Plant material used in this study consisted of the stem bark of B. aristata, collected from the local area of Rishikesh, Uttranchal. The plant was authenticated by Dr. H. J. Chaudhary, Director of Botanical Survey of India, Govt. of India, Dehradun Bsi/Nc/6(4)/06-07. The stem bark was dried in shade at room temperature. Dried stem bark was coarsely powdered by using grinder, and latter was packed into soxhlet column and that was defatted with petroleum ether then extracted with ethanolic and dried in vacuum. The presence of alkaloids were confirmed by qualitative examination.

Male Wistar albino rats (150–250 g) housed in a spacious cage for ten days after obtaining approval from ‘Institutes Ethical Committee' 997/c/06/CPCSEA. During the experiments rats were feeded with standard pellet diet. After randomization into various groups, the rats were acclimatized for 2–3 days in environment before initiation of experiment.

Rats were divided into four groups containing six animals in each group. All animals fasted before treatment. Group I was kept as vehicle control which received 5% Tween 80 p.o., group II received glucose only, group III received ethanolic extract 25 mg/kg and group IV received only extract only in a vehicle respectively .The rats of group II and III were loaded with glucose (3 g/kg, p.o.) 30 min after drug administration. Blood samples were collected from puncturing the retro orbital sinus just prior to drug administration, and 30, 90, 150 min after loading glucose. Serum glucose level was measured immediately by using glucose estimation kit (Span Diagnostic Pvt. Ltd. Surat, India) ₈ .

All the animals were randomly divided into five groups with six animals in each group. Group I was used as control. Group II, III, IV and V were made diabetic by single intraperitoneal injection of alloxan monohydrate (125 mg/kg; Rollex, India) and served as diabetic control, standard and treatment groups respectively. Rats exhibited in plasma glucose levels >250 mg/dl, 48 h after administration of alloxan were included in the study. Treatment for diabetes (B. aristata extract 25 and 50 mg/kg / p.o.) was started from 7 ^th day of alloxan administration.

Blood samples were collected retro-orbitally from the inner canthus of the eye under light ether anesthesia using capillary tubes (Micro Hematocrit Capillaries, Mucaps). Blood was collected in fresh micro centrifuge tube and plasma separated in a T8 electric centrifuger (Remi Udyog, New Delhi) at 2000 rpm for 15 min.

After 15 days of daily feeding of extracts orally the animals were killed by decapitation liver was collected.

Serum glucose, serum cholesterol serum total lipids, serum protein, serum urea, SGOT (Serum glutamate oxaloacetate transaminase) and SGPT (Serum glutamate pyruvate transaminase) were estimated by commercially available kits (Span Diagnostic Pvt. Ltd. Surat, India). Liver glycogen content was estimated by the method of Carrol et al.,1955 ₉ .

All results were expressed as the mean ± Standard Deviation (S.D) Statistical analysis was performed with Graph Pad Instat software (version 3.00, Graph Pad Software, San Diego, California, USA) by using one way analysis of variance (ANOVA) comparison was done by using Dunnett test. P value <0.05 was considered as significant.

The effect of extract of B. aristata (25 mg/kg) on glucose tolerance test are shown in Table 1. The supplementation of B. aristata improved the glucose tolerance in the fasted normal rats. After that serum glucose level was lowered significantly (P<0.05) at 90 mins and varied significantly (P<0.01) lowered at 150 mins. Extract also showed significant hypoglycemic effect after 90 mins of treatment.

