ANTIOXIDANTPROPERTIES AND HEPATOPROTECTIVE EFFECT OF ANDROGRAPHIS PANICULATAON PCM INDUCED HEPATOTOXICITY IN ALBINO RATS. Singh Aakansha

Singh Aakansha 1 and SinghS. K 2 . 1. Department ofKayachikitsa faculty of Ayurveda IMS, BHU,Varanasi. 2. Centre of Experimental Medicine &surgery,IMS, BHU Varanasi. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Methanolic extract ofAndrographispaniculata was evaluated for hepatoprotective and antioxidant activities in albino rats. The plant extract (250 and 500 mg/kg, bw.) exhibited a remarkable hepatoprotective and antioxidant activity againstparacetamol induced hepatotoxicity as judged from the serum marker enzymes and antioxidantlevels in liver tissues. Paracetamol induced a significant rise in aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), lipid peroxidase(LPO) and bilirubinwith a reduction of protein, superoxide dismutase (SOD). Treatment of rats with different doses of plant extract(250 and 500 mg/kg) significantly (P<0.001) alteredserum marker enzymes and antioxidant levels to near normal againstparacetamol induce albino rats. The activity of the extract at dose of 250 mg/kg was comparable to more effective than dose of 500, mg/kgbw.). Histopathological changes of liver sample were compared with respective control. Results indicate the hepatoprotective and antioxidant properties of Andrographispaniculataagainstparacetamol -induced hepatotoxicity in albino rats.

Introduction:-
Herbal medicines have recently attracted much attentionas alternative medicines useful for treating or preventinglife style related disorders and relatively very littleknowledge is available about their mode of action. Therehas been a growing interest in the analysis of plantproducts which has stimulated intense research on theirpotential health benefits. Liver, the key organ ofmetabolism and excretion has an immense task ofdetoxification of xenobiotics, environmental pollutantsand chemotherapeutic agents. Hence, this organ issubjected to variety of diseases and disorders. Severalhundred plants have been examined for use in a widevariety of liver disorders. Antioxidants play an importantrole in inhibiting and scavenging free radicals and thusproviding protection against infections and degenerativediseases.Andrographispaniculatais an herbaceous plant commonly known as "King of bitter" in the family Acanthaeae.Mostly the leaves and roots have been traditionally used over the centuries for different purposes.Andrographis has demonstrated a number of different pharmacological actions in in-vitro and/or animal studies. Anticancer, immunomodulatory, anti-inflammatory, antipyretic, hepatoprotective-hypotensive, hypoglycemic, antiplatelet and antithrombotic activity have all been reported.Andrographolide is considered one of the most active and important constituents in andrographis and is most concentrated within the leaf. The present study is aimedto evaluate the hepatoprotective and antioxidant activity of methanol extract ofAndrographispaniculata againstparacetamol -induced hepatotoxicity in albino rats.
Group V:-Paracetamol with A.paniculata 500 mg/kg body weight.-Animal received A.paniculata in the dose of 500mg/kgb.w for 12 days on 11 th day paracetamol-After 48 hours paracetamol administration, we sacrifice the rat, collect the serum for LFT (, SGOT, SGPT, Alkaline Phosphatase, Bilirubin, ProtienSOD,and LPO).At the end of the experiment (48h after paracetamol administration), all the animals were sacrificed by cervical decapitation. Blood samples were collected, allowed to clot. Serum was separated by centrifuging at 2500 rpm for 15 min and analyzed for various biochemical parameters.
The liver was immediately isolated and washed with normal saline, blotted with filter paper and weighed. Liver tissue homogenate (LTH) was prepared in normal saline and used for estimation of endogenous marker enzymes and biological antioxidants superoxide dismutase (SOD) activities.

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Histopathological studies:-Liver slices fixed for 12 hrs in Bouin's solution were processed for paraffin embedding following standard micro techniques (Galigher and Kozloff, 1971). 5μm sections of liver stained with alum haematoxylin and eosin, were observed microscopically for histopathological changes.

Statistical Analysis:-
The result were expressed as mean S.D of four animal from each group .The statistical analysis of variance were carried out by one way analysis of variance( ANOVA) P values<0.05 were consider significant.

Results:-
The effect of Andrographispaniculata on serum marker enzymes arepresented in table 1. The levels of serum AST, ALT,ALP, bilirubin, were markedly elevated andthat of protein decreased in paracetamole treatedanimals, indicating liver damage. Administration of Andrographispaniculata extract at the doses of 250 and 500 mg/kgremarkably prevented paracetamole induced hepatotoxicityin a dose dependent manner. Analysis of LPO levels by thiobarbituric acid reaction showed a significant (P<0.001) increase in the paracetamole treated rats. Treatment with Andrographispaniculata (250 mg/kg and 500 mg/kg) significantly (P<0.001) prevented the increase in LPO level which was brought to near normal. Paracetamole treatment caused a significant (P<0.001)decrease in the level of SOD, when compared with control group (table 2) The treatment of Andrographispaniculata at the doses of 250 and 500 mg/kg resulted in a significant increase of SOD,when compared to paracetamole treated rats. The high dose of combined extract of Picrorhizakurroa and Andrographispaniculata (500 mg/kg, b.w) was less effective in reducing PCM induced hepatotoxicity. All theseresults indicate a hepatoprotective potential of the extract.
Morphological observations showed an increased size and enlargement of the liver in acetaminophen treated groups. These changes were reversed by treatment with Andrographispaniculataat the doses tested (fig1). Histopathological studies, showed acetaminophen to produced extensive vascular degenerative changes and centrilobular necrosis in hepatocytes. Treatment with different doses of Andrographispaniculataextract produced mild degenerative changes and absence of centrilobular necrosis when compared with control ( fig. 2). All these results indicate a hepatoprotective potential of the extract.

