INTRODUCTION:

Liver is one of the largest organs in human body and the chief site for intense metabolism and excretion. So it has a surprising role in the maintenance, performance and regulating homeostasis of the body. Hepatotoxicity implies chemical-driven liver damage. The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Chemicals that cause liver injury are called hepatotoxins. More than 900 drugs have been implicated in causing liver injury.

Liver diseases are some of the fatal diseases in the world today. They pose a serious challenge to international public health. Modern medicines have little to offer for alleviation of hepatic diseases and it is chiefly the plant based preparations which are employed for the treatment of liver disorders. But availability of drug for the treatment of liver disorders is very less. Acetaminophen (Paracetamol, also known by the brand name Tylenol and Panadol) is usually well tolerated in prescribed dose but overdose is the most common cause of drug induced liver disease and acute liver failure worldwide.¹

The metabolism of alcohol (ethanol) to acetaldehyde proceeds in the hepatocyte via the alcohol dehydrogenase (ADH) as well as the microsomal ethanol oxidizing system (MEOS). Analogous to other hepatic microsomal drug metabolizing enzymes, MEOS consists of cytochrome P-450, NADPH-cytochrome 2 reductase and phospholipids. Chronic alcohol consumption increases the content of microsomal P-450 as well as phospholipids and enhances the activity of NADPH-cytochrome P-450 reductase, resulting in an induction of activities for MEOS and other microsomal drug metabolizing enzymes. The induction of MEOS activity is associated with an increased production of acetaldehyde which in turn may lead to an increased hepatotoxicity.

Silymarin is a flavonolignan that has been introduced fairly recently as a hepatoprotective agent. Many studies have demonstrated the beneficial hepatoprotective effects when treatment with Silymarin. Herbal drugs play a role in the management of various liver disorders most of which speed up the natural healing processes of the liver.

Tinospora cordifolia commonly known as ‘Guduchi’ is one of the most valuable medicinal herbs of ayurveda. The present study was undertaken to evaluate the hepato protective effect of Tinospora cordifolia.

MATERIALS AND METHODS:

Procurement of animals:

Male Wister albino rats (100 - 140g) used were procured from Sri Venkateshwara Enterprises, Bangalore and maintained under standard conditions, fed with standard diet and water ad libitum.

Procurement of diagnostic kits:

Diagnostic kits used for the estimation of TBARS, SGOT, SGPT, Catalase, Vitamin, and Protein were obtained from Agappe diagnostics, Maharashtra, India.

Procurement of Hepato toxic inducer and induction:

Ethyl alcohol and acetaminophen were used as the hepatotoxic inducer in rats and were procured from the Laboratories private limited and SISCOM, Tiruchirappalli, Tamilnadu, India (30ml/kg and 750 mg/ kg b.w. orally)².

Preparation of Ethanolic extract of Tinospora cordifolia:

Tinospora cordifolia plant materials were collected from Mannargudi, Tamilnadu, India. The leaves were air dried for 72 hours, powdered and used for extraction with 100ml of 99.9% of ethanol. The ethanolic mixture was evaporated at 55ºc and used for further studies. The extract was administered in different dose (10, 30 and 50mg/kg b.w.p.o)³ .

Procurement of hepato protective drug:

Silymarin was used to treat hepatotoxicity and was procured from Micro Labs Limited, India (25mg/kg b.w., orally).

Experimental Design:

Group1: Animals served as control and fed orally with normal saline 5ml/kg body weight daily for 7 days

Group2: Animals were treated with paracetamol (750mg/kg b.w) and (alcohol 30ml/kg/day) to induce toxicity for 7 days

Group3: Animals were induced with paracetamol, alcohol and treated with standard drug silymarin 25mg/kg for 7days as co-treatment for hepatoprotection.

Group4: Animals were induced with paracetamol, alcohol and treated with Tinospora cordifolia (10mg/kg b.w) as co-treatment for 7days

Group5: Animals were induced with paracetamol, alcohol and treated with Ttinospora cordifolia (20mg/kg b.w) as co-treatment for 7days

Group6: Animals were induced with paracetamol, alcohol and treated with Ttinospora cordifolia (30mg/kg b.w) as co-treatment for 7days

Study protocol:

The standard and test formulations were administered for 7 days using oral gavages once in a day. At the end of experiment, rats were sacrificed by cervical decapitation. Blood was collected to separate the serum and plasma. The liver tissue was dissected out, weighed and washed using ice cold saline solution. Tissues were homogenized with buffer solution, centrifuged and the resulting supernatant was used for various biochemicals and anti oxidant assay.