Figure 1

Table 1: Effect of ethanolic extract ethanolic extract on glucose tolerance test

Administration of alloxan monohydrates (125 mg/kg) led to elevation of blood glucose level. The anti-hyperglycemic effect of the ethanolic extract of stem bark of B. aristata (25 mg/kg, 50 mg/kg) and glibenclamide (5 mg/kg) on blood sugar levels of diabetic rats were shown in Table 2. The antidiabetic effect of the B. aristata extract 25 mg/kg, 50 mg/kg and Glibenclamide 5 mg/kg exhibited effect in dose dependent manner. The percent reduction of hyperglycemia was insignificant on the 1 ^st day of the treatment by B. aristata (25, 50 mg/kg) but it was reduced very significantly by 41% on 1 ^st day of glibenclamide (5 mg/kg) treatment. The percent reduction of hyperglycemia was significant (P<0.01) on 4 ^th , 7 ^th and 15 ^th day after treatment of the 25 mg/kg of B. aristata which was 36.6, 40.4, 60.4 % respectively. The percent reduction of hyperglycemia was significant (P<0.01) on 4 ^th , 7 ^th and 15 ^th day after treatment with the 50 mg/kg of B. aristata which was 43.30, 61.40, 75.46 % respectively. The percent reduction of blood glucose levels were significant (P<0.01) 45, 61.50, 74.3 % on 4 ^th , 7 ^th , and 15 ^th day respectively after treatment by glibenclamide 5 mg/kg (Table 2).

Figure 2

Table 2: Effect of ethanolic extract on serum glucose level diabetic rats

At the end of 15 days of treatment the body weight of the untreated diabetic rats was found to be significantly decreased and significant increase in the liver glycogen compared with the control rats. The administration of B. aristata and glibenclamide to diabetic rats restored the changes in the body weight, liver glycogen to near normal levels.

The normal function of the kidney was assessed as blood urea level in normal, diabetic and treated animals and it was altered from 31.83 mg/dl (normal) against 131.51 mg/dl (diabetic control) after the 15th day of alloxan treatment. The results of serum urea level indicates that the animals administered with B. aristata (25 and 50 mg/kg) in diabetic rats reduced the urea levels dose dependently (P<0.01). The percent reduction of blood urea level was 34.4 and 58.31 with the treatment of ethanolic extract of stem bark of B. aristata at the dose of 25 and 50 mg/kg respectively and glibenclamide was reduced 68.3% in comparison with the diabetic control group. The extent of gluconeogenesis and ketogenesis was assessed by estimation of serum protein, cholesterol, total lipids, SGOT and SGPT level in normal, diabetic and treated animals after 15 ^th day of test drug treatment. The levels of serum protein, cholesterol, total lipids, SGOT and SGPT level are shown in table-3. The levels of serum protein, cholesterol, total lipids, SGOT and SGPT were increased very significantly in diabetic rats as compared to normal rats. There was very significant (p<0.01) reduction in the levels of these on treatment with B. aristata and glibenclamide. The percent reduction of blood protein levels were 57 and 58.04 after treatment of alloxan induced diabetic rats by ethanolic extract of stem bark of B. aristata at the dose of 25 and 50 mg/kg respectively and glibenclamide was reduced 63.67% in comparison with the diabetic control group. The blood urea and protein levels were reduced significantly (P<0.01) but which was slightly lesser in comparison with standard.

The blood cholesterol and total lipid levels were reduced significantly (P<0.01) and their reduction was slightly higher than the standard, which reflects the potential hypolipidemic effect of the B. aristata. The percent reduction of blood cholesterol was 67.18 and 73.61 and total lipid levels were 68.23 and 69.29 after treatment of alloxan induced diabetic rats by the ethanolic extract of stem bark of B. aristata at the dose of 25 and 50 mg/kg respectively and glibenclamide was reduced blood cholesterol 65.82% and total lipids 63.62% in comparison with the diabetic group.

The treatment of alloxan induced diabetic rats by B. aristata decreased SGOT and SGPT levels significantly (P<0.01) and their reduction was slightly higher than the standard, which indicates the prevention of gluconeogenesis, ketogenesis and normal liver function. The percent reduction of SGPT was 61.52 and 63.46 and SGOT was 42.77 and 55.45 after the treatment of ethanolic extract of stem bark of B. aristata a dose of 25 and 50 mg/kg respectively and glibenclamide which reduced 57.68% of SGPT and 50.93% of SGOT in comparison with the diabetic group (Table 3A and 3B).

Figure 3

Table 3A:Effect of ethanol extract of stem bark on biochemical parameter in alloxan induced diabetic rats

Figure 4

Table 3B: Effect of ethanol extract of stem bark on biochemical parameter in alloxan induced diabetic rats

The authors wish to acknowledge the support and encouragement of Prof. Divakar Goli during the course of study.