The leaves of the plant Tectona grandis were collected from the Herbal Nature Park, Chuharpur, Forest Division, Yamunanagar, Haryana. Taxonomically, the plant was authenticated by Dr. H. B. Singh, Scientist Incharge and Head, Raw materials Herbarium and Museum, at National Institute of Science Communication and Information Resources (NISCAIR), New Delhi with a voucher specimen number NISCAIR/ RHMD/Consult/-2010-11/1619/217. After collection, the leaves were washed thrice with water to remove dust or debris and were air-dried. After drying completely, the leaves were ground to a coarse powder using a blender, then passed through the sieve of 40 mesh size and stored in a well-closed container.

Air-dried leaf powder of Tectona grandis (500g) was subjected to Soxhlet extraction by using methanol as solvent. The extract was further concentrated by using Buchi rotary vacuum evaporator (Gupta scientific store, Ambala) and then evaporating to dryness on the water-bath.

Phytochemical screening of extract was done to detect the presence of various chemical constituents like carbohydrates, amino acids, glycosides, alkaloids, lipids, fats, fixed oils, saponins, and flavonoids [15].

Adult Wistar rats of either sex (100-150g) were selected for the experimental study. The procurement of rats was done from the animal house of M.M. College of Pharmacy Mullana, (Ambala), Haryana. The animals were kept and maintained under laboratory control conditions of humidity (60 ± 1%), temperature (21.5°C ± 22°C) and 12 hour light or dark cycle. Free access to water and food was allowed to rats. Experiment protocols and procedures employed in the study were approved by the Institutional Animal Ethics Committee of M. M. University Mullana (Ambala), India (Reg. No. 1355/AC/10/ CPCSEA) and confirmed with the guideline of Committee for the purpose of Control and Supervision on experiments on animals.

The acute toxicity studies of methanolic extract of leaves of Tectona grandis were conducted by using adult Wistar rats of either sex (100-150g) maintained under approved husbandry conditions. In accordance with the up and down method of CPCSEA for toxicity studies, the animals fasted for 12 hours before the start of the experiment. Tectona grandis extract was administered orally at different doses (50-1000mg/kg). The experimental animals were kept under observation for behavioral changes from the first hour of drug intake up to seven days. The parameters, for example, hyperactivity, convulsions, hypothermia, sedation, mortality, and grooming were recorded for the doses of 200 mg/kg/day and 400 mg/kg/day [16].

Adult Wistar rats of either sex (100-150gm) were divided into five groups containing six animals in each group. Group I animals were considered as control who received only the vehicle for ten days at a dose of 1ml/kg (p.o.). Group II animals were considered as positive control and received a dose of carbon tetrachloride dispersed in sterile olive oil (1:1 v/v, 2ml/kg, i.p.) for ten days every 72 hours. Standard drug silymarin (100 mg/kg) was given to group III along with carbon tetrachloride dispersed in sterile olive oil (1:1 v/v, 2ml/kg, i.p.) for 10 days. Group IV-V received methanolic extract at the dose 200 and 400 mg/kg/day (dispersed in 0.5% sodium carboxymethyl cellulose) and carbon tetrachloride dispersed in sterile olive oil (1:1 v/v, 2 ml/kg) for 10 days. Overnight rats were fasted and sacrificed by using ethyl anesthesia at the end of the experiment. The blood samples were collected separately into the sterilized dry centrifuge tubes and separated serum was analyzed for determination of the liver enzymes markers. For histopathological studies, the liver is removed and kept in 10% formalin solution [17].

Reitman and Frankel's method is used for the estimation of SGOT and SGPT. Kind King’s method is used for the estimation of SALP and serum bilirubin. Jendrassik and Grof’s method and cholesterol oxidase/peroxidases method are used for the determination of total bilirubin and total protein, respectively [18-21].

10% neutral formalin solution was used to fix the liver from rats, further dehydrated with alcohol and embedded in paraffin. For light microscope analysis, fine sections mounted on glass slides were counterstained with hematoxylin and eosin.

Biochemical estimation results were marked for analysis of significant intergroup difference. Each parameter was analysed separately and one-way analysis of variance (ANOVA) was carried out. Dunnet's test was used for individual comparisons. Statistically significant results at P<0.05 were considered [22].

Phytochemical screening shows the presence of steroids, phenolic components, tannins, flavanoids and saponin in methanolic extracts of Tectona grandis leaves. The results are depicted in (Table 1).

Oral administration of the methanolic extract of Tectona grandis at a dose of 1000 mg/kg did not indicate any adverse effects or mortality when observed for 04 hrs and every-day for the next ten days. Data obtained from this and pilot study two doses i.e. 200 and 400 mg/kg were selected for further study.

CCl₄ treated Group II animals showed a steep boost in the levels of enzymes i.e. SGOT, SALP, SGPT, and serum bilirubin as compared to group I (untreated). Silymarin and Tectona grandis extract (200mg/kg and 400mg/kg) treated group (III-IV) showed a sharp fall in the levels of enzymes as compared to group II i.e. CCl₄ treated group (Table 2 & Fig. 1).

The microscopic histopathological evaluation of livers from the control group, CCl₄ treated group, standard drug silymarin, and methanolic extract have revealed the following observations.

The typical lobular arrangement is observed in rat liver. Hepatocytes are arranged in lobules as cords radiating around the terminal that is centrally placed hepatic veins. Hepatocytes seen are of uniform size having polyherbal shape and large nuclei centrally located. The cytoplasm is with a fine basophilic granularity is eosinophilic. Portal tracts comprising terminal branches hepatic portal vein and hepatic artery in fibrous stoma at the periphery are seen (Fig. 2).

The rat liver tissue with the disturbed lobular arrangement is observed. Early necrotic and degenerative alterations extending lobules are visible. There are steatotic alterations and ballooning degenerations in hepatocytes. Some amount of fibrosis seen in portal tracts. Toxic injury with fibrotic alterations and mild necrosis is observed in the liver (Fig. 3).

The rat liver showed liver tissue with the typical lobular arrangement. Variable size hepatocytes were observed and there is an increase in fibrous connective tissues. Mild hepatotoxicity is indicated in the liver (Fig. 4).

Rat liver showed liver tissue with the typical lobular arrangement. Few hepatocytes show steatotic accumulation. The liver with minimal sign of hepatotoxicity is observed. (Fig. 5 and Fig. 6).

Fig. (1). Effect of methanolic extract of Tectona grandis on biochemical parameters in experimental animal.

Fig. (2). Portal tracts comprising of terminal branches hepatic portal vein and hepatic artery in fibrous stoma at the periphery.

Fig. (3). Toxic injury with fibrotic alterations and mild necrosis is observed in the liver.

Fig. (4). Mild hepatotoxicity is indicated in the liver.

Fig. (5). Liver with minimal sign of hepatotoxicity.

Fig. (6). Liver with minimal sign of hepatotoxicity.