2.1 Collection and Identification of the Plant Material Fresh bark of P. rubra was collected from Jahangirnagar University residential area, Savar, Dhaka, Bangladesh. The plant was identified by the experts of Bangladesh National Herbarium (BNH). The specimen was preserved in BNH and Department of Pharmacy, North South University, Bangladesh and it has been assigned the accession number of DACB 35518.
2.2 Preparation of Extracts The plant bark was kept under sunshade for 5 days and then heated in an oven at below 40ºC for 24 hours to be fully dried. After drying, it was ground thoroughly to powdered form and was stored in cold conditions in an airtight container. The powder obtained was extracted via the method of cold extraction using ethanol and then kept for a period of 5 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material followed by a second filtration through whatman filter paper. The filtrate (ethanol extract) obtained was evaporated by rotary evaporator (Bibby RE-200, Sterilin Ltd., UK) at 5 to 6 rpm and at 68ºC temperature. It rendered a gummy concentrate of dark greenish black colour that was designated as crude ethanolic extract. The extract was finally dried by freeze drier and preserved.
2.3 Animal Used Young Swiss-Albino mice aged about 4-5 weeks with average weight of 25-35 gm and adult Long Evans Rats of either sex having average weight of 100-130 gm were used for the experiment and maintained in the animal house of the Department of Pharmacy, North South University for acclimation. The animals were originally obtained from International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B). They were housed in standard cages under standard environmental conditions of room temperature at 24 ± 1ºC and 55-65% relative humidity with 12 hour dark light cycle and provided with standard food for rodents and water ad libitum. All experiments involving animals were conducted according to the UK Home Office regulations (UK Animals Scientific Procedures Act 1986) and the ‘Principles of Laboratory Animal Care’ (National Institutes of Health publication no. 86-23, revised 1985).
2.4 Method for Phytochemical Analysis The freshly prepared extract of P. rubra was qualitatively tested for the presence of chemical constituents. Qualitative phytochemical tests for the identification of alkaloids, flavonoids, steroids, gum and carbohydrates, saponins, tannins and terpenoids were carried out for the extract by the method described previously.
2.5 Method for the Evaluation of Analgesic Effect
2.5.1 Hot plate test The hot-plate test (Hot/Cold Plate Model-35100-001, UGO Basile, Italy) was employed for measurement of analgesic activity as previously described by Lanhers et al. and modified by Ojewole. The temperature was regulated at 55º ± 1ºC. Mice of either sex were divided into four groups consisting of six animals in each group. The mice of each group were placed in the beaker (on the hot plate) in order to obtain its response to electrical heat induced pain stimulus. Licking of the paws or jumping out of the beaker was taken as an indicator of the animal’s response to heat-induced pain stimulus. The time for each mouse to lick its paws or jump out of the beaker was taken as reaction time (in second). Before treatment, the reaction time was taken once. The mean of this determination constituted initial reaction time before treatment of each group of mice. Each of the test mice was thereafter treated with either distilled water (DW), Diclofenac sodium (10 mg/kg BW) or ethanol extract of P. rubra at the doses of 250 and 500 mg/kg BW orally. Thirty minutes after treatment, the reaction times of each group mice were again evaluated five times individually in one hour interval on this occasion. Percent analgesic score was calculated as, PAS = Ta-Tb/Ta × 100
Where, Tb = Reaction time (in second) before drug administration; Ta = Reaction time (in second) after drug administration.
2.5.2 Acetic acid-induced writhing method The analgesic activity of the samples was evaluated using acetic acid induced writhing method in mice following the method of Koster et al. with slight modification. In this method, acetic acid is administered intraperitoneally to the experimental animals to create pain sensation. The animals were divided into four groups with six mice in each group. Group I animals received distilled water, Group II received Diclofenac sodium at 10 mg/kg while animals of Group III and Group IV were treated with 250 and 500 mg/kg of the ethanol extract of P. rubra 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. Animals were kept individually under glass jar for observation. Each mouse was observed individually for counting the number of writhing they made in 10 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 was used as a reference standard (positive control). The percentage inhibition of writhing was calculated as follows: % Inhibition = (1-VT/ VC) ×100 VT = number of writhing motions in drug-treated mice VC = number of writhing motions in the control group of mice.