Introduction

INTRODUCTION

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INTRODUCTION

Metal toxicity is a major medical concern. Of particular concern are “Heavy metals”

which occur naturally in the earth’s 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 100µg/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), Ngo-

Honglen, 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 anti-

inflammatory 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

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(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 anti-

inflammatory 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.

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LITERATURE REVIEW

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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. S-

adenosyl-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 con-

tamination 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, Parkinson’s disease, fibromyalgia, and chronic

fatigue/immune dysfunction syndrome. Therefore, accumulated data suggests an individual’s

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 individual’s environment, lifestyle, and

genetic uniqueness.

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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 down—if 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.

The gallbladder then secretes into the digestive tract with its bile, for removal from the body

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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, α-

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 UDP-

glucuronyl transferase. Normal serum total bilirubin varies from 2 to 21μmol/L. The indirect

(unconjugated) bilirubin level is less than 12μmol/L and direct (conjugated) bilirubin less

than 8μmol/L. The serum bilirubin levels more than 17μmol/L suggest liver diseases and

levels above 24μmol/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 μmol/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,

Crigler-Najjar syndrome and reabsorption of large hematomas and ineffective erythropoiesis.

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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 μmol/L

to 143.64μmol/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

of the metabolic syndrome in adults. ALT level is normally elevated during 2nd trimester in

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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, α-carotene, β-carotene, and β-cryptoxanthin are inversely

associated with alcohol-induced increase of serum GGT found in moderate and heavy

drinkers. GGT levels may be 2–3 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, menke’s 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

15

the Wilson’s 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 15μg/L. An AFP value above

400 - 500μg/L has been considered to be diagnostic for hepato-cellular carcinoma (HCC) in

patients with cirrhosis. A high AFP concentration ≥ 400μg/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 non-

Lens 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 HCC-

associated 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

16

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 Earth’s 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 mirror-

like 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 public’s 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 serpentine-

derived 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

μg/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 μg/day for “young men”

and 25 μg/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 earth’s 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).

21

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 anti-

inflammatory 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 Lady’s 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 it’s 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.

23

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 dehydro-

succinate sodium salt of silibinin is a powerful inhibitor of the oxidation of linoleic acid-

water 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,

24

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 pro-

oxidant 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 ß-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 benzyl-

penicillin (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-α (TNFα) in hepatic injury produced by α-amanitin has been investigated in

primary cultures of rat hepatocytes. At a concentration of 0.1 µmol/L, the toxin inhibits RNA

and protein synthesis within 12 hours, but cyto toxicity appears only much later (36 hours).

TNFα is not indispensable for the development of cyto toxicity, but exacerbatesitand

markedly increases lipid peroxidation. The addition of silibinin at a concentration of

25µmol/L to the culture medium prevented the effects of TNFα (50µg/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 carcinogenesis-

sensitive 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-κB, 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 100µmol/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 α-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 im-

mune 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 anti-

inflammatories 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, car-

bon 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, life-

threatening 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 ½ 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  include 

treatment  of  dyspepsia  (Similar 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, iridoid glycoside picrosides  and  

kutkin.   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 anti-

inflammatory 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 under-

stood, 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, 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 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, mac-

rophages, and mast cells. The author’ssug-gested Picrorhiza extract inhibited membrane-

mediated 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 Freund’s 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 re-

ported. 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 : Control group

Group II : Cr solution + Silymarin

Group III : Cr solution + Picrorhiza

Group IV : 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) (186µM)

3. Nitroblue tetrazolium (NBT) (300µM)

4. NADH (780µM)

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 30°C

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 40µl 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

(9.6mM−1cm−1) 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: I cm light path

Method: kinetic (increasing)

43

Pipette in curette

Standard Sample

Standard 100µl

Sample 100µl

Working reagent 1 ml 1 ml

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

C sample=Cstandard∗A 2 sample−A 1 sample

A 2 standard−A 1 standard

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: I cm light path

Reading: Against reagent blank

Assay type: End point

44

Pipetting in tubes:

Blank Standard Sample Unit

Reagent (R3) 1000 1000 1000 µL

R2 Drop 50µ/l Drop 50µ/l Drop 50µ/l

Standard 10 µL

Sample 10 µL

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

Add in same tubes:

Blank Standard Sample Unit

R4 200 200 200 µL

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

C sample=Cstandard∗A 2 sample−A 1 sample

A 2 standard−A 1 standard

A = Absorbance C = Concentration

45

RESULTS

46

RESULTS

Table 1: Descriptive and Anova

Control Silymarin Picrorhiza Silymarin and

Picrorhiza

Sig.

