Evaluation of Hepatoprotective Activity of Blumea mollis D. Don Merr. on Paracetamol-Induced Hepatotoxicity in Rats.

Brindha Devi G.B.¹* and Revathi K.²

¹Assistant Professor, Department of Zoology, Government College for Women (Autonomous) Kumbakonam, Tamil Nadu, India.

²Reader, Department of Advanced Zoology and Biotechnology, Ethiraj College, Chennai, Tamil Nadu, India

ABSTRACT:

The therapeutic values of numerous plants and their herbal formulations were tested against a few chemical induced subclinical levels of liver damages in rodents and experiments have clearly shown that plants such as Picorrhiza kurroa, Andrographis paniculata, Eclipta alba, Phyllanthus maderaspatensis and Trichopus zeylanicus are sufficiently active against certain hepatotoxins. Screening plants for antihepatitis activities remains in its infancy. The Methanol extract of Blumea mollis were studied for their hepatoprotective effects on paracetamol induced acute liver damage on Wistar albino rats. The degree of protection was measured by using biochemical parameters such as serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP and bilirubin Further, the effects of the extract on hepatic Glycogen (mg/100g tissue) GSH (nmlol/mg protein GST (u/g tissue) ,GPX (u/mg protein) GSH-R (mmol NADPH min^-1/g tissue) were estimated. The Blumea mollis extracts produced significant (P<0.05) hepatoprotection by decreasing the activity of serum enzymes and bilirubin while it significantly increased the levels of Hepatic glycogen reduced glutathione (GSH) Glutathione-S-transferase (GST) glutathione peroxidase (GPX) and GSH-R in a dose dependent manner.

KEYWORDS: Blumea: hepatoxicity: serum marker enzymes: liver diseases: herbal medicines

INTRODUCTION:

Liver has a pivotal role in regulation of physiological processes such as metabolism, secretion and storage. Furthermore, detoxification of a variety of drugs and xenobiotics occurs in liver. The bile secreted by the liver has, among other things, an important role in digestion. Liver diseases are among the most serious ailments. They may be classified as acute or chronic hepatitis (inflammatory liver diseases), hepatosis (non-inflammatory diseases) and cirrhosis (degenerative disorder resulting in fibrosis of the liver). Liver diseases are mainly caused by infections, autoimmune disorder, excessive consumption of alcohol and toxic chemicals (certain antibiotics, chemotherapeutics, peroxidised oil, aflatoxin, carbon-tetrachloride, chlorinated hydrocarbons, etc.). Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and other oxidative damages in liver (Recknagel et al., 1983; Wendel et al., 1987; Hiroshi et al., 1987; Dianzani et al., 1991). Enhanced lipid peroxidation produced during the liver microsomal metabolism of ethanol may result in hepatitis and cirrhosis (Smuckler, 1975).

Nearly 150 phyto-constituents from 101 plants have been claimed to possess liver protecting activity (Doreswamy and Sharma, 1995; Hand et al., 1989). Most of the studies on hepatoprotective plants were carried out using chemical-induced liver damage in rodents as models. In India, more than 87 medicinal plants are used in different combinations (Hikino and Kiso, 1989; Evans, 1996; Sharma et al., 1991). In most of these studies, marginal or moderate levels of hepatoprotective activities were observed.

Besides, most of the reported studies described the beneficial effects of drugs against a few hepatotoxic chemical-induced sub clinical level of hepatotoxicity.

Phyllanthus amarus also appears to be very effective against Hepatitis B (Karunakar et al., 1997). From the available data, few plants that are promising as hepatoprotective agents include P. kurroa (Picroliv), A. paniculata (Andrographolide), Silibum marianum (Silymarin) (Wang et al., 1996; Churang et al., 1997). Studies carried out in China and Japan resulted in the isolation of a hepatoprotective lignan, gomishin from the fruits of Chinese medicinal plant Schizandra chinensis. Gomishin is used for the treatment of chronic hepatitis (Wang et al., 1996; Churang et al., 1997). Studies carried out at Tropical Botanic Garden and Research Institute (TBGRI), Thiruvananthapuram have shown that Trichopus zeylanicus, Phyllanthus maderaspatensis, and P. kozhikodianus are extremely active against paracetamol-induced liver damage in rat (Asha and Pushpagandan, 1998; Subramaniam et al., 1998; Asha, 1996). Fumaric acid obtained from Sida cordifolia has significant anti-hepatotoxic activity in rats (Kumar and Mishra, 1997). Similarly ursolic acid which occurs in many plants also showed promising hepatoprotection against paracetamol and CCL₄ induced liver damage in rats (Shukla et al., 1992; Saraswat et al., 1996).

