Plant Materials The plants and their parts to be used for this study were selected based on their traditional uses. The fresh plant parts were collected from different areas of Bangladesh. Then the plant parts were kept under a shed till dried. The plant was identified by experts in Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh.
Preparation of Ethanolic Extract The different plant parts of three medicinal plants were freed from any of the foreign materials. Then the plant materials were chopped and air-dried under shed temperature followed by drying in an electric oven at 40º C. The dried plant materials were then ground into powder. About 100g of powdered material was taken in a clean, flat-bottomed glass container and soaked in 400 ml of 96% ethanol. The container with its contents was sealed and kept for a period of 4 days accompanying occasional shaking and stirring. The ethanolic extract was filtered by Buchner funnel and the filtrate was concentrated with rotary evaporator at bath temperature not exceeding 40? to have gummy concentrate of deep greenish-black extract (yield: D. blancoi 5.92%; A. nilotica 12.01%; Hibiscuss sabdariffa seed 4.68% and Hibiscuss sabdariffa calyxes 6.35%).
Test for Different Chemical Groups The crude ethanolic extract was tested for its different chemical groups as alkaloids, flavonoids; reducing sugars, saponins, steroids and tannins.14 In each test 10% (w/v) solution of the extract in ethanol was taken.
Test Animals and Drugs Young Swiss-albino mice either sex, 3-4 weeks of age, weighing 20 -25 g, were used for in vivo pharmacological screening. Mice were purchased from the Animal Research Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR,B). They have housed Pharmacology Laboratories, Pharmacy discipline, Khulna University, Khulna. Animals were maintained under standard environmental conditions (temperature: (24.0±1.0°C), relative humidity: 55-65% and 12hrs light/12 hrs dark cycle) and had free access to feed and water ad libitum. The cages were cleaned once daily. These studies were carried out following approval from the ethical committee comprising pharmacologist and toxicologist expert on the use and care of animals of Pharmacy discipline, Khulna University, Khulna.
The standard drug loperamide was used for antidiarrhoeal activity testing and the drug was purchased from Square Pharmaceuticals Ltd, Bangladesh.
Antidiarrhoeal Activity Antidiarrhoeal activity was tested by using Castor oil induced method in mice15, 16. Twenty Swiss albino mice were randomly divided in to four groups (n=5). Control group received only distilled water 2ml/mice, positive control group received loperamide 3mg/kg body weight as standard and test groups received the extracts at the doses of 250mg and 500mg/kg body weight. Mice were housed in separate cages having paper placed below for collection of fecal matters. Diarrhea was induced in the mice by oral administration of castor oil (1.0ml/mice). Extract and drugs were given orally 1hr before the administration of castor oil. The time for first excretion of feces and the total number of fecal output by the animals were recorded. Normal stool was considered as numerical value 1 and watery stool as numerical value 2. Percent inhibition of defecation in mice was calculated by using the following equation: % inhibition = {(Mo–M)/Mo}x100; where, Mo = Mean defecation of control and M = Mean defecation of test sample
Statistical Analysis Data were presented as mean ± Standard Error Mean (S. E. M). Statistical analysis for animal experiment was carried out using one-way ANOVA followed by Dunnet’s multiple comparisons. The results obtained were compared with the control group. p values < 0.01 were considered to be statistically significant (p indicates probability).