Catalase mg/ml 32.383 ± 0.265 36.996 ± 7.861 46.070 ± 0.000 45.733 ± 0.583 0.05≥ 0.007

MDA mg/ml 4.226 ± 2.953 0.776 ± 0.137 0.213 ± 0.164 0.303 ± 0.154 0.05≤0.514

SOD mg/ml 0.201 ± 0.001 0.353 ± 0.100 0.523 ± 0.063 0.312 ± 0.295 0.05≤0.371

GSH mg/ml 0.287 ± 0.066 0.423 ± 0.151 0.386 ± 0.159 0.409 ± 0.045 0.05≤ 0.322

Creatinine mg/dl 4.836 ± 1.188 3.096 ± 2.893 2.796 ± 3.055 6.354 ± 0.882 0.05≤ 0.253

Urea mg/dl 23.196 ± 4.878 24.586 ± 11.848 19.900 ± 3.884 8.764 ± 6.648 0.05≤ 0.111

Chromium µg/g 1.306 ± 0.720 1.680 ± 0.511 0.806 ± 0.633 0.410 ± 0.036 0.05≤ 0.086

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 (0.05≤0.514) decreased MDA level as compared to control

group.

The mice given silymarin or Picrorhiza alone or silymarin and Picrorhiza combinedly

were insignificantly (0.05≤0.371) 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

Picrorhiza were insignificantly decreased as compared to control mice. Urea level decrease

47

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 MDA SOD GSH Creatinin

e

Urea Chromium

Catalase 1 -.175

.587

-.270

.397

.183

.569

-.131

.684

-.184

.567

-.474

.120

MDA 1 .269

.397

-.316

.317

.023

.943

.276

.386

.678

.015

SOD 1 .139

.666

.222

.488

.056

.863

.267

.401

GSH 1 .438

.154

-.841

.001

-.208

.157

Creatinine 1 -.673

.017

-.153

.635

Urea 1 .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

p≤0.684), urea (r=-0.184 and p≤0.567) and chromium metal (r=-474 and p≤0.120). On the

other hand result showed positive correlation of Catalase with GSH (r=0.183 and p≤0.569).

Results in table 2 showed negative correlation of serum MDA with GSH (r=-0.316

and p≤0.317). On the other hand positive correlation of serum MDA with SOD (r=-0.269 and

p≤0.397) creatinine (r=0.023 and p≥0.943), urea (r=0.276 and p≤0.386) and Chromium metal

(r=0.678 and p≥0.0.15).

Data represented in table 2 showed positive correlation of serum SOD with GSH

(r=0.139 and p≤0.666), creatinine (r=0.222 and p≥0.488), urea (r=0.056 and p≤0.863) and

chromium metal (r=0.267 and p≤0.401).

48

The data represented in table 2 showed negative correlation of serum GSH with urea

(r=-0.841 and p≥0.001) and chromium metal (r=-0.208 and p≤0.517). On the other hand

result showed positive correlation of serum GSH with creatinine (r=0.438 and p≤0.154).

Results in table 2 showed negative correlation of serum creatinine with urea (r=-0.673

and p≥0.017) and chromium metal (r=-0.153 and p≤0.635).

Table 2 showed positive correlation of serum urea with chromium (r=0.420 and

p≤0.174).

49

Table 3: Multiple ComparisonsDependent Variable

(I) (J)Groups Groups

Sig.

Catalase G1 G2 0.190G1 G3 0.003G1 G4 0.003G2 G3 0.023G2 G4 0.027G3 G4 0.919

MDA G1 G2 0.967G1 G3 0.762G1 G4 0.206G2 G3 0.794G2 G4 0.219G3 G4 0.319

SOD G1 G2 0.341G1 G3 0.102G1 G4 0.235G2 G3 0.428G2 G4 0.794G3 G4 0.588

GSH G1 G2 0.669G1 G3 0.676G1 G4 0.176G2 G3 0.406G2 G4 0.090G3 G4 0.324

Creatinine G1 G2 0.367G1 G3 0.295G1 G4 0.429G2 G3 0.873G2 G4 0.111G3 G4 0.087

Urea G1 G2 0.826G1 G3 0.604G1 G4 0.046G2 G3 0.465G2 G4 0.032G3 G4 0.105

Chromium G1 G2 0.425G1 G3 0.293G1 G4 0.078G2 G3 0.085G2 G4 0.021G3 G4 0.398

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 (p≤0.190), G3 and

G4 (p≤0.0.919) but there was a significant difference between G1 and G3 (p≥0.003), G4

(p≥0.0.003), G2 and G3 (p≥0.0.023), G4 (p≥0.027) of catalase values.

