1. INTRODUCTION:

Liver is the vital organ of metabolism and excretion. Liver diseases are the most serious ailment and are mainly caused by toxic chemicals (Excess consumption of alcohol, high doses of paracetamol, carbon tetrachloride, chemotherapeutic agents, peroxidised oil, etc). Liver diseases such as jaundice, cirrhosis and fatty liver are very common in worldwide.

About 20,000 deaths found every year due to liver disorders. Liver diseases such as jaundice, cirrhosis and fatty liver are very common in worldwide (Gupta and Misra et al.,2006). In spite of tremendous strides in modern medicine, there are hardly any drugs that stimulate liver function, offer protection to the liver from damage or help Herbal drugs play a role in the management of various liver disorders most of which speed up the natural healing processes of the liver (Handa SS et al.,1989; Hikino H, et al., 1988; Evans WC et al.,1996; Sharma A, et al., 1991). Numerous medicinal plants and their formulations are used for liver disorders in ethnomedical practice as well as traditional system of medicine in India. Some of these plants have already been reported to posses strong antioxidant activity.

Liver damage is associated with cellular necrosis, increase in tissue lipid peroxidation and depletion in the tissue GSH levels.In addition serum levels of many biochemical markers like SGOT, SGPT,triglycerides, cholesterol,bilirubin,alkaline phosphatase are elevated (Mascolo N et al 1998).

Butea monosperma (Fabaceae) better known as Palash by the locals is rich in flavanoids. Most flavanoids are bioactive compounds due to the presence of phenolic group in their molecule. Among the various medicinal properties attributed, the flower is of greater therapeutic value in the treatments of liver disorders and indigestion (Shrivastava et al., 2003 and Gunakkunru et al., 2005)

2. MATERIAL AND METHOD:

2.1. Drugs and Chemicals Paracetamol (Karnataka Antibiotic and Pharmaceuticals Ltd, Bangalore) and Silymarine (Micro labs, Bangalore.). All other chemicals were obtained from local sources and were of analytical grade.

2.2. Plant Materials The barks of Butea monosperma were procured from campus of Guru Ghasidas Vishwavidyalaya, Bilaspur (C.G). The Plant material was authenticated by Dr. H.B. Singh, Head, Raw Material Herbarium & Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. Reference No NISCAIR/RHMD/consult/-2010-11/1482/80 dated 11 August 2010 and was deposited in the department of pharmacology, Slt. Inst. of pharm. Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur (C.G) for future reference. After authentication, seeds were cleaned and the material was dried in shade and milled into coarse powder by a mechanical grinder. 1kg powdered barks were extracted with ethanol in a Soxhlet extractor for 72 hrs. Extract was evaporated under low pressure by using Buchi type evaporator.

2.3. Animals Adult male wistar strain albino rats weighing 180-220g purchased from Ghose Biological, Cuttack were maintained in the animal house of School of Pharmaceutical Sciences, S.O.A University, Bhubaneswar for experimental purpose. Then all the animals were acclimatized for seven days under standard husbandry conditions, i.e. room temperature of 25±1 ⁰C; relative humidity 45-55% and a 12:12 hrs. light/ dark cycle. The animals had free access to standard rat pellet (Pranav Agro Industries Ltd, Baroda, India), with water supplied ad libitum under strict hygienic conditions. The institutional animal ethical committee (Reg.no OCP/2010/2271/ac/10/ CPCSEA/1349) permitted the study.

2.4. Experimental Design and pharmacological screening Five groups of six animals were used for the study. Group-I (Control) received single daily dose of distilled water (5 ml/Kg, p.o.) for 7 days. Group -2 (Toxic control) were treated with vehicle (0.5% of Tween 40, 1 ml/kg, p.o.) for 7 days. Group -3( Standard) received silymarin (25 mg/kg, p.o.) for 7 days. Group 4, 5 pretreated with ethanolic extract of Butea monosperma bark (EEBM) 250 mg and 500 mg/kg body weight respectively for 7 days orally. Food was withdrawn 16 hrs before paracetamol administration to enhance the acute liver toxicity. Group 2, 3, 4 and 5 were treated with paracetamol (750mg/kg i.p) diluted with propylene glycol (12.5% solution) was administered on 7^th day after 1 hrs of extracts treatment and sacrificed 18 hours after administration of paracetamol.

