Plant material collection and extraction: The seeds of H. suaveolens were collected from Khulna, Bangladesh and were identified by the experts at Bangladesh National Herbarium, Mirpur, Dhaka (Accession No. DACB- 33879). About 100 gm of powdered material was taken in a clean, flat bottomed glass container and drenched in 500 ml of 90% ethanol. The container with its contents was sealed and kept for a period of 7 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material. The filtrate thus obtained was evaporated in the open air to get a viscous mass. The viscous mass was then dried to get crude ethanolic extract (yield: 10% w/w). The extract thus obtained was used for experimental purposes.
Drugs: Diclofenac sodium (Drug International Ltd.), Loperamide (Drug International Ltd.) and Chloramphenicol (Square Pharmaceuticals Ltd.).
Preliminary phytochemical screening: The ethanolic extract of the seeds was subjected to preliminary phytochemical test for the detection of major chemical groups. In each test 10% (w/v) solution of extract in ethanol was taken unless otherwise mentioned in individual test (Evans, 1989; Wagner et al., 1984, Ghani, 2003).
Animals: The experimental animals, Swiss-albino mice of either sex (weighing 20-25 g) were purchased from the Animal Research Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). The animals were kept at animal house (Pharmacy Discipline, Khulna University) for adaptation after their purchase under standard laboratory conditions (relative humidity 55- 65%, room temperature 21.0 ± 2.0 °C and 12 h light/dark cycle) and fed with standard diets and had free access to tap water.
Analgesic activity: Analgesic activity of the ethanolic extract of the seeds of H. suaveolens was tested using the model of acetic acid induced writhing in mice (Whittle, 1964; Ahmed et al., 2004). Experimental animals were randomly selected and divided into four groups denoted as group-I, group-II, group-III, group- IV consisting of 5 mice in each group. Group I was treated as ‘control group’ which received 1% (v/v) Tween-80 in water at a dose of 10 mg/kg of body weight; group II was treated as ‘positive control’ and given the standard drug diclofenac sodium at a dose of 25mg/kg of body weight; group III and group IV were test groups and treated with the extract at the doses of 250 and 500 mg/kg of body weight respectively. Control vehicle, standard drug and extract were administered orally, 30 minutes prior to acetic acid (0.7%) intraperitoneal injection. After an interval of 5 minutes, the number of wriths (squirms) was counted for 15 minutes.
Antidiarrhoeal Activity: Antidiarrhoeal activity of H. suaveolens was tested by using the model of castor oil induced diarrhoea in mice (Chatterjee, 1993). The mice were all screened initially by giving 0.5 ml of castor oil and only those showing diarrhoea were selected for the final experiment. The test animals were randomly chosen and divided into three groups having five mice in each. Group-I was kept as ‘control’ and received only distilled water containing 1% Tween-80; group-II was treated as 'positive control' and received standard antimotility drug loperamide at a dose of 50 mg/ kg; group III was 'test group' and treated with EtOH extract at the dose of 500 mg/kg. Control vehicle, standard drug and the extract were administered orally, 1 h prior to the oral administration of castor oil at a dose of 0.5 ml per mouse. Individual animals of each group were placed in separate cages having adsorbent paper beneath and examined for the presence of diarrhoea every hour in 5 h study after the castor oil administration. Number of stools or any fluid material that stained the adsorbent paper was counted at each successive hour during the experiment (5 h). The latent period of each mouse was also counted. At the beginning of each hour new papers were placed for the old ones.
Cytotoxic activity: The method of Meyer et al. (1982) with some modifications was adapted to study the general toxicity of seeds of H. suaveolens. The brine shrimp eggs were hatched in a conical flask containing brine shrimp medium (300 ml). The flasks were well aerated with the aid of an air pump, and kept in a water bath at 29 – 30o C. A bright light was left on. The nauplii hatched within 48 h. The extract was dissolved in brine shrimp medium with addition of few drops of 5% DMSO to obtain a concentration of 5, 10, 20, 40, 80 and 160 µg/ml. Each preparation was dispensed into clean test tubes in 10 ml volumes and tested in duplicates. For control, same procedure was followed except test samples. A series of the same concentration as of the sample was prepared for positive control, chloramphenicol (Rahman et al., 2007). After marking the test tubes properly, 10 living shrimps were added to each of the test tubes with the help of a Pasteur pipette. The test tubes containing the sample, control and positive control were then incubated at 29°C for 24 h in a water bath, after which each tube was examined and the surviving brine shrimps counted and recorded. From this, the percentage of mortality was calculated at each concentration. The LC50 values were calculated with best fit line by using Microsof Excel 2007.