The hepatoprotective effects of a crude aqueous extract of Wedelia chinensis were investigated against acute hepatitis induced by 3 hepatotoxins: Carbon tetrachloride in mice and D (t) galactosamine in rats. After treatment with Wedelia chinensis (300 mg) at 2, and 10 hours after hepatotoxin administration, a reduction in the elevation of serum glutamate oxaloacetic transaminase (SGOT), aspartate amino transferase (AST), glutamate pyruvic transaminase (SGPT), and alanine transaminase (ALT) levels were observed at 24 hr. The serological observations were confirmed by histological examinations (Lin et al., 1994). A combination of different herbal fractions is likely to provide desired activities to cure severe liver diseases. Development of such medicines with standards of safety and efficacy can revitalize treatment of liver disorders with hepatoprotective activity.

MATERIAL AND METHODS:

Collection of Plant Material:

The whole plant was obtained from the fields near mudichur road, tambaram, Chennai. The taxonomical identification of the plant was authenticated in the Plant Anatomy and Resrarch center (PARC), Mudichur, Tambaram.

Preparation of Extract:

The whole plant parts of Blumea mollis were rinsed with distilled water and dried under shade. The dried plants were ground into powder with an electric blender. One hundred gram of the dried sample was macerated in 600ml of 80% ethanol, agitated for 10 minutes with an electric blender and left overnight in a refrigerator at 4°C. The mixture was filtered with a cheese cloth and the filtrate obtained concentrated under reduced pressure using a rotary evaporator (at 37°C) to about 10% of its original volume. The concentrate was then allowed in a water bath at 37OC for complete evaporation to dryness yielding 38.94g (9.7%) of the extract.

Animals:

Animals - Albino rats of Wistar strain weighing about 150-200 g were obtained from Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences Univeristy, Chennai and kept under standard laboratory conditions at 12:12 hrs L:D cycles at 25°-28°C and 60-80% RH. Animals were reared with robust health by providing pellet diet (Lipton, India) and water ad libitum.

2.2 Experimental protocol:

The rats were divided into 3 groups of 6 rats each. The animals in group 1 served as control and given distilled water, po, for 10 days in succession. The group 2 rats served as test and were administered distilled water similarly followed by oral administration of paracetamol @ 3g/kg body weight, 1 hr after distilled water administration. The animals in group 3 served as experimental and treated orally with Methanol extract of Blumea mollis (10mg/kg body weight) once in a day for 10 days in succession followed by a single oral administration of paracetamol (3g/kg body weight), 1 hr after Blumea mollis administration.

2.3 Assessment of liver function:

After 24hr of paracetamol administration rats of all groups were sacrificed by cervical dislocation, blood was collected from the carotid arteries in the neck blood vessels, and centrifuged at 2000 rpm for 10 min to separate the serum, which was kept at 4°C to assay the activities of serum enzymes. Serum glutamate oxaloacetate transaminase (SGOT) serum glutamate pyruvate transaminase (SGPT) and alanine transaminase (ALT) (King 1965b) aspartate transaminase (AST) (King 1965b), were estimated.

After the collection of blood, the liver was immediately excised, washed with cold saline, blotted and weighed. A piece of 1g of liver from each rat was taken and homogenized to make liver homogenate; this was then subjected to biochemical analysis. Hepatic glycogen reduced glutathione (GSH) Glutathione-S-transferase (GST) glutathione peroxidase (GPX) and GSH-R were determined.

2.4 Statistical analysis:

Results of the biochemical estimations are reported as mean ± SD. Total variations, present in a set of data were estimated by one - way analysis of variance (ANOVA), Student’s t-test was used for determining significance. The percent age of the protection is calculated as 100 x (values of paracetamol control - values of sample)/(values of paracetamol control—values of normal control).

RESULTS AND DISCUSSION:

Rats treated with a single dose of paracetamol alone developed significant hepatocellular damage as evidenced from a significant (P<0.05) increase in the serum SGOT, SGPT, ALP, AST, ACP and LDH when compared with control. When liver is damaged liver enzymes such as glutamate pyruvate transaminase (GPT), glutamate oxaloacetate transaminase (GOT) and alkaline phosphatase enter into the circulation. An increase in the levels of these marker enzymes in the serum is an indication of liver damage (Subramonium et al., 1998). Other effects of induced liver damage such as reduction of prothrombin synthesis giving an extended prothrombin time and reduction in clearance of certain substances such as bromsulphthalein can be used in the evaluation of hepatoprotective effect of plant extracts. The hepatoprotective effect of a drug against different hepatotoxins differs especially when the mechanisms of action of the toxins are different (Chungo et al., 1997).

Pretreatment of rats with BM extract reduced the elevated serum levels of these hepatospecific enzymes, in a dose responsive manner. Treatment with paracetamol caused a reduction in hepatic glycogen and GSH levels. Pretreatment of rats with BM (100 mg/kg body weight) exhibited a high degree of protection by reversing the altered levels of glycogen and GSH. The activities of GST, GPX, and GSH-R showed significant reduction in liver of paracetamol treated rats as compared to the control group. Pretreatment of rats with BM significantly increased the enzyme activities.