After 18 hrs of paracetamol administration all the animals were anesthetized using anesthetic ether, and blood sample were collected by cardiac puncture method and serum was used for estimation of SGOT and SGPT ( Reitman S, et al1957), ALP ( Bessey OA et al 1964 ), Albumin and Total protein (Gornall et al., 1949) and total bilirubin (Malloy et al., 1937). Immediately after the collection of blood, the animals were euthanized with an over dose of ether; their livers removed. The liver was washed by normal saline were preserved in 10% formalin for histopathological studies.

2.5 Statistical Analysis The experimental results were expressed as the Mean ± SEM for six animals in each group. The biochemical parameters were analysed statistically using one-way analysis of variance ANOVA, followed by Dunnett’s multiple comparison test (DMCT). P value of < 0.05 was considered as statistically significant.

3. RESULTS

Preliminary phytochemical studies revealed the presence of phenolic compound and flavonoids were noticed in ethanolic bark extract. The effects of EEBM on serum transaminase, alkaline phosphatase, total protein, albumin and bilirubin levels in paracetamol-induced liver damage in rats are summarized in Table-1 and 2. Administration of paracetamol (750mg/kg; body weight), after 18 hours of intoxication resulted a significant (P<0.05) elevation of hepatospecific serum markers SGOT, SGPT, SALP, Total bilirubin and significant (P<0.05) decrease of total protein and albumin in paracetamol-treated group, in comparison with the normal control group. On administration of EEBM (Group IV and V,Table-1 and 2) and Silymarin at the dose of 25mg/kg (Group III, Table-1and 2) the level of these enzymes and biochemicals were found retrieving towards normalcy.

Table 1: Effect of Butea monosperma bark extracts on serum enzyme in paracetamol induced hepatic damage in rats.

Group	Treatment	Dose	SGOT (U/L)	SGPT (U/L)	ALP (U/L)
I	Solvent control	5ml/kg	74.69±2.58	68.37±2.39	98.43±1.78
II	PCM	---	187.47±1.53	154.16±2.18	154.08±2.03
III	PCM+Silymarin	25mg/kg	84.61±1.36***	76.38±1.09***	82.74±2.13***
IV	PCM+Ethanol ext	250mg/kg	114.92±2.57***	107.38±2.29***	124.57±1.48***
V	PCM+Ethanol ext	500mg/kg	97.78±2.09***	92.23±3.49***	89.33±2.28***

Values expressed as mean±SEM, from six observations, **p<0.01, ***p<0.001, when compared with PCM control.

Table 2: Effect of Butea monosperma bark extracts on serum biochemical parameters in paracetamol induced hepatic damage in rats.

Group	Treatment	Dose	Total protein (mg/dl)	Albumin (mg/dl)	Total bilirubin (mg/dl)
I	Solvent control	5ml/kg	9.64±1.31	5.27±0.13	1.14±0.04
II	PCM	-----	5.39±0.92	4.16±0.08	2.69±0.37
III	PCM+Silymarin	25mg/kg	8.86±0.56**	5.12±0.06***	1.26±0.09**
IV	PCM+Ethanol ext.	250mg/kg	8.49±1.01**	4.24±0.31	1.61±0.24*
V	PCM+Ethanol ext	500mg/kg	9.28±1.04***	4.98±0.36*	1.26±0.43*

Values expressed as mean±SEM, from six observations, *p<0.05, **p<0.01, ***p<0.001, when compared with PCM control.

Histopathological studies of rat liver tissue from Group I animals show normal hepatic cells with central vein (V) and sinusoidal dilation (Fig. 1). In paracetamol treated group (Group II), severe hepatotoxicity was observed by severe necrosis (N) with disappearance of nuclei (Fig. 2). The liver taken from Group III animals treated with standard drug Silymarin showed the normal hepatic cells with portal vein (V) and portal artery (Fig. 3). Mild degree of necrosis (N) with areas of inflammation adjacent to necrosised area was observed in Group V animals, treated EEBM (250mg/kg/day) (Fig. 4) while normal hepatocytes with regenerating hepatocytes and mild inflammation in the portal area (M) was observed with Group V animals at the dose of 500mg/kg/day of EEBM (Fig.5).

