the addition of 0.2ml of NADH. The mixture was incubated at 30C for 90 seconds and arrested by the addition of 1.0ml of glacial acetic acid. The reaction mixture was then shaken with 4.0ml of n-butanol, allowed to stand for 10 minutes and
41

centrifuged. The intensity of the chromogen in the butanol layer was measured at 560nm in a spectrophotometer. One unit of enzyme activity is defined as the amount of enzyme that gave 50% inhibition of NBT reduction in one minute.

ASSAY OF CATALASE (CAT):

Catalase activity was assayed following the method of Luck (1974).

PRINCIPLE:
The UV absorption of hydrogen peroxide can be measured at 240nm, whose absorbance decreases when degraded by the enzyme catalase. From the decrease in absorbance, the enzyme activity can be calculated.

REAGENTS
1. Phosphate buffer: 0.067 M (pH 7.0) 2. Hydrogen peroxide (2mM) in phosphate buffer

PROCEDURE PREPARATION OF ENZYME EXTRACT

A 20% homogenate of the different parts of B. Monnieri was prepared in phosphate buffer. The homogenate was centrifuged and the supernatant was used for the enzyme assay.

ASSAY
H2O2-phosphate buffer (3.0ml) was taken in an experimental curette, followed by the rapid addition of 40l of enzyme extract and mixed thoroughly. The time required for a decrease in absorbance by 0.05 units was recorded at 240nm in a spectrophotometer. The enzyme solution containing H2O2-free phosphate buffer served as control. One enzyme unit was calculated as the amount of enzyme required to decrease the absorbance at 240nm by 0.05 units.

ASSAY OF GLUTATHIONE S-TRANSFERASE (GST)

Glutathione S-transferase was assessed by the method of Habig et al. (1974).

PRINCIPLE
The enzyme is assayed by its ability to conjugate GSH and CDNB, the extent of conjugation causing a proportionate change in the absorbance at 340nm.

REAGENTS
1. Glutathione (1mM) 2. 1-chloro-2,4-dinitrobenzene (CDNB) (1mM in ethanol) 3. Phosphate buffer (0.1M, pH 6.5)

PROCEDURE
42

PREPARATION OF ENZYME EXTRACT

The samples (0.5g) were homogenized with 5.0ml of phosphate buffer. The homogenates were centrifuged at 5000rpm for 10 minutes and the supernatants were used for the assay.

ASSAY
The activity of the enzyme was determined by observing the change in absorbance at 340nm. The reaction mixture contained 0.1ml of GSH, 0.1ml of CDNB and phosphate buffer in a total volume of 2.9ml. The reaction was initiated by the addition of 0.1ml of the enzyme extract. The readings were recorded every 15 seconds at 340nm against distilled water blank for a minimum of three minutes in a spectrophotometer. The assay mixture without the extract served as the control to monitor non-specific binding of the substrates. GST activity was calculated using the extinction co-efficient of the product formed (9.6mM1cm1) and was expressed as n moles of CDNB conjugated/minute.

Estimation of Creatinine concentration

Creatinine forms with alkaline picrate a colored creatinine picrate complex containing ionic bonds. The rate of formation of the colored complex is proportional to the creatinine concentration.

Procedure Preparation of working reagent

Mix (R2) + (R3) in a ratio of 1:1.

Stability of working reagent

Reagent remains stable 20 to 25oC for 2 days. If the absorbance of working reagent is higher than 0.4 at 492 nm the reagent can not be used.

Assay conditions
Wavelength: 492 (480-520) nm Temperature: 37oC Curette: Method: I cm light path kinetic (increasing)

43

Pipette in curette
Standard Standard Sample Working reagent 1 ml 100l 100l 1 ml Sample

Mix and after 30 seconds read the absorbance against distilled water (AI). After 2 minutes incubation read the absorbance against (A2). The reagent kit is suitable for two0reagent method too; Reagents (R2) and (R3) can also be pipette separately (0.5-0.5 ml). Calibration frequency Two points calibration is recommended: After reagent lot change As required following quality control procedures.

Calculation using calibration

A = Absorbance C = Concentration

Estimation of Urea concentration

Urea is hydrolyzes by unease forming ammonia carbonic acid. Carbonic acid spontaneously decomposes into ammonia and carbon dioxide. The released ammonium, in the presence of salicylate and nitroferricyanide react with alkaline solution of sodium hypochlorite, to form a green dye compound. The intensity of green color produced is directly proportional to the amount of urea concentration.

