Introduction

The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Hepatotoxicity implies chemical-driven liver damage. Certain medicinal agents when taken in overdoses and sometimes even when introduced within therapeutic ranges may injure the organ. Other chemical agents such as those used in laboratories and industries, natural chemicals (e.g. microcystins) and herbal remedies can also induce hepatotoxicity. Chemicals that cause liver injury are called hepatotoxins. More than 900 drugs have been implicated in causing liver injury and it is the most common reason for a drug to be withdrawn from the market. Chemicals often cause subclinical injury to liver which manifests only as abnormal liver enzyme tests. Drug induced liver injury is responsible for 5% of all hospital admissions and 50% of all acute liver failures.1

The plant Adhatoda vasica Nees (AV) of the Acanthaceae family has been used for thousands of years in India. . Extracts of the leaves of AV are extensively used in cough, asthma, bronchitis, tuberculosis, inflammation and allergy.2-4  AV has been reported to posses, hepatoprotective activity. 5 The major bioactive chemical constituents of AV are the alkaloids like Vasicine, Vasicinone, Vasicinol, Vasicol, Vasicinolone, Vasicinine and adhatodine etc.6-7 The extract of A. vasica, also contain  high amount of flavonoid. 8  Keeping this in view, the present study was aimed at evaluating the hepatoprotective effect of Adhatoda vasica leaves Extract against Paracetamol Induced Hepatic Damage in Rats.

Materials and Methods

Plant material and extraction

The fresh leaves of AV were collected in the month of April to May from nagaon, Assam, India and duly authenticated. . The plant material was authenticated by Dr S.S.Dhawan, Professor of food and nutrition department, HAU, Hisar, Haryana.

The fresh leaves of AV were manually plucked, air dried, powdered (800 g) and aqueous extracts were prepared using sufficient water by percolation method followed by steam evaporation. A final yield of 20 g of the extract was obtained.9

Animals

Healthy adult Wistar albino rats (Rattus norvegicus) weighing 200—250 grams each were used for the study. All the animals were taken care of under ethical consideration as per the guidelines of the CPCSEA with due approval from the Institutional Animal Ethical Committee (Registration no: 634/02/a/CPCSEA; dated 19/5/2002).

Chemicals used

Crude powder of silymarin was obtained from MicroLabs India, Paracetamol was procured from Nulife Pharmaceuticals, India. Enzymes like alanine transaminase (ALT), aspartate transaminase (AST), total protein and albumin/globulin ratio were assayed using standard kits available. 

Experimental design for hepatoprotective study 10

A total of thirty animals were equally divided into five groups with six animals in each group:
Group–A: Normal Control - Received normal saline, 2 ml/kg/day                                                   

Group- B: Normal saline (2ml/kg) for 6 days + single dose of Paracetamol (2gm/kg) orally on  day 7

Group–C: Standard group treated with Silymain 25 mg/kg orally for 6 days + single dose of  Paracetamol (2gm/kg) orally on day 7                                                                                                                                                                                           Group–D: Test group treated with aqueous extract (200 mg/kg) orally for 6 days + single dose of Paracetamol (2gm/kg) orally on day 7.                                                                                                      

Group–E: Test groups treated with aqueous extract (100 mg/kg body weight) orally for 6  days + single dose of Paracetamol (2gm/kg) orally on day 7.

Induction of hepatotoxicity 10

Leaving aside six rats for Normal Control Group, 24 rats were induced hepatotoxicity by a single intraperitoneal injection of paracetamol in the dose of 2 gm/kg body weight. After 48 hours of treatment, blood was collected by intracardiac puncturing for biochemical markers.

Statistical analysis

The data was statistically analysed using One-way ANOVA.11 Values of p < 0.01 were considered significant.

Results

Paracetamol induced liver toxicity

The data was statistically analysed using One-way ANOVA. After 48 hr of administration of PCM, the serum levels of ALT, AST, were markedly increased whereas Plasma protein and Albumin/Globulin ratio were decreased when compared to normal control. Pretreatment with low dose of AV (100 mg/kg, p.o), medium dose of AV (200 mg/kg)  and silymarin significantly reduced the serum levels of ALT, AST, and increased  Plasma protein and Albumin/Globulin ratio when compared to PCM control (P<0.01)-(Table-1)

Table 1

Hepatoprotective effect of AV pretreatment on paracetamol induced in rats

Values are expressed as Mean ± SEM; n=6 rats in each group. One-way ANOVA done.  p<0.01 when compared to Normal Control Group.

Discussion

From the study, it was seen that pretreatment with low dose of AV (100 mg/kg, p.o), medium dose of AV (200 mg/kg) and silymarin significantly reduced the serum levels of ALT, AST, and increased Plasma protein and Albumin/Globulin ratio when compared to PCM control.

PCM is a commonly used as analgesic and antipyretic drug and is safe in therapeutic doses but produces fatal hepatic necrosis with toxic doses. 12 The toxic effect of PCM is due to oxidative damage induced by its metabolite, N-acetyl-p-benzoquinoneimine, produced by the action of cytochrome P-450 in the liver. This metabolite reacts with reduced glutathione (GSH) to yield non-toxic 3-GS-yl-PCM. Depletion of GSH causes the remaining quinone and other natural endogenous oxygen species to bind to cellular macromolecules leading to cell death.13  Damage induced by PCM is accompanied by an increase in the levels of serum biomarker enzymes.     

Flavonoids, tannins and microelements have been suggested to act as antioxidants and exert their antioxidant activity by scavenging the lipid peroxidation.14  The high scavenging property of Adhatoda vasica may be due to hydroxyl groups existing in the phenolic compound’s chemical structure that can provide the necessary component as a radical scavenger.15  Earlier studies conducted, revealed that vasicine content in Adhatoda vasica decreases significantly lipid peroxidation and similarly increases in antioxidants like superoxide dismutase, catalases, glutathione peroxidation and reduced glutathione.16

Conclusion

The present study reveals the hepatoprotective activity of AV against PCM induced hepatic damage in rats. The results show that AV is effective in low and medium doses (100 mg/kg, p.o and 200 mg/kg, p.o. Though the aqueous extract of Adhatoda vasica leaves showed significant hepatoprotective activity against Paracetamol induced hepatotoxicity, it is also needed further research to isolate the compound and exact mechanism responsible for hepatoprotective activity of the plant to rationalize its use as a drug to give more emphasis on this plant for the development of medicinal value.