The experimental works were done in the department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh after the collection of plant materials.
Collection of experimental rats: For the purpose of subacute toxicity studies, 12 Long Evan’s male rats of about five weeks old weighing 120-140 g were collected from the Animal Branch of International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B).
Maintenance of the rats: The rats were kept in properly numbered iron cages individually in a hygienic animal house with an optimum temperature (25-30°C) and were given standard laboratory diet. The animals were maintained in this way for 15 days before drug administration and continued up to the end of the experiment.
Grouping of the rats: Individual weight of the rats was taken and they were grouped into three. The rats of group B (4 rats, average weight 56 g) and C (4 rats, average weight 58 g) were used for experiment while those of group A were used as control (4 rats, average weight 51 g).
Administration of the sample: The compounds 1 and 2 were dissolved separately in distilled water using tween-20 as co-solvent, so that 0.3 mL contained 300 μg of the compound. The rats in group A, B and C were injected intraperitoneally with vehicle (300 μL isotonic solution), compound 1 and 2, respectively at a dose of 300 μg/day/rat for 14 consecutive days. On the 15th day blood was collected from external jugular vein under mild ether anaesthesia for the estimation of haematological and biochemical parameters. Then all the rats were sacrificed and liver, kidney, heart and lung were removed for histopathological study.
Gross general observation: During the whole experiment period their behaviour, CNS excitation and depression, reflexes, muscular weakness, salivation, diarrhoea and food intake were observed. The body weight of each rat of groups A, B and C were measured before drug administration, after completion of the treatment and prior to sacrificing the rats.
Investigation of haematological profiles: The haematological parameters like Total Count (TC) of RBC and WBC, Differential Count (DC) of WBC, platelet count and haemoglobin percentage were performed just before drug intake and also on 14th day pf drug administration. For TC, DC and platelet count blood was drawn from the tail veins of the experimental and the control rats of either groups and blood smears were made on glass slides followed by staining with Leisman reagent. Blood was also drawn from each rat with the help of capillary tube for estimating the haemoglobin percentage by Van Kampen-Ziftra’s method.
Biochemical parameters of blood: To determine biochemical parameters such as SGOT (Serum Glutamate Oxaloacetate Transaminase), SGPT (Serum Glutamate Pyruvate Transaminase), SALP (Serum Alkaline Phosphatase), serum bilirubin, creatinine and urea blood samples were collected from each rat of either group from their throat vein while they were sacrificed after 14 days of drug administration. The samples were analysed using the usual procedures.
Histopathological investigation: For histopathological investigation, the liver, kidney, lung and heart of all experimental and control rats were isolated after sacrificing them at the end of 14 days of drug administration. These tissues were separately sliced in pieces, fixed in 10% formaline for 3 days, processed, stained, mounted on glass slides and observed under power microscope.