MATERIALS AND METHODS

Collection of seaweed: Gracilaria corticata were collected from Mandapam station which is located in the Gulf of Mannar region, Rameswaram, India during June to December, 2003. It was authenticated by Dr. N. Kaliaperumal and J.R. Ramalingam, Senior scientists at CMFRI, Mandapam, Ramanathapuram District, Tamilnadu. Algae samples were collected by handpicking. They were washed and freed from extraneous matter such as epiphytes, sand particles, pebbles and shells and brought to the laboratory in plastic bags. The samples were then, thoroughly washed with fresh water. They were shade dried, powered, sieved and preserved for further analysis.

Extraction: The powdered material was subjected to Soxhlet extraction using water for 6 h. The solvent was removed in vacuo to give an appropriate yield of 12%. The extract was used for the treatment.

Production of AFB₁ and dosage: Aspergillus parasiticus NRRL-2000 obtained from Central Clinical Laboratory, Madras Veterinary College, Chennai was used as the source of inoculum for the production of aflatoxin. The fungus was maintained by subculturing it on potato-dextrose-agar slants (Shotwell et al., 1966). AFB₁ was produced, characterized and estimated by the method of Romer (1975).

Experimental animal: Male albino Wistar rats weighing about 125 to 150 g (45-60 days old) were maintained under standard experimental conditions (Temperature 27±2°C, relative humidity 60±5% and 12 h light/dark cycle) and had free access to water ad libitum. Before the commencement of actual experiment the rats were acclimatized in the above condition for 15 days.

Experimental protocol: Rats of same age and equal weight were selected after acclimatization and divided into 4 groups of 6 animals in each. The animals were fed with the respective experimental feed and water through out the experimental period. All the managemental practices were followed to all animals identically.

Group I :Control group, feed alone for 30 days

Group II:Feed with AFB₁ (1 ppm) for 30 days

Group III:Feed with AFB₁ (1 ppm) + Aqueous extract of G. corticata (250 mg kg^-1) for 30 days

Group IV:Feed with AFB₁ (1 ppm)+Sylimarin (100 mg kg^-1) for 30 days

Morphological parameters: Body weights of individual animals were recorded before and after the experimental period. Six rats from each treatment group were sacrificed after 30 days along with their control group. The animals were fasted overnight, sacrificed by cervical decapitation. Liver was immediately excised after perfusing with physiological saline, the liver was blotted, dried and weighed in the electronic balance.

Biochemical parameters: At the end of the 30 days of experimental duration, the blood samples of each animal were collected in separate tubes, kept at room temperature for 1 h and then centrifuged at 1000 g for 10 min to obtain serum and used for the assay of protein (Lowry et al., 1951), Bilirubin (Gupta et al., 2007), diagnostic marker enzymes viz., SGOT, SGPT (Sengottuvelu et al., 2007), LDH (Ahamed et al., 2003) and ALP (Tiez et al., 1983).

Statistical analysis: Results are expressed as Mean±SD. One-way ANOVA was carried out and the statistical comparisons among the groups were performed with using a statistical software package program SPSS 7.5.

RESULTS AND DISCUSSION

At the end of the 30 days of the experimental duration, the body weight gain, liver weight, total protein and albumin levels were decreased in group-II rats due to the AFB₁ toxicity. Concentration of serum bilirubin and the activities of serum marker enzymes viz., SGOT, SGPT, LDH and ALP were significantly elevated when compared to the normal (group-I). Co-administration of G. carticata extract at a dose of 250 mg kg^-1 body weight exhibited a significant improvement in body weight gain and liver weight and alters the total protein and albumin to almost normal level (Table 1). Serum levels of bilirubin and marker enzymes were also restored towards normal following the extract treatment (Table 1, 2). The activity of the extract against the AFB₁ induced toxicity was compared with a standard hepatoprotective drug, sylimarin. From the results it is evident that the effect of aqueous extract of G. carticata and sylimarin was comparable in all parameters tested.

