Sample collection and identification: The plant Hygrophila spinosa was collected from Bankra, Jesssore, Bangladesh during June 2009. After collection the plants were washed by water to remove soils and dusts, identified by Bangladesh National Herbarium, Mirpur; Dhaka DACB Accession No: 34474 and a voucher specimen also deposited there.
Preparation of extract: The collected plants were shade-dried for four week. The plants were ground into a coarse powder with the help of a suitable grinder (Capacitor start motor, Wuhu motor factory, China). The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. About 150 gm of powdered material was taken in a clean, flat-bottomed glass container and soaked in 800 ml of 95% 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 obtain is then evaporated by rotary evaporator.
Animals: Mice of random sex (Swiss-webstar strain, 20-25 g body weight) collected from animal resources branch of the International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) were used for the experiments. The animals were kept at animal house (Pharmacy Discipline, Khulna University, Khulna) for adaptation after collection under standard laboratory conditions (relative humidity 55-65%, room temperature 25 +2 0 C and 12 hour light: dark cycle) and fed with standard diets (ICDDR, B formulated) and had free access to tap water.
Chemicals: Diclofenac-Na and loperamide were provided by Square Pharmaceuticals Ltd. Bagladesh. Molisch’s, Mayer’s, Dragendroff’s, Fehling’s and Benedict’s reagent.
Phytochemical tests: Small amount of dried extract was appropriately treated to prepare sample solution and then subjected to the specific phytochemical tests (Ghani, 2003). LibermannBurchard test was performed to identify steroids. Mayer’s reagent and Dragendroff’s reagent test was performed to identify alkaloids. Ferric chloride test was performed to identify tannin. Molisch’s test, Fehling’s test and Benedict’s test were performed to investigate the presence of reducing sugar. For saponin, flavonoid and glycosides general identifying test were performed (Evans, 1989).
Determination of analgesic activity: The method of Whittle (1964) and Ahmed et al. (2004) was adopted with minor modification. The experimental animals were randomly divided into four groups, each consisting of five animals. Group I was treated as 'control group' which received 1% (v/v) Tween-80 in water at the dose of 10 mL/kg of body weight; group II was treated as 'positive control' and was given the standard drug diclofenac sodium at the dose of 25 mg/kg of body weight; group III and group IV were test groups and were treated with the extract of Hygrophila spinosa at the doses of 250 and 500 mg/kg of body weight respectively. Control vehicle, standard drug and extracts were administered orally, 30 minutes prior to the intraperitoneal injection of 0.7% acetic acid; after an interval of 15 minutes, the number of writhes (squirms) was counted for 5 minutes. The number of writhings in the control was taken as 100% and percent inhibition was calculated as follow:
% Inhibition of writhing = 100 – (treated mean / control mean) × 100
Antidiarrhoeal Activity: The method of Chatterjee (1993) with minor modification was adapted to study the Antidiarrhoeal activity of the ethanolic extract of Hygrophila spinosa using castor oil induced diarrhoea in mice. 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 four groups having five mice in each. Group-I was kept as control and received 1% Tween-80 at the dose of 10 mL/kg of body weight; group-II was ‘positive control’ and was treated with standard antimotility drug loperamide at the dose of 50 mg/kg of body weight. Group III and Group IV were ‘test group’ and treated with the extract at the doses of 250 and 500 mg /kg of body weight respectively. Control vehicle and the extract were administered orally, 40 min prior to the oral administration of castor oil. Individual animals of each group were placed in separate cages having adsorbent paper beneath and examined for the presence of diarrhoea every hour in five hours study after the castor oil administration. Number of stools and any fluid material that stained the adsorbent paper were counted at each successive hour during the experiment (5 hour). The latent period of each mouse was also counted. At the beginning of each hour new papers were placed for the old ones.