1. INTRODUCTION:

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). Inspite of the tremendous advances made in allopathic medicine, no effective hepatoprotective medicine is available. Plant drugs are known to play a vital role in the management of liver diseases. There are numerous plants and polyherbal formulations claimed to have hepatoprotective activities. In India, more than 87 medicinal plants are used in different combinations in the preparation of 33 patented herbal formulations (Handa SS et al., 1989)

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).

A methanolic extract of the seeds of Hygrophila auriculata at a dose of 200 mg/kg/p.o exhibited potent hepatoprotective activity against paracetamol-and thioacetamide-induced liver damage in rats (Anubha Singh and Handa 1995).

Hygrophila auriculata have been reported to possess anti oxidant (Sunil Kumar, Klausmuller 1999), Hypoglycemic activity (Fernando et al., 1991), Anti-inflammatory and antipyretic activity (Patra et al., 2009) and Antibacterial activity (Boily and Vampuyvelde 1986). In the present study we have evaluated the hepatoprotective activity of this plant against CCl4 induced hepatotoxicity in rats.

2. MATERIALS AND METHODS:

2.1. Chemicals:

Nitro blue tetrazolium (NBT), Carbon tetrachloride (CCl4), Thiobarbituric acid (TBA), 5,5’-dithio-bis (2-nitrobenzoic acid), glutathione (reduced), Glutathione (oxidized), and L-ascorbic acid were purchased from Sigma Chemical Company (St. Louis, MO, USA). All other chemicals used were of analytical grade and were obtained from Glaxo Laboratories, Mumbai, India, and Sisco Research Laboratories, Mumbai, India.

2.2. Plant material and preparation of drug:

The leaves of - Hygrophila auriculata was purchased from local Traditional medical shop at Thanjavur, Tamil Nadu. The leaves were dried and make a fine powder. The fine powder of Hygrophila auriculata leaves was dissolved in distilled water just before oral administration.

2.3. Animals:

Male albino rats of Wistar strain approximately weighing approximately 150-200g were used in this study. They were healthy animals and housed in spacious polypropylene cages bedded with rice husk. The animal room was well ventilated and maintained under standard experimental conditions (Temperature 27±2ºc and 12 hours light / dark cycle) throughout the experimental period. All the animals were fed with standard pellet diet (Gold Mohur, Mumbai, India) and water ad libitum. They were acclimatized to the environment for 1 week prior to experimental use.

2.4. Experimental Design:

Body weights of the animals were recorded and they were divided into 3 groups of 6 animals each as follows; Group 1- Normal control rats were fed with standard diet and served as a vehicle control, which received liquid paraffin at the dose of 3.0ml/kg intraperitonially. Group 2- Rats were induced with hepatocellular damage by receiving suspension of Carbon tetrachloride (CCl4) in liquid paraffin (1:2,v/v, 1ml of CCl4 i.p./kg body weight) once in every day for 7 consecutive days. Group 3: Rats were treated with Hygrophila auriculata orally (through intragastric tube) at the dose of 500 mg.

2.5. Biochemical Study:

Animals were sacrificed by cervical dislocation.The blood samples were collected by direct cardiac puncture. The blood samples were allowed to clot and serum were separated and the serum was used for the assay of maker enzymes viz., Glutamate oxaloacetate transaminase (SGOT), Glutamate pyruvic transaminase (SGPT) (Reitman S, et al., 1957), alkaline phosphatase (ALP) (Kind and King’s , 1954), Reduced glutathione (Moron et al 1979) and total bilirubin (Malloy and Evenlyn, 1937)

2.6. Estimation of Liver Lipid Peroxides and Protein:

Estimation of liver lipid peroxides malondialdehyde (the product of lipid peroxidation) in the liver homogenate was measured as described Beuge and Aust (1978). Protein in the liver homogenate was measured according to the method of lowery et al (Lowry O et al 1951).

2.7. Statistical Analysis:

The results were expressed as mean ± SEM of six animals from each group. The statistical analysis were carried out by one way analysis of variance (ANOVA)

P values < 0.05 were considered significant.