Procedure
Wavelength: 578 (480-630) nm Temperature: 25-37oC Curette: Reading: Assay type: I cm light path Against reagent blank End point

44

Pipetting in tubes:
Blank Reagent (R3) R2 Standard Sample 1000 Drop 50/l Standard 1000 Drop 50/l 10 10 Sample 1000 Drop 50/l L L Unit L

Mix and incubate for 3 minutes at 37oC or for 5 minutes at 20-25oC.

Add in same tubes:

Blank R4 200 Standard 200 Sample 200 Unit L

Mix, incubate for 5 minutes at 37oC or for 10 minutes at 25oC and read sample and standard absorbance against blank. Volume can be proportionally modified. This methodology can describe the manual procedure to use the kit.

Calibration frequency
Two point calibrations are recommended: After reagent lot change As required following quality control procedures.

Calculation using calibration

A = Absorbance

C = Concentration

45

RESULTS

46

RESULTS
Table 1: Descriptive and Anova
Control Silymarin Picrorhiza Silymarin and Picrorhiza Catalase mg/ml MDA mg/ml SOD mg/ml GSH mg/ml Creatinine mg/dl Urea mg/dl Chromium g/g 32.383 0.265 4.226 2.953 0.201 0.001 0.287 0.066 4.836 1.188 23.196 4.878 1.306 0.720 36.996 7.861 0.776 0.137 0.353 0.100 0.423 0.151 3.096 2.893 24.586 11.848 1.680 0.511 46.070 0.000 0.213 0.164 0.523 0.063 0.386 0.159 2.796 3.055 19.900 3.884 0.806 0.633 45.733 0.583 0.303 0.154 0.312 0.295 0.409 0.045 6.354 0.882 8.764 6.648 0.410 0.036 0.05 0.007 0.050.514 0.050.371 0.05 0.322 0.05 0.253 0.05 0.111 0.05 0.086 Sig.

Anova is significant at 0.05 level.

The changes in the activities of enzymatic antioxidants namely SOD, catalase GSH and MDA in liver and creatinine, Urea and nikel metal of control and experimental animals are shown in Table 1. In this experiment mice given either silymarin or Picrorhiza alone and combined silymarin and Picrorhiza were significantly (0.05 0.007) increase catalase level as compared to control group mice. Table 1 shows that the mice given silymarin or Picrorhiza alone or combine silymarin and Picrorhiza were insignificantly (0.050.514) decreased MDA level as compared to control group. The mice given silymarin or Picrorhiza alone or silymarin and Picrorhiza combinedly were insignificantly (0.050.371) increase SOD level as compared control mice. The mice given either silymarin or Picrorhiza alone or combine silymarin and Picrorhiza were insignificantly (0.05 0.322) increased the level of GSH as compared to control mice. The mice given either silymarin or Picrorhiza alone were insignificantly (0.05 0.253) decreased the level of creatinine as compared to mice of control group. However, combine silymarin and Picrorhiza were insignificantly increasing the creatinine level. The mice given silymarin alone were insignificantly (0.05 0.111) increased urea level than control mice. However, the mice given Picrorhiza alone or combine silymarin and
47

Picrorhiza were insignificantly decreased as compared to control mice. Urea level decrease more in those mice which are treated with combine silymarin and Picrorhiza than mice treated with Picrorhiza alone. The mice given silymarin alone were insignificantly (0.05 0.086) increased the level of chromium metal than control mice. However, the mice given Picrorhiza alone or combine and silymarin were insignificantly decrease chromium metal level. Table 2: Correlation
Catalase Catalase 1 MDA -.175 .587 MDA 1 SOD -.270 .397 .269 .397 SOD 1 GSH .183 .569 -.316 .317 .139 .666 GSH 1 Creatinine Urea -.131 .684 .023 .943 .222 .488 .438 .154 Creatinine 1 -.184 .567 .276 .386 .056 .863 -.841 .001 -.673 .017 Urea 1 Chromium -.474 .120 .678 .015 .267 .401 -.208 .157 -.153 .635 .420 .174 Chromium 1

The data represented in table 2 shows negative correlation of serum Catalase with MDA (r = -0.175 and p 0.587), SOD (r=-0.270 and p 0.397), creatinine (r=-131 and p0.684), urea (r=-0.184 and p0.567) and chromium metal (r=-474 and p0.120). On the other hand result showed positive correlation of Catalase with GSH (r=0.183 and p0.569). Results in table 2 showed negative correlation of serum MDA with GSH (r=-0.316 and p0.317). On the other hand positive correlation of serum MDA with SOD (r=-0.269 and p0.397) creatinine (r=0.023 and p0.943), urea (r=0.276 and p0.386) and Chromium metal (r=0.678 and p0.0.15). Data represented in table 2 showed positive correlation of serum SOD with GSH (r=0.139 and p0.666), creatinine (r=0.222 and p0.488), urea (r=0.056 and p0.863) and chromium metal (r=0.267 and p0.401).
48