There was an insignificant difference between G1 and G2 (p≤0.0.967), G3 (p≤0.762)

G4 (p≤0.206), G2 and G3 (0.794), G4 (p≤0.219), G3 and G4 (0.319) of MDA values.

There was an insignificant difference between G1 and G2 (p≤0.341), G3 (p≤0.102)

G4 (p≤0.235), G2 and G3 (0.428), G4 (p≤0.794), G3 and G4 (0.588) of SOD values.

There was an insignificant difference between G1 and G2 (p≤0.669), G3 (p≤0.676),

G4 (p≤0.176), G2 and G3 (p≤0.406), G4 (p≤0.090), G3 and G4 (p≤0.324) of GSH values.

There was an insignificant difference between G1 and G2 (p≤0.367), G3 (p≤0.295),

G4 (p≤0.429), G2 and G3 (p≤0.873), G4 (p≤0.111), G3 and G4 (p≤0.087) of creatinine

values.

There was an insignificant difference between G1 and G2 (p≤0.826), G3 (p≤0.604),

G2 and G3 (p≤0.465), G3 and G4 (0.105) but significant difference between G1 and G4

(p≥0.046), G2 and G4 (p≥0.032), of urea values.

There was an insignificant difference between G1 and G2 (p≤0.425), G3 (p≤0.293),

G4 (p≤0.078), G2 and G3 (p≤0.085), G3 and G4 (p≤0.398) but significant difference between

G2 and G4 (p≤0.021), 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

REFERENCES

Abdul Salam, Al-Abrash, Faizeh A., Al-Quobaili, Ghada N. Al-Akhras, 2000. Catalase

evaluation in different human disease associated with oxidative stress. Saudi Medical

Journal, 21(9): 826-830.

Anjali Uniyal, Gopal S. Rawat, Sanjay Kr. Uniyal, 2011. Extraction of Picrorhiza kurrooa.

Current science, 100(11): 1055-1059.

Assem L. and Zhu H, 2007. Chromium Toxicological Overview. Institute of environment and

health Cranifield University, 1: 1-14.

Baro , V.P and Muriel, 1999. Role of glutathione, lipid peroxidation and antioxidants on

acute bile-duct obstruction in the rat. Biochim. Biophys. Acta, 1472: 173–180.

Bedosa, P., K. Houglum, C. Trautwein, A. Holstege and M. Chojkier, 1994. Stimulation of

collagen al(I) gene expression is associated with lipid peroxidation in hepatocellular

injury, A link to tissue fibrosis. Hepatology, 19: 1262–1271.

Boigk, G., L. Stroedter, H. Herbst, J.Waldschmidt, E. O. Riecken & D. Schuppan, 1997.

Silymarin retards collagen accumulation in early and advanced biliary fibrosis

secondary to complete bile duct obliteration in rats. Hepatology, 26: 643–649.

Chidambaram A., Sundaramoorthy P., Murugan A., Sankar Ganesh K., Baskaran L., 2009.

Chromium induced cytotoxicity in blackgram. Iran. J. Environ. Health science, 6(1):

17-22.

Deann J. Liska, 1998. The detoxification enzyme system. Alternative Medicine Review, 3(3):

187-198.

Debasis Bagchi, Manashi Bagchi and Sidney J. Stohs, 2001. Chromium(VI)-induced

oxidative stress, apoptotic cell death and modulation of p53 tumor suppressor gene.

Molecular and cell biochemistry, 222: 149-158.

Fraschini F. and Demartini G. , 2002. Pharmacology of sliymarin Clin drug invest. 22(1): 51-

65.

Girish C., Suresh Chandra Pradhan , 2009. Indian Journal of Experimental Biology. 47: 257-

263.

Gramenzi A., Caputo F., Biselli M., Kuria F. 2006. Review article: alcoholic liver disease-

pathophysiological aspects and risk factors. Aliment pharmacol ther, 24: 1151-1161.