3. RESULTS:

CCl₄-Induced Hepatotoxicity

The toxic effect of CCl4 was controlled in the animals treated with Hygrophila auriculata crude aqueous extract, it significantly decreased in the level of MDA and increased in the level of GSH when compared to untreated (Table 1). It also significantly restore the levels of marker enzymes like SGOT, SGPT and ALP when compared to untreated (Table 1), it also significantly increased in the level of protein and decrease the level of bilirubin in serum.

Table 1 Effect of Hygrophila auriculata on Biochemical parameters

Parameters

Group I

Group II

Group III

MDA (nmol /L)

GSH (mg/dl)

SGOT (IU/dl)

SGPT (IU/dl)

ALP (IU/dl)

Protein (gm/dl)

Albumin(gm/dl)

Bilirubin(gm/dl)

2.2 ± 0.74

12.52 ± 1.5

31.97 ± 0.51

23.17 ± 0.21

41.366 ±1.35

6.18 ± 0.62

4.04 ± 0.45

0.75 ± 0.09

15 ± 1.48

2.42 ± 0.39

60.109 ±0.58

57.1 ± 0.26

54. ± 1.303

3.52 ± 0.52

2.29 ± 0.46

1.51 ± 0.06

2.4 ± 0.94 *

10.87 ± 1.04 *

40.12 ± 0.47 *

23.18 ± 0.51*

42.48 ± 1.08

5.78 ± 1.03 *

3.23 ± 0.36 *

0.74 ± 0.06*

Values were expressed as mean ± SD for six rats in each group.

*Significantly different from Group II.

4. Discussion:

The findings of the present investigations are based on the hepato protective ability of Hygrophila auriculata to reduce the injurious effects caused by CCl4 because hepatic cells appear to participate in a variety of enzymatic metabolic activities. Metabolically, CCl₄ is a well known model compound for producing chemical hepatic injury, requires biotransformation by the heaptic microsomal cytochrome P 450 to produce hepatotoxic metabolites, namely trichloromethyl free radicals (CCl₃– and/or CCl₃OO–) (Brent and Rumack, 1993).

Malondialdehyde (MDA) is a commonly used biomarker of lipid peroxidation, which arises from the breakdown of lipid peroxyl radicals, is one of the indicators of oxidative stress. Measured levels of MDA can be considered a direct index of oxidative injuries associated with lipid peroxidation (Halliwell, 1991). In this context a marked increase in the concentration of MDA in liver and serum indicates oxidative stress in CCl4 intoxicated rats when compared to control rats. Administration of Hygrophila auriculata significantly decreased the level of MDA demonstrate the reduction of oxidative stress in Hygrophila auriculata and CCl4 intoxicated rats (Table 1). Glutathione status is a highly sensitive indicator of cell functionality and viability. Perturbation of GSH status of a biological system has been reported to lead to serious consequences (Pastore et al., 2003). Decline GSH content in liver and serum of CCl4 intoxicated rats (Table 1), and its subsequent return towards near normalcy in CCl4 and Hygrophila auriculata treated rats reveal antioxidant effect of Hygrophila auriculata. Damage caused to hepatic cells, the leakage of plasma (Zimmerman and Seef, 1970) causing an increased levels of hepato specific enzymes in serum. The elevated serum enzyme levels like AST and ALT are indicative of cellular leakage (Table 1) and functional integrity of cell membrane in liver (Achliya et al., 2004; Drotman and Lawhorn, 1978). Treatment with Hygrophila auriculata decreased the serum levels of AST ALT and ALP towards their respective normal value that is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage caused by CCl4.

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Received on 16.09.2012 Accepted on 25.10.2012

Asian J. Res. Pharm. Sci. 2(4): Oct.-Dec. 2012; Page 140-142

PG & Research Department of Biochemistry, Enathi Rajappaa College, Enathi, Pattukkottai, Thanjavur Dt., Tamil Nadu, India - 614 615