The data represented in table 2 showed negative correlation of serum GSH with urea (r=-0.841 and p0.001) and chromium metal (r=-0.208 and p0.517). On the other hand result showed positive correlation of serum GSH with creatinine (r=0.438 and p0.154). Results in table 2 showed negative correlation of serum creatinine with urea (r=-0.673 and p0.017) and chromium metal (r=-0.153 and p0.635). Table 2 showed positive correlation of serum urea with chromium (r=0.420 and p0.174).

49

Table 3: Multiple Comparisons

Dependent Variable Catalase (I) (J) Groups Groups G1 G2 G1 G3 G1 G4 G2 G3 G2 G4 G3 G4 G1 G2 G1 G3 G1 G4 G2 G3 G2 G4 G3 G4 G1 G2 G1 G3 G1 G4 G2 G3 G2 G4 G3 G4 G1 G2 G1 G3 G1 G4 G2 G3 G2 G4 G3 G4 G1 G2 G1 G3 G1 G4 G2 G3 G2 G4 G3 G4 G1 G2 G1 G3 G1 G4 G2 G3 G2 G4 G3 G4 G1 G2 G1 G3 G1 G4 G2 G3 G2 G4 G3 G4 Sig. 0.190 0.003 0.003 0.023 0.027 0.919 0.967 0.762 0.206 0.794 0.219 0.319 0.341 0.102 0.235 0.428 0.794 0.588 0.669 0.676 0.176 0.406 0.090 0.324 0.367 0.295 0.429 0.873 0.111 0.087 0.826 0.604 0.046 0.465 0.032 0.105 0.425 0.293 0.078 0.085 0.021 0.398

MDA

SOD

GSH

Creatinine

Urea

Chromium

50

Multiple Comparisons The data represented in table 3 interprets the following results of multiple comparisons between group 1 (G1), group 2 (G2), group 3 (G3) and group 4 (G4) of Catalase, MDA, SOD, GSH, Creatinine, Urea and Nickel metal. There was an insignificant difference between G1 and G2 (p0.190), G3 and G4 (p0.0.919) but there was a significant difference between G1 and G3 (p0.003), G4 (p0.0.003), G2 and G3 (p0.0.023), G4 (p0.027) of catalase values. There was an insignificant difference between G1 and G2 (p0.0.967), G3 (p0.762) G4 (p0.206), G2 and G3 (0.794), G4 (p0.219), G3 and G4 (0.319) of MDA values. There was an insignificant difference between G1 and G2 (p0.341), G3 (p0.102) G4 (p0.235), G2 and G3 (0.428), G4 (p0.794), G3 and G4 (0.588) of SOD values. There was an insignificant difference between G1 and G2 (p0.669), G3 (p0.676), G4 (p0.176), G2 and G3 (p0.406), G4 (p0.090), G3 and G4 (p0.324) of GSH values. There was an insignificant difference between G1 and G2 (p0.367), G3 (p0.295), G4 (p0.429), G2 and G3 (p0.873), G4 (p0.111), G3 and G4 (p0.087) of creatinine values. There was an insignificant difference between G1 and G2 (p0.826), G3 (p0.604), G2 and G3 (p0.465), G3 and G4 (0.105) but significant difference between G1 and G4 (p0.046), G2 and G4 (p0.032), of urea values. There was an insignificant difference between G1 and G2 (p0.425), G3 (p0.293), G4 (p0.078), G2 and G3 (p0.085), G3 and G4 (p0.398) but significant difference between G2 and G4 (p0.021), of chromium metal values.