At a dose of 250 mg/kg, the effect was only marginal whereas at higher dose (500mg/kg) the drug effectively prevented the paracetamol induced liver damage.

Fig. 1. Histopathology of solvent control group(Group-I)

Fig. 2. Histopathology of Toxic Control(Group-II)

Fig. 3. Histopathology of Standrad drug (Group-III)

Fig. 4. Histopathology of Test drug EEBM (Group-IV)

Fig. 5. Histopathology of Test drug EEBM (Group-V)

4. DISCUSSION:

Paracetamol (N-acetyl-p-aminophenol, acetaminophen), a widely used analgesic and antipyretic drug is known to cause produces hepatic necrosis in high doses in experimental animals and humans (Prescott et al., 1971; Mitchell, 1988; Kuma and Rex, 1991; Eriksson et al., 1992; Thompsen et al., 1995). It is established that following an oral therapeutic dose, a fraction of paracetamol is converted via the cytochrome P-450 pathway to a highly toxic metabolite, N-acetyl-pbenzoquinone- imine (NAPQI) (Dahlin et al., 1984), which is normally conjugated with glutathione and excreted in the urine as conjugates. Overdoses of paracetamol deplete glutathione stores, leading to accumulation of NAPQI, mitochondrial dysfunction (Parmar et al., 1995), and the development of acute hepatic necrosis. Also depletion of glutathione enhances the expression of tumor necrosis factor alpha (TNFa) (Agarwal and Piersco, 1994). TNFQ primes phagocytic NADPH oxidase to the enhanced production of oxygen free radicals and contributes to liver damage (Gupta et al., 1992). Paracetamol-induced hepatotoxicity in rodents is a widely used animal model to assess hepatoroptective activity of new compounds (McLean, 1975 and Handa et al., 1986).

The study of different enzyme activities such as SGOT, SGPT, SALP, Albumin, Total Protein and Total Bilirubin have been found to be of great value in the assessment of clinical and experimental liver damage. In the present investigation it was observed that the animals treated with paracetamol resulted in significant hepatic damage as shown by the elevated levels of serum markers. These changes in the marker levels will reflect in hepatic structural integrity. The rise in the SGOT is usually accompanied by an elevation in the levels of SGPT, which play a vital role in the conversion of amino acids to keto acids24. The pre-treatment with EEBM both at the dose of 250mg/kg and 500mg/kg, significantly attenuated the elevated levels of the serum markers. The normalization of serum markers by EEBM suggests that they are able to condition the hepatocytes so as to protect the membrane integrity against paracetamol induced leakage of marker enzymes into the circulation. The above changes can be considered as an expression of the functional improvement of hepatocytes, which may be caused by an accelerated regeneration of parenchyma cells. Serum ALP and bilirubin levels, on the other hand are related to hepatic cell damage. Increase in serum level of ALP is due to increased synthesis in presence of increasing billiary pressure. Effective control of bilirubin level and alkaline phosphatase activity points towards an early improvement in the secretory mechanism of the hepatic cell.

The histopathological observations in paracetamol-treated rats showed severe necrosis, with disappearnce of nuclei. This could be due to the formation of highly reactive radicals because of oxidative threat caused by paracetamol. All these changes were very much reduced histopathologically in rats treated with EEBM. Based on the above results and phytochemical analyses of ethanolic extract of Butea monosperma barks contain phenolic compound and flavonoids. Therefore there is a possibility that the Butea monosperma bark extract may possess hepatoprotective activity. It could be concluded that EEBM exerts significant hepatoprotection against paracetamol-induced toxicity.

5 ACKNOWLEDGEMENT:

The authors are grateful to Dr. Durga Madhav. Kar , Head of the department of pharmacology, School of Pharmaceutical sciences, SOA University, kalinga Nagar, Bhubaneswer, orissa for providing all the necessary facilities to carry out this work..

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Received on 10.02.2011 Modified on 23.02.2011

Research J. Pharm. and Tech. 4(5): May 2011; Page 771-774