Discussion:-
Paracetamol (Acetaminophen) a widely used antipyretic analgesic drug produces acute hepatic damage on accidental over dosage (Hazai et al. 2002). It is established that, a fraction of acetaminophen is converted via the cytochrome P450 pathway to a highly toxic metabolite; N-acetyl-p-benzoquinamine (NAPQI) (Dahlinet al., 1984) which is normally conjugated with glutathione and excreted in urine. Overdose of acetaminophen depletes glutathione stores, leading to accumulation of NAPQI, mitochondrial dysfunction (Parmar, and Kandakar, 1995) and the development of acute hepatic necrosis. Several P450 enzymes are known to play an important role in APAP bioactivation to NAPQI. P450 2E1 have been suggested to be primary enzymes for acetaminophen bioactivation in liver microsomes (Raucyet al., 1989). Studies demonstrated that acetaminophen induced hepatotoxicity can be modulated by substances that influence P450 activity. In the assessment of liver damage by paracetamole the determination of enzyme levels such as AST, ALT is largely used. Necrosis or membrane damage releases the enzyme into circulation and hence it can be measured in the serum. High levels of AST indicates liver damage, such as that caused by viral hepatitis as well as cardiac infection and muscle injury, AST catalyses the conversion of alanine to pyruvate and glutamate and is released in a similar manner. Therefore ALT is more specific to the liver, and is thus a better parameter for detecting liver injury. Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver (Drotman and Lawhan, 1978).
Serum ALP, bilirubin and protein levels on other hand are related to the function of hepatic cell. Increase in serum level of ALP is due to increased synthesis, in presence of increasing biliary pressure (Muriel and Garcipiana, 1992).Administration of paracetamole caused a significant (P<0.001) elevation of enzyme levels such as AST,ALT, ALP, bilirubin and decrease in protein when compared to control. There was a significant (P<0.001) restoration of these enzyme levels on administration of the extract in a dose dependent manner .The reversal of increased serum enzymes in paracetamole induced liver damage by the extract may be due to the prevention of the leakage of intracellular enzymes by its membrane stabilizing activity. This is in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes (Thabrew and Joice, 1987). Effective control of ALP, bilirubin and total protein levels points towards an early improvement in the secretary mechanism of the hepatic cells. The efficacy of any hepatoprotective drug is dependent on its capacity of either reducing the harmful effect or restoring the normal hepatic physiology that has been distributed by a hepatotoxin. The plant extract decreased paracetamole induced elevated enzyme levels in tested groups, indicating the protection of structural integrity of hepatocytic cell membrane or regeneration of damaged liver cells. The increase in LPO level in liver induced by paracetamole suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanism to prevent formation of excessive free radicals. Treatment with Andrographispaniculata significantly reverses these changes. Hence it is likely that the mechanism of hepatoprotection of Andrographispaniculata is due to its antioxidant effect.Decrease in Normal Alcoholic Paracetamol A.paniculata 250mg A.paniculata 500mg liver weight 1419 enzyme activity of superoxide dismutase (SOD) is a sensitive index in hepatocellular damage and is the most sensitive enzymatic index in live injury .SOD has been reported as one of the most important enzymes in the enzymatic antioxidant defense system. It scavenges the superoxide anion to form hydrogen peroxide and thus diminishing the toxic effect caused by this radical. In Andrographispaniculata causes a significant increase in hepatic SOD activity and thus reduces reactive free radical induced oxidative damage to liver,number of deleterious effects due to the assimilation of superoxide radical and hydrogen peroxide.
Extensive vascular degenerative changes and centrilobular necrosis in hepatocytes was produced by paracetamole. Treatment with different doses of water extract of Andrographispaniculata produced only mild degenerative changes and absence of centrilobular necrosis, indicating its hepatoprotective efficiency. Free radical mediated process has been implicated in pathogenesis of most of the diseases. The protective effect of Andrographispaniculata onparacetamole induced hepatotoxicity in rats appears to be related to inhibition of lipid peroxidation and enhancement of antioxidant enzyme levels in addition to free radicals scavenging action.

Conclusion:-
Based on results of our study, it can be concluded that paracetamol might disturb the balance between reactive oxygen species production and antioxidant protection in liver in paracetamolhepatotoxicity.The low dose of Andrographispaniculata (250 mg/kg bw.) possess good hepatoprotective activity against PCM induce liver damage, whereas high dose (500 mg/kg, bw.)of Andrographispaniculata failed to produce similar effect. The protective effect of the extract may be due to its antioxidant activity