51

DISCUSSION

52

DISCUSSION
The present study was design to investigate the combined impact of silymarin and Picrorhiza kurroa on chromium (Cr) induced hepatotoxicity in the mice. Silymarin is used as standard drug in various experimental and clinical studies due to its proven hepatoprotective effect (Dhiman and Chawla; 2005 and Singh et al., 2012). The Picrorhiza kurroa was also used as hepatoprotective drug because it increase the activities of antioxidant enzymes or it was used to counter action of free redical by the presence of the electrophilic picroside I, picroside II, and kutkoside (Jeyakumar et al., 2008). The administration of chromium to mice result in enhanced lipid peroxidation, decrease glutathione peroxidase activity, increased MDA and decrease catalase level. The carcinogenicity of Cr compound may be related to enhanced production of reactive oxygen species, presumably through the formation of oxidative tissue damage including damage to DNA (Stohs and Bagchi; 1994). The toxic changes associated with Cr induced liver damage are similar to that of acute viral hepatitis clinically (Sidhu et al., 2004). Therefore, the Cr induced hepatotoxicity was selected as experimental model of liver injury in the present investigation. In present study Cr intoxication cause significant increase of hepatic enzymes and this was probably due to the consequences of oxidative stress and necrotic cell death (Kyle et al., 1987). Treatment with silymarin significantly attenuated the increase level of serum markers in a dose dependant manner as compared to hepatotoxicant control. This ability of silymarin may be due to its free redical scavenger activity (Singh et al., 2012). In earlier report the combination of silymarin (50mg/kg) with Picrorhiza kurroa extract offered significant hepatoprotective against CCl4 induced liver damage (Yadav et al., 2008). In this study it was also confirmed that silymarin with Picrorhiza extract significantly hepatoprotective against Cr. The observed effect in our studies due to the combined action of silymarin with Picrorhiza in improving the hepatic cell functioning upon experimental liver damage. Catalase is an enzyme present in most of the aerobic cells; it protects them from oxidative stress by catalyzing the rapid decomposition of hydrogen peroxide (H2O2) in two types of reactions depending on its peroxidatic and catalytic activities. (Salam et al., 2000). In this experiment mice given either silymarin or Picrorhiza alone and combined silymarin and Picrorhiza were significantly (p 0.05) increase catalase level as compared to control group mice. These results from study indicate that the potentional role of silymarin, Picrorhiza alone and in combination pretreatment was prevent oxidative stress and strengthening antioxidant defense mechanism.
53

The reactive oxygen species (ROS) superoxide radical (O2), hydrogen peroxide (H2O2) and hydroxyl radical (OH-) was increased after discovery of their critical role in many diseases. Their increased level and decreased antioxidant defense can cause DNA damage and direct inhibition of proteins. The main target substrates for free oxygen radical activity are polyunsaturated fatty acids in membrane phospholipids, the modification of which results in disorganization of self frame work and function. The end product of these reactions is malondialdehyde (MDA). It is excreated in urine, blood and other fluids and therefore serves as a marker of lipidperoxidation and the presence of oxidative stress respectively (Todorova et al., 2005). It means that oxidative stress increased with the increased level of MDA. Table 1 shows that the mice given silymarin or Picrorhiza alone or combine silymarin and Picrorhiza were insignificantly (p 0.05) decreased MDA level as compared to control group. The result indicates that silymarin; Picrorhiza alone and in combination state was effective in reducing hepatoxicity. But Picrorhiza alone was more effective and lower the level of MDA. Superoxide Dismutase (SOD) catalyzes the reduction of superoxide anions to hydrogen peroxide. It plays a critical role in the defense of cells against the toxic effects of oxygen radicals. SOD competes with nitric oxide (NO) for superoxide anion, which inactivates NO to form peroxynitrite. Therefore, by scavenging superoxide anions, SOD promotes the activity of NO. SOD has suppressed apoptosis in cultured rat ovarian follicles, neural apoptosis in neural cell lines, and transgenic mice by preventing the conversion of NO to peroxynitrate, an inducer of apoptosis. The SOD level decrease oxidative stress produced by toxicity (Tilly et al., 1995). The mice given silymarin or Picrorhiza alone or silymarin and Picrorhiza combinedly were insignificantly (p 0.05) increase SOD level as compared control mice. Results indicates that silymarine , Picrorhiza alone and combined silymarin and Picrorhiza are effective and increase the level of SOD. Picrorhiza alone was more effective as it increase the level of SOD more as compared to silymarin alone and combination of silymarin and Picrorhiza. Glutathione is a small protein composed of three amino acids: cysteine, glutamic acid and glycine. It is involved in detoxification of the liver and the body. Glutathione to toxins, such as heavy metals, solvents, and pesticides, and transforms them into a form that can be excreted in urine or bile. Glutathione is also an important antioxidant, counteracting the effects of free radicals produced in the body by oxidation reactions. Increase level of GSH reduced the oxidative stress (James Holly; 2011). The mice given either silymarin or Picrorhiza alone or combine silymarin and Picrorhiza were insignificantly (p 0.05) increased the level of GSH as compared to control mice. Therefore, it can be concluded from result that
54