Biochemical analysis

Biochemical parameters such as ALT, AST, Urea, Creatinine, Bilirubin, TBARS, SOD, CAT, GGT, Vit-C and Vit-E were evaluated using standard procedures.

Statistical analysis

The data obtained in present investigation was subjected to statistical analysis. All results are expressed as Mean ± S.D. Student ‘t’ test was performed using SPSS soft ware.

Results and Discussion

A large number of studies are in progress aiming to identify substances that would be effective in reducing the severity of alcoholic and drug induced liver diseases. Paracetamol induced hepatic failure is the second leading cause of liver transplantation ⁴

Hepatic injury is directly associated with altered metabolic functions ⁵. In past, several studies have been carried out to examine the effect of plants used traditionally by herbalists to support normal liver function and treat diseases of liver. In the present study Tinospora cordifolia has been chosen to identify the efficacy of this plant as hepato protective agent.

Silymarin, is a herbal drug which is mostly used in liver diseases and has been used as standard drug for hepato cellular injury. It has a regulatory action on cellular and mitochondrial membrane permeability in association with an increase in membrane stability against xenobiotic injury ⁶ .In the present investigation, paracetamol and alcohol were used for dual intoxication, silymarin was used as standard drug and Tinosporia cordifolia as herbal drug. The efficacy of herbal drug Tinosporia cordifolia at different doses was compared with standard drug silymarin. Administration of paracetamol and alcohol resulted in elevated activities of AST, ALT, ALP and LDH (Table I) in serum in Group II against their respective control (Table 1). Similarly, serum bilirubin level was also found to be increased significantly as a result of paracetamol and alcohol induced toxicity (Table 2). On the other hand, total serum protein level was lowered in response to paracetamol and alcohol administration when compared with control (Table 2).

Abnormally higher activities of serum AST, ALT, ALP and LDH after paracetamol and alcohol administration as observed in the present study is an indication of the development of hepatic injury, which is responsible for leakage of cellular enzymes into the blood. When liver plasma membrane gets damaged, a variety of enzymes normally located in the cytosol are released into the circulation ⁵. The rise in the AST is usually accompanied by an elevation in the levels of ALT, which play a vital role in the conversion of amino acids to keto acids⁷ . Increase in serum level of ALP is due to increased synthesis in presence of increasing billiary pressure. Besides these marker enzymes, there was also significant increase in LDH activity. Being a cytosolic enzyme increase in LDH might be due to tissue injury and leakage of cytosolic enzymes into plasma.

Table 1 : Effect of Tinospora cordifolia on liver marker enzymes

Groups	ALT U/L	AST U/L	ALP U/L	LDH U/L
Group 1	74.5±2.645	81±1.92	127±2.6	314±13.69
Group 2	202±3.65^*	185.6±3.0^*	251.75±9.0^*	710±25.81^*
Group 3	100.8±4.86^**	94±2.9^**	149.25±3.5^**	447±25.1^**
Group 4	104.8±7.42^ns	121±5.3^**	184±4.32^**	510±29.43^ns
Group 5	95±4.2^ns	114.8±2.5^ns	172.5±8.58^ns	415±12.9^**
Group 6	87.5±2.1^ns	88±5.51^**	136.25±4.924^**	356±16.1^ns

Values are mean±S.E ( n = 6 ), * statistically significant when compared with group 1 , ** statistically significant when compared with group 2, ns - not significant

Table 2 : Effect of Tinospora cordifolia on biochemical parameters

Groups	Bilirubin mg/dL	Protein g/dL
Group 1	0.58±0.17	6.3±0.182
Group 2	1.25±0.13 *	2.95±0.24*
Group 3	0.8±0.08 **	5.8±0.22 **
Group 4	0.78±0.09 ^ns	5.0±0.26 *
Group 5	0.75±0.12 ^ns	5.2±0.43 ^ns
Group 6	0.75±0.13 ^ns	6.02±0.25 **

Values are mean±S.E ( n = 6 ), * statistically significant when compared with group 1 , ** statistically significant when compared with group 2, ns - not significant

Table 3: Effect of Tinospora cordifolia on Lipid peroxidation and Enzymatic anti oxidants

