Drugs and chemicals Acetic acid was obtained from Merck, Germany. Tween-80 was obtained from BDH Chemicals, UK. Normal saline solution was purchased from Beximco Infusion Ltd., Bangladesh. Diclofenac sodium was obtained from Square Pharmaceuticals Ltd., Bangladesh.
Plant material For this present investigation, the S. dulcis and F. racemosa were collected from Village: Milua, Thana: Doulatpur, District: Manikgong, Bangladesh in July 2008 and were identified at the Bangladesh National Herbarium, Mirpur, Dhaka where the Voucher specimen no: 32766 and 32767 for Scoparia dulcis L. and Ficus racemosa Linn. respectively were deposited. The collected plant parts were dried for one week and ground into a coarse powder with the help of a suitable grinder. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced.
Preparation of the extract About 180 gm of powdered material was taken in a clean, flat bottomed glass container and soaked in 500 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. Then it was filtered through Whatman filter paper (Bibby RE200, Sterilin Ltd., UK). The filtrate (ethanol extract) obtained was evaporated using rotary evaporator. It rendered a gummy concentrate of reddish black color. The gummy concentrate was designated as crude extract of ethanol. The extract was transferred to a closed container for further use and protection.
Animals Young Swiss-albino mice of either sex aged 4- 5 weeks, average weight 20-25 gm were used for the experiment. The mice were purchased from the animal Research Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDRB). They were kept in standard environmental condition (at 24.0±0°C temperature & 55-65% relative humidity and 12 hour light/12 hour dark cycle) for one week for acclimation after their purchase and fed ICDDRB formulated rodent food and water ad libitum. The set of rules followed for animal experiment were approved by the institutional animal ethical committee.
Phytochemical screening Phytochemical screening of the prepared extracts was conducted with various qualitative tests to identify the presence of chemical constituents. To perform the tests the following chemicals and reagents were used: Carbohydrates with Molisch’s test, glycoside with water and sodium hydroxide solution, saponins with the capability of producing suds, steroids with chloroform and sulphuric acid, flavonoids with Mg and HCl, tannins with ferric chloride solution, gum with Molish reagents and concentrated sulfuric acid. Alkaloids were tested with Mayer’s reagent, Hager’s reagent and Dagendorff’s reagent. These were identified by characteristic color changes using standard procedures.
Analgesic activity Hot plate method Experimental animals of either sex were randomly selected and divided into four groups designated as group-I, group-II, group-III and group-IV consisting of five mice in each group for control, positive control and test sample group respectively. Each group received a particular treatment i.e. control (1% Tween-80 solution in water, 10ml/kg, p.o.), positive control (Diclofenac sodium 10 mg/kg, p.o.) and the test sample (ethanolic extract of 100 mg/kg, p.o. & 200 mg/kg, p.o. respectively). The animals were positioned on Eddy’s hot plate kept at a temperature of 55±0.5 0C. A cut off period of 15 s (Franzotti et al., 2000) was observed to avoid damage to the paw. Reaction time was recorded when animals licked their fore or hind paws, or jumped prior to and 0, 30, 60 and 90 min after oral administration of the samples (Eddy et al., 1953; Kulkarni, 1999; Toma et al., 2003).
Acetic acid induced writhing method The analgesic activity of the samples was evaluated using acetic acid induced writhing method in mice (Ahmed et al., 2004). In this method, acetic acid is administered intraperitoneally to the experimental animals to create pain sensation. As a positive control, any standard NSAID drug can be used. In the present study Diclofenac sodium was used to serve the purpose. The plant extract was administered orally in two different doses (100 and 200 mg/kg body weight) to the Swiss Albino mice after an overnight fast. Test samples and vehicle were administered orally 30 minutes prior to intraperitoneal administration of 0.7% v/v acetic acid solution (0.1ml/10g) but Diclofenac sodium was administered 15 minutes prior to acetic acid injection. Then the animals were placed on an observation table. Each mouse of all groups were observed individually for counting the number of writhing they made in 15 minutes commencing just 5 minutes after the intraperitoneal administration of acetic acid solution. Full writhing was not always accomplished by the animal, because sometimes the animals started to give writhing but they did not complete it. This incomplete writhing was considered as half-writhing. Accordingly, two half-writhing were taken as one full writhing. The number of writhes in each treated group was compared to that of a control group while Diclofenac sodium (10 mg/kg) was used as a reference substance (positive control).
Statistical analysis The results of statistical analysis for animal experiment were expressed as mean ± SEM and were evaluated by ANOVA followed by Dunnet’s multiple comparisons. The results obtained were compared with the vehicle control group. The p<0.05, 0.001 were considered to be statistically significant.