2.1 Collection of Plant Materials The whole plant of V. cinerea was collected from the Bhatiyari and Pahartoli, Chittagong, Bangladesh in June, 2012. The plant was identified by the experts of Bangladesh Forest Research Institute Herbarium, Chittagong where voucher specimen has been deposited.
2.1.1 Drying and grinding: After collection, the whole plant was washed with running tap water and dried at room temperature not exceeding 50ºC. The dry materials were ground into a coarse powder with the help of a grinder and kept in airtight container in a cool and dark place until extraction commenced.
2.1.2 Hot extraction by soxhlet extractorAbout 130gm of powder was subjected to hot extraction with 700ml of methanol (99.98%) with a Soxhlet apparatus (Quickfit, England). A gummy residue (yield 16.70%) was obtained after the evaporation of the plant extract with a rotary evaporator (Heidolph, Germany) under reduced temperature and pressure.
2.1.3 ChemicalsStandard drugs such as paracetamol, diclofenac sodium, acetyl salicylic acid were obtained from Square Pharmaceuticals Ltd as gift samples. Solvents used in this experiment were of analytical grade and purchased from Merck, Germany.
2.1.4 Experimental animals: For the experiment Swiss albino mice of either sex, 6-7 weeks of age, weighing between 25-30g, were collected from the Animal Resources Branch of the International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR,B). The mice were maintained under standard environmental conditions of temperature: (27.0±1.0ºC), relative humidity: 55-65% and 12h light/12hr dark cycle and had free access to ICDDR,B formulated diet and water ad libitum. Appropriate measures were taken to minimize the pain or discomfort of animals and the mice were acclimatized to laboratory condition for one week prior to experiments. All protocols for animal experiment were approved by the institutional animal ethical committee.
2.2 Preliminary Phytochemical Investigation For preliminary phytochemical investigation, the crude methanol extract of V. cinerea was subjected to various tests to determine the chemical nature of the extract. The presence of alkaloid content was determined by performing Mayer’s test; white precipitate (ppt) indicated the presence of alkaloids. The formation of intense yellow coloration upon the addition of few drops of sodium hydroxide and the subsequent loss in color upon the addition of dilute acetic acid indicated the presence of flavonoids. The existence of glycoside in the sample was identified by performing Salkowski’s test as well as Libermann-burchard’s test; Orange-reddish color at the junction of 2 layers confirms the presence of glycosides. The presence of both the reducing sugar and gums were confirmed by Fehling’s test and Molisch’s reagent, respectively.
2.3 Test for Antipyretic Activity The crude extract of V. cinerea was subjected to test for the antipyretic activity using Swiss albino mice (25–30g) of either sex. Before the start of experiment the selected healthy animals were acclimatized to laboratory conditions. The animals were randomized into three groups, each group containing seven mice. The normal body temperature of each mouse was recorded using digital clinical thermometer and then by injecting 20% aqueous suspension of Brewer’s yeast (10ml/kg, s.c.) pyrexia was induced in all mice [17]. All groups were fast overnight but free access to drinking water was provided. After 24h rectal temperature of each mouse was recorded again. The induction of pyrexia was confirmed by rise in temperature of more than 32.9ºF, while animals showing less than 32.9ºF rise of temperature were excluded from experiment. Group-I received saline (10ml/kg) as a negative control, group-II received paracetamol (150mg/kg) as a standard drug while the remaining group-III received 500mg/kg body weight of the plant extract, respectively. Rectal temperature was recorded periodically at 1, 2 and 3hr after drugs administration.
2.4 Test for Analgesic Activity The analgesic activity of the crude extract was evaluated using formalin-induced writhing method in mice. Experimental animals (Swiss albino mice) were randomly selected and divided into three groups denoted as group-I, group-II, and group-III consisting of 7 mice in each group. Each group received a particular treatment i.e. control, standard and the two doses of the extract. Test samples (about 200 and 400mg/kg body weight of the plant extract), control and diclofenac sodium were given orally by means of a feeding needle. An interval of thirty minutes was given to ensure proper absorption of the administered substances. Then the writhing inducing chemical, formalin solution (5%) was administered intraperitoneally to each of the animals of all groups. After an interval of 10mins, which was given for absorption of formalin, number of squirms (writhing) was counted for 5mins.
2.5 Test for Anti-inflammatory Activity To determine the anti-inflammatory activity of the methanol extract of V. cinerea, 15 clean centrifuge tubes (three for positive control, acetyl salicylic acid, three for negative control, 99.8% ethanol and nine for crude extract) were used. 1.0ml of 5% egg albumin solution was added to all test tubes. Later on 1ml of acetyl salicylic acid (0.1mg), 1ml of methanol and 1ml of crude extract (1000mg/kg) were added to the positive, negative control and test groups, respectively. The pH (5.6±0.2) of all the reaction mixtures was adjusted by 1NHCl. These were heated, cooled and after filtration, the absorbance was measured spectrophotometrically at 660nm.
3. STATISTICAL ANALYSIS Results are expressed as the mean±SEM. Statistical analysis for the animal experiments was carried out using one-way ANOVA followed by Dunnett’s multiple comparisons. The results obtained were compared with the vehicle control group; p=0.05 was considered as statistically significant.