The leaves and bark of Cassia glauca was collected during November 2007 from the Western ghats, Karnataka, India. The plant was identified by Dr. Harsha Hegde, Research Officer, RMRC, ICMR, Belgaum, Karnataka, India. A voucher specimen (SA-01) has been kept in herbarium of Department of Pharmacognosy, KLES College of Pharmacy, Belgaum, Karnataka, India.

Cassia glauca leaves and bark was air dried in the shade and cut into small pieces. Hundred grams each of leaves and bark was extracted with 1000 ml of water by the method of hot extraction at 60ºC for 6 h with occasional shaking and the extracts were filtered and filtrate were evaporated to dryness under vacuum to yield dry residue_. Animals in the control group received only the vehicle (Normal saline 5 ml/kg, b.w.) (Jain, 1968; Latha and Pari, 2004; Aslan et al. 2007).

Wistar rats (180-200 g) were obtained from animal house, Department of Livestock Production, Government Veterinary College, Hebbal, Bangalore, India. Rats were kept in groups of six in plastic cages at controlled temperature (25°C) and humidity (about 40%) with a 12 h light cycle beginning at 7 am. Rats were provided with standard diet and tap water ad libitum. All experiments were carried out between 8 and 12 am at an ambient temperature of 23-25°C. The study was approved by Institutional Animal Ethical Committee of Jawaharlal Nehru Medical College, Belgaum, India (Resolution No. 1/16/2007).

The acute oral toxicity study was carried out as per the guidelines set by OECD. The animals (n=3 in each group) were observed continuously for 2 h under the following profiles:

(i) Behavioral profile. Alertness, restlessness, irritability and fearfulness.

(ii) Neurological profile. Spontaneous activity, reactivity, touch response and pain response.

(iii) Autonomic profile. Defecation and urination.

After a period of 24 and 72 h animals were observed for any lethality or death.

Fasting blood sugar level of each animal was determined, after overnight fasting for 16 h. Rats divided in to three groups (n=6) were administered saline to control group and 500 mg/kg aqueous extract of Cassia glauca leaves and bark to second and third group respectively. Blood samples were collected at 30, 60 and 120 min after the oral administration of aqueous extract (Shirwaikar and Rajendran, 2006; Aslan et al., 2007).

The oral glucose tolerance test was performed in overnight fasted (18 h) normal rats. Rats divided in to three groups (n=6) were administered saline to control group and 500 mg/kg aqueous extract of Cassia glauca leaves and bark second and third group respectively. Glucose (2 g/kg) was fed 30 min after the administration of extract. Blood samples were withdrawn from sinus retrorbital at 30, 60 and 120 min of glucose administration (Shirwaikar and Rajendran, 2006; Aslan et al., 2007). and glucose levels were estimated using a glucose oxidase–peroxidase reactive strips and a glucometer (Sugarcheck, Wockhardt Ltd., Mumbai, India).

Rats were fasted for 16 h prior to STZ injection. Diabetes was induced in rats by intraperitoneal (i.p.) injection of streptozotocin (STZ) (Spectrochem Pvt.Ltd. Mumbai, India) at a dose of 70 mg/kg b.w., (Brain et al., 1997; Kadnur et al., 2005) dissolved in 0.1 M cold citrate buffer (pH= 4.5). Seven days after the injection, the blood glucose concentration level above 200 mg/dl was considered to be diabetic and used in the experiments. To prevent the hypoglycemia 5% glucose solution was orally given to the diabetic rats. Only rats found with permanent diabetes were used for the antidiabetic study.

Diabetic rats were divided into 3 groups as follows:

Group 1: Diabetic control. Received only saline.

Group 2: Diabetic rats given (500 mg/kg b.w. p.o.) aqueous extract of leaves.

Group 3: Diabetic rats given (500 mg/kg b.w. p.o.) aqueous extract of bark.

The oral glucose tolerance test was performed in overnight fasted (18 h) diabetic rats. The blood glucose concentrations of the animals were measured at the beginning of the study and Glucose (2 g/kg p.o.) was fed 30 min after the administration of extracts of Cassia glauca leaves and bark (500 mg/kg p.o.). Blood samples were withdrawn at 30, 60 and 120 min of glucose administration and glucose levels were estimated using a glucose oxidase–peroxidase reactive strips and a glucometer (Sugarcheck, Wockhardt Ltd., Mumbai, India).

Rats were divided in to five groups of six rats (n=6) each. Groups 1 served as normal healthy control group and group 2 as diabetic untreated control and saline was given to both groups. Group 3 served as standard and was treated with glibenclamide (Sun Pharmaceuticals Ltd, India) (0.25 mg/kg/day p. o.) (Shirwaikar et al., 2004; Bairy et al., 2005; Shirwaikar and Rajendran, 2006). Group 4 and 5 was treated with dose of 500 mg/kg p.o. per day aqueous extract of Cassia glauca leaves and bark respectively for 21 day. Blood glucose levels of animals were measured on day 1, 7, 14 and 21 day of the study (Latha and Pari 2004; Chakrabarti et al., 2005)

Values are presented as mean ± S.E.M. Statistical difference between the treatment and the controls were tested by using one-way analysis of variance (ANOVA) followed by Student's 't ' test (500 mg/kg drug treated group was compared with diabetic control). A difference in the mean P value <0.05 was considered significant.

Experiments were carried out on normal healthy rats. The behavior of the rats treated with aqueous extract of Cassia glauca leaves and bark appeared normal. No toxic effect was seen even with the dose of 5 g/kg b.w. and there were no lethality in any of the group.

Since no toxicity was observed with the extract in toxicity studies, the aqueous extract of Cassia glauca leaves and bark was prepared and administered to normal, glucose loaded model and STZ-induced diabetic rats at a dose of 250 and 500 mg/kg b.w. (p.o.) to determine the effect of aqueous extract on the blood concentrations upon acute administration. Changes in the blood glucose level of each group of animal were followed during a 2 h period (Table 1). It was observed that the aqueous extract of Cassia glauca leaves and bark at a dose of 250 and 500 mg/kg p.o. failed to show any hypoglycemic activity in normal and OGTT. However, aqueous extract of Cassia glauca leaves and bark at a dose of 500 mg/kg p.o. showed significant hypoglycemic activity in acute model of STZ-diabetic rats, while no remarkable effect was observed on normoglycemic rats. The improvement in diabetic-glucose tolerance in aqueous extract of bark treated (500 mg/kg) group was brought by lowering the initial blood glucose level of 437.2± 4.19 mg/dl significantly (p< 0.001) to 288.7±9.95 mg/dl at 2 h, where as the leaves extract showed significant (p< 0.001) hypoglycemic effect by lowering the initial fasting blood glucose level of 430.2 ± 31.9 mg/dl to 241.5 ± 2.0 mg/dl at 2 h. While in the untreated diabetic group the fasting blood glucose increased from the initial value of 380.5 ± 5.3 mg/dl to 399.2 ± 6.9 mg/dl. From the result it is clear that the aqueous extract of Cassia glauca leaves and bark (250 and 500 mg/kg b.w. p.o.) did not showed any significant effect on normoglycemic rats (Table 1).

Legends of Table 1: Values are given as mean ± S.E M from six rats in each group

***P < 0.001 highly significant from diabetic control animals and initial values.

Aq. E: Aqueous extract

Figure 1

Table 1: Hypoglycemic effect of aqueous extract in different models of rats

Since significant hypoglycemic effect was observed at 500 mg/kg in diabetic acute model, it was decided to determine the chronic effect of the extract of Cassia glauca leaves and bark by using 500 mg/kg/day for 21 days. From the results of chronic model study (21 days), it was observed that there was significant decrease (P<0.001) of serum glucose level on day 21 (260.3 ± 12.4 mg/dl)) from day 1 (354.5 ± 7.3 mg/dl) with the treatment of aqueous extract of Cassia glauca bark, which is comparable to the effect produced by standard drug glibenclamide (Table 2). Glibenclamide lowered the serum glucose level significantly to day 21 (212.2 ± 6.8 mg/dl) from day1 (294.5 ± 11.0 mg/dl). The aqueous extract of leaves failed to produce any significant changes in fasting blood glucose levels on day 21 from day1. However the aqueous extract of leaves produced significant effect by lowering the fasting blood glucose level of day 7 (305.3 ± 13.5 mg/dl) as compared to fasting blood glucose level of day 21 (284.5 ± 10.5 mg/dl).These finding suggest that the aqueous extract of Cassia glauca bark possessed a significant hypoglycemic effect at 500 mg/kg dose in diabetic rats.

Legends of Table 2: Values are given as mean ± S.E M from six rats in each group

***P < 0.001 highly significant from diabetic control animals and initial values.

Aq. E: Aqueous extract

Figure 2

Table 2: Effect of aqueous extracts on serum glucose level on chronic administration

The authors sincerely thank Dr. F. V. Manvi, Principal, KLES’s College of Pharmacy for providing the necessary facilities for this work.