MATERIALS AND METHODS

Plant materials: The traditional medicinal plants viz. Enicostemma littorale Blume (Family: Gentianaceae) and Eclipta alba (Family: Asteraceae) were identified, in and around Vellore District, Tamilnadu, India. The plant leaves were collected during the months, September-January in 2004. The leaves were authenticated, cleaned, shade dried and voucher specimens were deposited in the laboratory for future reference.

Preparation of plant extracts: The shade dried leaves were powdered separately in an electrical blender and stored at 5°C until further use. A 100 g of each plant leaf powder was taken and mixed with 500 mL of distilled water and stirred magnetically in separate containers overnight at room temperature. The residue was removed by filtration and the aqueous leaf extracts were concentrated under vacuum to get each about 20% solid yield. The leaf extracts were combined in 1:1 tested for hepatoprotective and in vivo antioxidant effects in the male albino rats at the selected optimum dosage of 250 mg kg^-1 b.wt. and administered orally in aqueous solution.

Experimental animals: Studies were carried out using Wistar strain male albino rats (160-180 g). The rats were obtained from Tamilnadu Veterinary and Animal Science University, Chennai, India. The animals were acclimatized to the laboratory conditions, fed with standard pellet diet supplied by Hindustan Lever Ltd., Bangalore, India and had free access to water. The experiments were designed and conducted in accordance with guideline of Institutional Animal Ethics Committee.

Experimental design: Distilled ethyl alcohol was obtained from Hayman Ltd., England. The hepatotoxic dose was standardized as 5 mL of 20% ethanol (7.9 g kg^-1 b.wt.) orally, using an intra-gastric tube daily as described earlier (Rajakrishnan et al., 1997) for 45 days.

Thirty rats were randomly divided into 5 groups of 6 each.

Estimation of biochemical parameters: The activity levels of hepato-specific marker enzymes viz. aspartate transaminase (AST), alanine transaminase (ALT) in serum were estimated by the method of Reitman and Frankel (1957) and the activity level of alkaline phosphatase (ALP) in serum was estimated by the method of King and Armstrong (1934). The lipid peroxidation was assessed in tissue by measuring the levels thiobarbituric acid reacting substances (TBARS) by the method of Niehaus and Samuelson (1968). The tissue antioxidant enzymes superoxide-dismutase (SOD) and catalase (CAT) activity levels were estimated by the methods of Kakkar et al. (1984) and Sinha (1972), respectively. Serum total protein was estimated by the method of Lowry et al. (1951). Analysis of variance (ANOVA) and Student t-test were carried out using SPSS package for statistical analysis.

RESULTS

Administration of ethanol at the dose level of 7.9 g kg^-1 b.wt., daily for 45 days to albino rats resulted a significant elevation of hepato-specific serum markers AST, ALT, ALP and total protein in comparison with normal animals (Table 1). On administration of aqueous leaf extracts of Ens+Ecl in 1:1 revealed a significant depletion in the activity levels of AST, ALT and ALP.

The ethanol induced oxidative stress in the liver was assessed by measuring the levels of thiobarbituric acid reactive substances (TBARS) and the activity levels of tissue antioxidant defense enzymes, SOD, CAT. Elevated levels of TBARS and a significant reduced activity level of SOD and CAT were recorded in ethanol administered animals (Table 2). The aqueous leaf extracts of Ens, Ecl and Sylamarine (reference drug) administered animals revealed significant depletion in the levels of TBARS and elevation in the activity levels of tissue SOD and CAT (Table 3).

Table 1:	Activity levels of AST, ALT, ALP, SOD, CAT and levels of TBARS ethyl alcohol induced hepatotoxicity
Values are mean of 6 individual observations in each group±SD. p denotes statistical significance, + and – indicate % of changes over the normal group; AST: Aspartate tranaminase, ALP: Alkaline phosphatase, ALT: Alanine transaminase, TBARS: Thiobarbituric reactive substances, SOD: Superoxide dismutase, CAT: Catalase. Unit1: One unit of activity was taken as the enzymes reaction which gives 50% inhibition of NBT reduction in one minute.Unit2: μmoles of hydrogen peroxide consumed per min

