Chemicals and drugs: DPPH (1, 1-diphenyl, 2-picryl hydrazyl), was obtained from Sigma chemical co. USA. