2.1. Plant material and extraction P. guajava leaves were collected from the Chittagong University Campus, Chittagong, Bangladesh in August 2012. The plant was taxonomically identified by Professor Dr. Mostafa Kamal Pasha, Department of Botany, University of Chittagong and the voucher specimen (Pharma-0024/2012) was deposited at Pharmacological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories, Chittagong, Bangladesh. The collected leaves were washed, chopped, dried, powdered and extracted with 98% ethanol and concentrated by using rotary vacuum evaporator. The yield of the extract was 3.52% (w/w, in terms of dried starting material) which was kept in refrigerator at 4 °C
2.2. Experimental animals Male Wister rats weighting (180?10) g were procured from animal house of BCSIR Laboratories Chittagong to assess the antidiabetic and the antidiarrhoeal activity. All animals were kept under standard laboratory conditions. The animals were fed with standard diet and allowed to drink water ad libitum. All aspects of animal care complied with the ethical guidelines of Pharmacology Research Division, BCSIR Laboratories Chittagong, Bangladesh.
2.3. Chemicals All chemicals and drugs of this experiments such as castor-oil (Shengyang Kaiyingsheng Chemical Co. Ltd., China), Tween 80 [Polyoxyethylene (20), Loba Chemie Pvt. Ltd., India], alloxan tetrahydrate (Merck, India), glibenclamide (Marion Roussel Ltd., Aventis, Bangladesh), loperamide (Opsonin, Bangladesh), glucose powder (dextrose monohydrate, GlaxoSmithKline, Chittagong, Bangladesh Ltd.), barium sulfate (BaSO4) (Merck, India Ltd.) and diethyl ether (Sigma-Aldrich, India) were obtained commercially and were of analytical grade.
2.4.1. Oral glucose tolerance test (OGTT) model The OGTT was performed in overnight fasted (18 h) normal rats as per reported method. Firstly, blood glucose level (BGL) of all fasted rats was withdrawn from the tip of tail and measured as “0 min” with the help of a blood glucose meter (Accu-Chek Active, Roche Diagnostics, Germany). Then, they were divided into four groups (n=5). Control animals received only distilled water (2 mL per rat) as Group I. Group II rats were given glibenclamide orally at a dose level 4.15 mg/kg body weight as positive control. Wister rats in Group III and IV were treated orally with EEPGL at doses of 1.0 and 0.5 g/kg body weight, respectively. After 30 min, BGL of all groups was measured. Then these animals were given glucose solution (10 g/kg body weight) orally and the concentration of BGL was estimated at 30, 60, 120 min subsequently. 2.4.2. Alloxan induced diabetic test model After fasts of 18 h, forty two male Wister rats were induced by alloxan tetrahydrate (100 mg/kg i.p.) in sterile saline. Diabetes was confirmed after 24 h with BGL of >10 mmol/L. Then the diabetic animals were separated and used for the study. Fifteen diabetic rats were selected and randomly divided into three experimental groups (marked as Group II to IV). Each group contains five rats. Group II as diabetic control received only distilled water. Group III as positive control was treated antidiabetic drug glibenclamide (4.15 mg/kg). Group IV was treated with ethanolic extracts of P. guajava leave (EEPGL) at 750 mg/kg. Group I was previously selected as control group which was non diabetic. After extract/drug administration, BGL of samples was determined at 0 h, 3 h, 6 h and 9 h by aforesaid system. All the animals were anesthetized with diethyl ether during blood collection. Room temperature and humidity were maintained in order to increase the survival capacity of treated rats.
2.4.3. Castor oil-induced diarrhoea (COID) model To determine antidiarrhoeal activity of EEPGL, COID model was conducted by following described method[16]. Twenty five Wister rats were randomly divided into five equal groups (n=5) namely control group, positive control group and three treated groups. The control group received only distilled water 2 mL per rat while positive control group received loperamide 2 mg/kg as standard and three treated groups received EEPGL at the dose of 750 mg/kg, 500 mg/kg and 2 50 mg/kg body weight, respectively. Rats were housed in separate cages with paper placed below for collection of fecal matters. Firstly, extract and drug were given orally to treated groups and positive control group respectively. In control group, only distill water was given orally. Then, one hour later castor oil (2 mL per rat) was induced orally to all rats initiating diarrhoea. The number of both hard and soft pellets was counted at every hour over 6 h period for each rat. Diarrhoea was defined as the presence of stool with fluid material that stained the paper placed beneath the cages.
2.4.4. Gastrointestinal motility test with BaSO4 milk (BSM) model for diarrhoea BSM model was carried out by reported method. Overnight fasted (18 h) twenty five Wister rats were randomly divided in to five equal groups (n=5). Control group received only distilled water 2 mL per rat orally. Positive control group received commercially available anti diarrhoeal drug loperamide 2 mg/kg orally. Treated groups received EEPGL 250 mg/kg, 500 mg/kg and 750 mg/kg orally. After thirty minutes, all groups of rats were administered with 2 mL of 10% BaSO4 solution. Lastly, after 30 min rats were sacrificed. Finally, the distance traveled by BaSO4 milk was measured and expressed as a percentage of the total length of small intestine (from pylorus to the ileo-cecal junction).
2.5. Statistical analysis All the values of antidiarrhoeal, tests were expressed as mean' SEM (standard error of the mean). Statistical differences between the mean of the various groups were analyzed by using student’s t test. All the graphical presentation and statistical calculations were prepared using “Microsoft Excel-2007”. Mean values were considered significantly different if P<0.05, P<0.01 and P<0.001.