Groups	TBARS Mm/mg	SOD U/L	CAT U/L	GGT U/L
Group 1	0.45±0.129	65.5±4.43	85±2.58	87.2±1.7
Group 2	1.275±0.1*	25.25±2.21*	45±2.16*	21±2.58*
Group 3	0.8±0.08**	56.5±3.41**	66.5±1.29**	67±1.71**
Group 4	0.9±0.08^ns	49±2.58^ns	57±1.83**	53±1.825**
Group 5	0.66±0.053^ns	58±2.82^ns	67.5±2.38**	63±2.58**
Group 6	0.59±0.06^ns	62±1.63^ns	72±2.16**	73.5±2.64**

Values are mean±S.E ( n = 6 ), * statistically significant when compared with group 1 , ** statistically significant when compared with group 2, ns - not significant

Table 4: Effect of Tinospora cordifolia on non Enzymatic anti oxidants

Groups	VIT-C mg/dL	VIT-E mg/dL
Group 1	63 ±2.58	169±3.9
Group 2	34.25±4.34*	74±4.32*
Group 3	55.25±3.86**	143.5±1.914**
Group 4	42.75±2.21**	127±2.16**
Group 5	53.75±2.63^ns	137.5±2.1^ns
Group 6	57.75±3.86**	153±2.58^ns

Values are mean±S.E ( n = 6 ), * statistically significant when compared with group 1 , ** statistically significant when compared with group 2, ns - not significant

Oral administration of silymarin, the standard drug and various doses of Tinospora cordifolia treatment (Group III, IV, V and VI) to paracetamol and alcohol intoxicated rats resulted in gradual normalization of the activities of AST, ALT, ALP and LDH. Serum bilirubin is considered as an index for the assessment of hepatic function and any abnormal increase indicates hepatobiliary disease and severe disturbance of hepato cellular architecture .Treatment with Tinospora cordifolia ethanolic extract and standard drug silymarin significantly decreased the elevated level of total bilirubin in serum towards normalcy indicating its hepatoprotective efficacy. Hepatotoxin impairs the capacity of liver to synthesize albumin ⁸ . Subsequent treatment of paracetamol and alcohol intoxicated rats with standard drug and ethanolic extract of Tinospora cordifolia increased the total serum protein level.

Hepatic lipid peroxidation, expressed as TBARS (thiobarbituric acid reacting substances), increased significantly in paracetamol toxicity. While, the activities of protective enzymes such as Superoxide dismutase (SOD) and catalase (CAT) and GGT content in liver tissue were lowered after paracetamol and alcohol administration (Table 3). Enhanced TBARS and reduced activities of SOD and CAT is an indication of generation of free radical stress as a mark of hepatic damage due to paracetamol and alcohol toxicity. Marked reductions in the activities of these free radical scavenging enzymes, SOD and CAT, associated with paracetamol and alcohol toxicity were significantly reversed to normal on oral treatment with standard drug silymarin and ethanolic extract of Tinospora cordifolia in a dose dependent manner conferring the antilipid peroxidative ability of the extract.

The non-enzymatic antioxidant defense system protects from the deleterious effects of reactive oxygen metabolites. Vitamin E is one of the major chain breaking lipophilic antioxidants. It inhibits ROS-induced generation of lipid peroxyl radicals thereby protecting cells from lipid peroxidation. Decrease in vitamin E in paracetamol and alcohol induced toxicity indicates increased oxidative stress. Treatment with Standard drug silymarin and Tinospora cordifolia significantly increased the level of vitamin E indicating its ameliorative function (Table 4). Vitamin C is a water soluble antioxidant present in the liver tissue. It is an essential cofactor for the enzymes prolyl hydroxylase and lysyl hydroxylase, which catalyze the hydroxylation of proline and lysine residues, during collagen biosynthesis Vitamin C is a potent scavenger of reactive oxygen species in plasma and extracellular compartments of the kidney. It scavenges and destroys free radicals in combination with vitamin E and glutathione. The drastic decrease of vitamin C in paracetamol and alcohol induced toxicity indicates increased oxidative stress, free radical formation and simultaneous damage of the plasma membrane (Table 4). Treatment with Standard drug silymarin and Tinospora cordifolia significantly reduced the level of vitamin C indicating its ameliorative function.

It was concluded that Tinospora cordifolia indeed has a high potential in healing liver parenchyma and regeneration of liver cells. Thus it may act even in humans as potent liver tonic, thus has equivalent therapeutic value with the standard drug Silymarin.

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Received on 21.12.2011 Modified on 11.01.2012

Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 281-284