Habib-ur-Rehman M., Tariq Mehmood, Tahir Salim, Naeema Afzal, Nasir Ali, Javed Iqbal,

Muhammad Tahir, Asif Khan, 2009. Effect of silymarin on serum level of ALT and

62

GGT in ethanol induced hepatotoxicity in Albino rats. Med Call Abbottabad, 21(4):

73-75.

Hatice Akkaya, Okkes Yilmaz, 2012. Antioxidant Capacity and Radical Scavenging Activity

of Silybum marianum and Ceratonia siliqua Ekologi, 21(28): 9-16.

Jacques Guertin, 2004. Toxicity and Health Effect of Chromium (All oxidation states),

Chromium (VI) Handbook, L1608-C06 fm: 214

Jane M. Murphy, RNC, MS, PNP, Mary Caban, BS, MPH, and Kathi J. Kemper, MD, MPH,

2000. Milk thistle (Silybum marianum). Longwood Herbal Task Force, The center for

holistic pediatric education and research, 1-25.

Jayshree Aiyar, Holly J. Berkovits, Robert A. Floyd and Karen E. Wetterhahn, 1991.

Reaction of Chromium (VI) with Glutathione or with hydrogen peroxide:

Identification of reactive intermediates and their role in Chromium(VI) induced DNA

damage. Environmental health precpectives, 92: 53-62.

Jennifer A, Biser, Laura A. Vogel, Joel Berger, Brien Hjelle and Sabine S. Loew, 2004.

Effect of heavy metals on immunocompetence of white-footed mice (Peromyscus

Leucopus). Journal of wildlife diseases, 40(2): 173-184.

Jeyakumar R., Rajesh R., Meena B., Rajaprabhu D., Ganesan B., Buddhan S., Anadan, 2008.

Antihepatotoxic effect of Picrorhiza kurroa on mitochondrial defense system in

antitubercular drugs (isoniazid and rifampicin)-induced hepatitis in rats. Journal of

medicinal plants research, 2(1): 017-019.

John B., Vincent, 1999. Mechanism of chromium action: Low molecular weight chromium

binding substance. Journal of American college of nutrition, 18(1): 6-12.

Kumar Anil, 2012. International Journal of Research in Pharmacy and Chemistry, 2(1): 92-

103.

Kuppanan Gobianand, Sivanesan Karthikeyan 2007. Silymarin modulates the oxidant–

antioxidant imbalance during diethylnitrosamine induced oxidative stress in rats.

European J. of Pharmacology, 560: 110-116.

Lily Dara, Jennifer Hewett, Joseph Kartaik Lim, 2008. Hydroxycut hepatotoxicity : A case

series and review of liver toxicityfrom herbal weigh loss suppliments. World

gastroenterol, 14(45): 6999-7004.

Mayer K. E., Mayers R. P. and Lee S. S., 2005. Silymarin treatment of viral hepatitis: a

systematic review. Journal of Viral Hepatitis, 12: 559-567.

Mourelle, M., P. Muriel, L. Favari & T. Franco, 1989. Prevention of CCl4 induced liver

cirrhosis by silymarin. Fund. Clin. Pharmacol, 3: 183–191.

63

Murad, S. D. Grove, K. A. Lindberg, G. Reynolds, A. Sivarajah & S. R. Pinnell, 1981.

Regulation of collagen synthesis by ascorbic acid. Proc. Natl. Acad. Sci. USA, 78:

2879–2882.

Muriel, P. & M. Mourelle, 1990. Prevention by silymarin of membrane alterations in acute

liver damage. J. Appl. Toxicol, 10: 275–279.

Muriel, P. and Mourelle, M., 1990. The role of membrane composition in ATPases activities

of cirrhotic rats. Effect of silymarin. J. Appl.Toxicol. 10: 281–284.

Muriel, P. and Suarez, O. R., 1994. Role of lipid peroxidation on biliary obstruction in the rat.

J. Appl. Toxicol, 14: 423–426.

Muriel, P., Garciapin T., Perez Alvarez V. and Mourelle, M., 1992. Silymarin protects

against paracetamol-induced lipid peroxidation and liver damage. J. Appl. Toxicol, 12:

439–442.