silymarin, Picrorhiza alone and in combined form are effective for liver detoxification because these increase the level of GSH. But silymarin alone and in combination with Picrorhiza were more effective than Picrorhiza alone. Creatinine is a breakdown product of creatine, which is an important part of muscle. In the increase serum creatinine level were certainly as a result of liver and kidney tissue damage and dysfunction. It is suggested that it may be a result of the oxidative stress, which had been occurred in the two metal toxicity. In other words the reduced antioxidant production was due to the increased oxygen metabolites and the elevated free radicals, oxygen metabolites and the elevated free radicals, which cause a decrease in the activity of the antioxidant defense system (Kechrid et al., 2006). The present study in which mice given either silymarin or Picrorhiza alone were insignificantly (p 0.05) decreased the level of creatinine as compared to mice of control group. However, combine silymarin and Picrorhiza were insignificantly increasing the creatinine level. So, we can conclude from the result that silymarine and Picrorhiza alone are effective in detoxification as compared to combine silymarin and Picrorhiza treatment. Urea, also called carbamide, is an organic chemical compound, and is essentially the waste produced by the body after metabolizing protein. Naturally, the compound is produced when the liver breaks down protein or amino acids, and ammonia; the kidneys then transfer the ureafrom the blood to the urine. Low serum urea concentration is not an independent risk factor for hepatotoxicity after paracetamol overdose (Waring et al., 2007). The present study showed that mice given silymarin alone were insignificantly (p 0.05) increased urea level than control mice. However, the mice given Picrorhiza alone or combine silymarin and Picrorhiza were insignificantly decreased as compared to control mice. Urea level decrease more in those mice which are treated with combine silymarin and Picrorhiza as compared to mice treated with Picrorhiza alone. It was concluded that combine silymarin and Picrorhiza was very effective in detoxification. Chromium is an essential trace metal in human diet is also a major component of alloy. Its excess amount causes toxicity. It cause organ toxicity and DNA breaks (Kechrid et al., 2005). Particles of nickel may cause some morphological transformation in numerous cellular system and chromosomal aberration (Coen et al., 2001). The salt of nickel as particle of nickel can be allergens and carcinogens in man while forming the oxygenated redicals (Lansdown; 1995). The study showed that mice given silymarin alone were insignificantly (p 0.05) increased the level of chromium metal than control mice. However, the mice given
55

Picrorhiza alone or combine and silymarin were insignificantly decrease chromium metal level. It was concluded that picrorhiza alone and in combination with silymarin was more effective than silymarin alone but combine treatment of Picrorhiza and silymarin was more effective than all in reducing chromium level. Thus it is concluded that Picrorhiza kurroa alone and combined silymarin and picorrhiza was more effective than silymarin alone.

56

SUMMARY

57

SUMMARY
The present study was design to explore The combined impact of silymarin and pcrorrhiza on chromium induced hepatotoxicity in mice. 12 albino mice were taken from Tolinton market of Lahore. The animals were randomly divided into 4 groups of 3 animals each. The protocol was approved by Institute of Molecular Biology and Biotechnology of University of Lahore. The group I was control group i.e. it was never given any treatment. The group I was control group i.e. it was never given any treatment. Group II was treated with1ml of 100mg/L Cr solution dissolved in 250ml of water for 12 days. After 12 days they were treated with 200mg of silymarin extract mixed with their feed. Group III was treated with 1ml of 200mg/L Cr solution dissolved in 250ml of water for 12 days. After 12 days they were treated with 200mg of picrorohiza kurroa extract mixed with their feed. Group IV was treated with 1ml of 300mg/L Cr solution dissolved in 250ml of water for 12 days. After 12 days they were treated with 200mg of picrorohiza kurroa extract combined with 200mg of silymarin extract mixed with their feed. After 24 days the animals were slaughtered, blood was collected, and serum was separated by centrifugation of blood at 4000 rpm. The serum samples were analyzed for liver marker enzymes (i.e. catalase, MDA, GSH. SOD) urea and creatinine concentration in blood for their antioxidant status. The results of these tests were analyzed by Anova, Multiple comparisons and Correlation and the result concluded that Picrorhiza kurroa alone and combined silymarin and picorrhiza was more effective than silymarin alone.

58

CONCLUSION

59

CONCLUSION
The present study is The combined impact of silymarin and Picrorhiza kurroa on the chromium induced hepatotoxicity in mice. The liver biomarker tests determine the

oxidative stress was catalase, MDA, GSH, and SOD. However, serum urea and serum craetinine was also measured to determine the hepatoxiciy. From the result and discussion of the present study it was concluded that Picrorhiza kurroa alone and combine silymarin and Picrorhiza was more effective than silymarin in detoxification of liver toxicity.

60

LITERATURE CITED

61

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