Table 1:	Effect of aqueous extract of G. corticata on body weight gain, liver weight, total protein, albumin and total bilirubin
Values are expressed as Mean±SD (For six animals in each group), *Values are statistically significant at p<0.05 when compared with control (group I)

Table 2:	Effect of aqueous extract of G. corticata on serum marker enzymes
Values are expressed as Mean±SD (For six animals in each group). *Values are statistically significant at p<0.05 when compared with control (group I)

The present study demonstrates, the hepatoprotective effect of G. corticata on AFB₁ induced liver injury in rats. The liver weight decreased in aflatoxin induced hepatotoxic conditions may largely be due to protein degradation during tumour growth. Protein metabolic perturbations in host, although causing tissue waste may themselves favour the growth of tumour itself. The tumour growth elicited marked loss of body weight in growing asitic hepatoma bearing rats (Tessitore et al., 1987). This may be due to decreased food intake and/or absorption, which contribute to muscle waste in tumour Lachexia (Pain et al., 1984). In this study, rats treated with AFB₁, body weight gain and liver weight were significantly reduced and increased after treatment with aqueous extract and sylimarin.

The results in the present study indicate that 250 mg kg^-1 of aqueous extract of G. corticata was able to reduce all the elevated biochemical parameters due to the hepatotoxin intoxication. The reduced levels of total protein and albumin due to aflalotoxin induced hepatotoxicity. Hypoalbuminemia is most frequent in advanced chronic liver diseases. Hence decline in total protein content can be deemed as an useful index of severity of cellular dysfunction in chronic liver disease (Balamurugan and Muthusamy, 2008). The reduction is attributed to the initial damage produced and localized in the endoplasmic reticulum which results in the loss of P₄₅₀ leading to its functional failure with a decrease in protein synthesis and accumulation of triglycerides leading to fatty liver (Pattanayak and Priyashree, 2008). In rats administered with aqueous extract of G. corticata and sylimarin along with the AFB₁, the levels of protein and albumin were resumed to near normal levels. The total protein level was raised suggesting the stabilization of endoplasmic reticulum leading to protein synthesis.

Determining the activities of serum marker enzymes like SGOT, SGPT, LDH and ALP can make assessment of liver function. When liver cell plasma membrane is damaged, a variety of enzymes normally located in the cytosol are released in to the blood stream and their determination in the serum is a useful quantitative marker of the extent and type of hepatocellular damage (Balamurugan and Muthusamy, 2008). The rise in SGOT activity is almost always due to hepatocellular damage and is usually accompanied by raise in SGPT (Ravikumar et al., 2005). In the present study, the data in Table 2 reveals that, the animals of toxic control group recorded high level of serum marker enzymes viz., SGOT, SGPT, LDH and ALP indicating severe hepatic damage due to toxic effect of AFB₁, the serum marker enzyme levels in the standard drug treated rats showed almost normal values indicating the protective effect of silymarin. The animals treated with aqueous extract of G. corticata at the dose of 250 mg kg^-1 recorded significant reduction in the serum marker enzyme levels which is in comparison to the values of silymarin treated group indicating the effect of the algae in stabilizing the plasmamembrane and in protecting the hepatocytes from the toxic effect of aflatoxin. Alkaline phosphatase (ALP) is the prototype of these enzyme that reflects the pathological alteration in biliary flow (Bhakta et al., 1999). Determination of serum bilirubin represents as an index for the assessment of hepatic function and any abnormal increase in the levels of bilirubin in the serum indicate hepatobiliary diseases and severe disturbance of hepatocellular function (Martin and Friedman, 1992). Stabilization in the levels of serum bilirubin in the G. corticata treated groups as compared to aflatoxin alone fed group clearly indicates the improvement in the functional status of the liver.

The present investigation indicates the aqueous extract of G. corticata had therapeutic and preventive efficiencies in AFB₁ induced hepatotoxicity in rats. Hence, it is possible that the mechanism of hepatoprotuction by G. corticata may be due to its antioxidant action. Further studies are recommended to elucidate the antioxidant activity and mechanism of the hepatoprotective action of this algae.