Muriel, P., 1996. Alpha-interferon prevents liver collagen deposition and damage induced by

prolonged bile duct obstruction in the rat. J. Hepatol, 24: 614–621.

Neuran Ercal, Hande Gurer-Orhan and Nukhet Aykin-Burns, 2001. Toxic metals and

oxidative stress Part I: Mechanism involved in metal induced oxidative damage.

Current topic in medicinal chemistry, 1: 529-539.

Nitin Dixit, Sanjula Baboota, Kanchan Kohli, S. Ahmed, Javed Ali, 2007. Silymarin: A

review of pharmacological aspects and bioavailability enhancement approaches.

Journal Pharmacol, 39(4): 172-179.

Pablo Morlel and Mario Moreno, G., 2004. Effect of silymarin and vitamins E and C on liver

damage induced by prolonged biliary obstruction in the rat. Basic and Clinical

Pharmacology & Toxicology, 94: 99-104.

Pandey Govind and Sahni, Y. P., 2011. A review on hepatoprotective activity of silymarin.

IJRAP, 2(1): 75-79.

Patrizia Russo, Alessia Catassi, Alfredo Cesario, Andrea Imperatori, Nicola Rotolo, Massimo

Fini, Pierluigi Granone and Lorenzo Dominioni, 2005. Molecular mechanisms of

hexavalent Chromium induced apoptosis in human bronchoalveolar cells. Am. J.

Respir cell molecular biology, 33: 589-600.

Pechova A. and Pavlata, 2007. Chromium as an essential nutrient: a review. Veterinarni

medicine, 52(1): 1-18.

Peter Grevatt, C., Robert Benson, Charles Hiremath, Annie Jarabek, Winona Victery, 1998.

Toxicological review of trivalent chromium. Cas No.16065-83-1.

64

Peter Grevatt, C., Robert Benson, Charles Hiremath, Annie Jarabek, Winona Victery, 1998.

Toxicological review of hexavalent chromium. Cas No.18540-29-9.

Quanren He, Jiyoung Kim and Raghubir Sharma, P., 2004. Silymarin protects against liver

damage in BALB/c mice exposed to fumonisin B1 despite accumulation of free

sphenoid basses. Toxicological Science, 80: 335-342.

Rajaprabhu D., Rejesh R., Jeyakumar R., Buddhan S., Ganesan B. and Anadan R., 2007.

Protective effect of Picrorhiza kurrooa on antioxidant defense status in adriamycin-

induced cardiomyopathy in rates. Journal of medicinal plant research, 1(4): 080-085.

Russel Flegal, Jerold Last, Earnest E., McConnell, Marc Schenker, Hanspeter Witschi, 2001.

Scientific review of toxicological and human health issue related to the development

of a public health goal for Cr (VI), Report prepared by the Chromate Toxicity Review

Committee. 2(1): 05-020.

Seokjoo Yoon, Sang Seop Han and Rana, S.V.S., 2008. Molecular markers of heavy metal

toxicity- A new paradigm for health risk assessment. Environmental Biology, 29(1):

1-14.

Shaker, E., Mahmud, H. and Mnaa, S., 2010. Silymarin, the antioxidant component and

Silybum marianum extracts prevent liver damage. Food and Chemcial Toxicology,

48: 803-806.

Silva, R. F., Lopes, R. A., Sala, M. A., Vinha, D., Regalo, S. C. H., Souza, A. M. Gergorio,

Z. M. O., 2006. Action of trivalent chromium on rate liver structure. Histometric and

hematological studies. 24(2): 17-203.

Silvio De Flora, 2000. Threshold mechanism and site specificity in chromium(VI)

carcinogenesis. Carcinogenesis, 21(4): 533-541.

Srinivasan, R. and Ramprasath, C., 2012. Beneficial role of silibinin in monitoring the

cadmium induced hepatotoxicity in Albino Wistar rats. RRST, 4(1): 46-52.

Stohs, S. J. and Bagchi D., 1995. Oxidative mechanism in toxicity in metal ions. Free redical

biology and medicine, 18(2): 321-336.

Wahsha, M., Al. Jassabi S., 2009. The role of silymarin in theprotection of mice liver damage

against microcystin-LR toxicity. JJBS, 2(2): 63-68,

Yong-Ping Pe, Jian Chen, Wei-Lin Li, 2009. Progress in research and application of

silymarin. Medical and Aromatic Plant Science and Biotechnology, 3(1): 1-8.

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