Table 2:	Hepatoprotective effect of the aqueous leaf extracts of Eclipta alba and Enicostemma littorale on ethanol induced hepatotoxicity
Values are mean of 6 individual observations in each group±SD. - indicate % of changes over the ETOH fed rats. *The group compared with ETOH fed rats by using Student t-test; **p denotes statistical significance of ANOVA to the difference between the ETOH fed and plant extract, Unit1: One unit of activity was taken as the enzymes reaction which gives 50% inhibition of NBT reduction in one min. Unit2: μmoles of hydrogen peroxide consumed per min

Table 3:	Synergistic hepatoprotection of Enicostemma littorale and Eclipta alba during ethanol induced hepatotoxicity
Values are mean of 6 individual observations in each group±SD. - indicate % of changes over the ETOH fed rats; *The combined (Ecl+Ens) compared with silymarin fed model by using student ‘t’test; **The group compared with ETOH fed rats by using Student t test; Unit1: One unit of activity was taken as the enzymes reaction which gives 50% inhibition of NBT reduction in one min.Unit2: μmoles of hydrogen peroxide consumed per min

DISCUSSION

Measurement of the enzymes activity in the body fluids is a useful monitor of disease state (Porchezhian, 2005). An obvious sign of hepatic injury is leakage of cellular enzymes into serum (Savides and Oehme, 1983). In the present study the elevated levels of serum AST, ALT and ALP in ethanol administered animals might be due to the damage of the liver tissue and/or changes in cell permeability that all AST, ALT and ALP to leak into serum (Paul and Giboney, 2005). Similar observations during ethanol induced hepatotoxicity were elsewhere recorded (Daniell et al., 2002).

Intake of alcohol results in excessive generation of free radicals (Isayama et al., 2003). Free radicals are the Reactive Oxygen Species (ROS) are known to cause oxidative damage to number of molecules in cell, including membrane-lipids, proteins and nucleic acids. In the present study the hepatic cellular injury in ethanol administered animals might be due to increased oxidative stress leading to lipid per oxidation, cell membrane damage and thereby leakage of the cellular enzymes. The level of lipid per oxidation was assessed by measuring the levels of thiobarbituric acid reactive substances (TBARS) in the liver tissue (Rukkmani et al., 2004). The significant increase of the tissue TBARS levels in the ethanol administered animals revealed lipid peroxidation in the liver. The depleted activity levels of cellular antioxidant enzymes viz. The SOD and CAT recorded in the present study revealed reduced cellular antioxidant defense mechanism and thereby leading to increased lipid peroxidation. However, the administration of aqueous leaf extracts of the Ens and Ecl revealed hepatoprotective effect during ethanol induced hepatotoxicity. The significant decrement in the levels of AST, ALT and ALP in plant extract fed animals might be due to decreased leakage from liver cells. This suggests that the plant extracts could be able to repair the probable hepatic injury and/or restore the altered cellular permeability. Thus, reducing the toxic effect of ethanol in the liver tissue. The significant depletion in tissue levels of TBARS in the plant extracts fed animals might be due to reduced lipid peroxidation and/or elevation of tissue antioxidant defense enzymes activity levels indicating that the plant extracts could reduce the free radical generation and increased free radicals scavenging mechanism. The significant increment in the activity levels of SOD and CAT in the plant extract fed animals are in corroboration with the increased free radicals scavenging mechanism. Similar studies reported that significant decrease in the activity of liver SOD, CAT in ethyl alcohol intoxicated rat was observed and the therapeutic treatment with A. indica herbal drug promoted the hepatoprotection by elevating free radical scavenging activity (Maruthappan and Sakthi, 2009).

In the present study, a significant higher hepatoprotective and antioxidant effect were revealed in the plant extract simultaneously fed along with ethanol supplemented group animal (1st to 45th day) when compared to that of the plant extract fed group after 45th day onwards (45th to 90th day).

The results of the study envisaged that alcohol induced oxidative stress leading to hepatic tissue damage. The aqueous leaf extract of Ens and Ecl supplementation recorded significant hepatoprotective effect against ethanol induced oxidative stress, when compared with sylimerin, the reference hepato protective drug. The above results suggest further scope of studies on isolation of the potent hepatoprotective ingredients from leaf of Ens+Ecl to combat the ethanol induced hepatotoxicity.