Paracetamol (N-acetyl p-amino phenol, acetamino phen) a widely used analgesic and antipyretic drug is known to cause hepatotoxicity in experimental animals and humans at high doses It is mainly metabolized in the liver to excretable glucuronide and sulphate conjugates. However, hepatotoxicity of paracetamol has been attributed to formation of toxic metabolites when a part of paracetamol is activated by hepatic Cyt-p 450 (Savides MC & Ochma FW (1983) to a highly reactive metabolite N-acetyl-p-benzoquinoneimine which is normally conjugated with GSH and excreted in the urine as conjugates. Overdose of paracetamol leads to mitochondrial dysfunction followed by acute hepatic necrosis. Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver. Damage to liver cells cause leakage of cellular enzyme into serum. A significant rise in SGOT, SGPT could be taken as an index of liver damage. The reversal of increased serum transaminases. Returns to normal by Blumea mollis supplementation with healing of hepatic parenchyma and regeneration of hepatocytes.

Table –I-Effect of Blumea mollis extract on serum and liver biochemical parameters in paracetamol induced hepatic damage in rats (Values are mean ± SD from 6 animals in each group).

	*Biochemical parameters* *Serum*
	*SGOT (IU/l)*		*SGPT (IU/l)*		*ALT (IU/l)*		*Bilirubin (mg/dl)*
Control (Gr.1)	94.2 ± 5.1		58.40 ± 2.4		126 ± 8.2		0.89 ± 0.02
Test Paracetamol(Gr.2)	252.8 ± 4.03		172.5 ± 3.6		275.2 ± 1.4		3.48 ± 0.12
Experimental (10 mg/kg bw) + Paracetamol (Gr.3)	193.4 ± 6.2		128.4 ± 4.8		176.4 ± 4.2		1.3 ± 0.04
	*Biochemical parameters - Liver*
	*Glycogen (mg/100g tissue)*	*GSH (nmlol/mg protein)*		*GST (u/g tissue)*		*GPX (u/mg protein*	*GSH-R (*mmol NADPH min^-1/g tissue)
Control (Gr.1)	6.76 ± 0.97	30.5 ± 1.8		107.8 ± 2.1		8.8 ± 0.8	172 ± 3.2
Test Paracetamol(Gr.2)	2.18 ± 0.29	14.4 ± 2.4		91.6 ± 1.8		5.6 ± 0.6	110 ± 4.8
Experimental (10 mg/kg bw) + Paracetamol (Gr.3)	3.62 ± 0.7	23.2 ± 0.8		95.2 ± 1.2		7.24 ± 0.8	142.6 ± .28

Effect of Blumea mollis extracts on serum biochemical parameters in paracetamol induced hepatic damage in rats.

ALP and ACP concentration have been used to evaluate chemically induced hepatic injury. More than 90% of ALP activity has been found to be elevated in serum of common laboratory animals used in toxicity studies Blumea mollis prevented the paracetamol effect on ALP activity in serum. It is reasonable to suggest that Blumea mollis limited the severity of liver injury. Stabilization of serum bilirublin levels through the administration of Blumea mollis extract is further a clear indication of the improvement of the functions of the liver cells.

The present results support that the recovery of hepatic glycogen content was observed in the pretreatment of Blumea mollis treatment, while signs of biochemical recuperation were present in the liver of rats treated with Blumea mollis.

GSH in the cytosolic pool consists of 85% hepato cellular GSH and 15% mitochondrial GSH. Hepatic GSH depletion or even extra hepatic GSH depletion can provide useful information on the protective role of GSH against toxic foreign compounds. Thus GSH, be regarded as an endogenous protective agent against drugs In the present study decreased level of reduced GSH in liver was decrease in paracetamol induced animals, while pretreatment of BC clearly enhanced the GSH levels. GST is a soluble protein located in cytosol, which plays an important role in the detoxification of excretion of xenobiotics. It increases the solubility of hydrophobic substances and metabolises toxic compounds to non-toxic ones, which mean they have an increasing protective activity of the liver. The increased hepatic GST activity induced by B can, therefore, reduce the paracetamol hepatotoxicity. There was a decrease in GPX activity in animals administered with paracetamol, which could be due to the higher production of toxicity. In presence of Blumea mollis, GPX levels were restored back to control levels. The increase in hepatic GSH-R activities were shown in Blumea mollis supplemented rats as compared with the liver of paraceta mol-induced rats.

These results suggest the hepatoprotective action of Blumea mollis which protect hepatic cells from paracetamol induced damage and the degree of hepatoprotection improved with increasing dosage.

Further these data provide information regarding the possible use of Blumea mollis a hepatoprotectant in Indian systems of medicine.

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Received on 05.05.2012

Modified on 20.05.2012

Accepted on 30.05.2012

Research J. Pharmacology and Pharmacodynamics. 4(4): July –August, 2012, 206-209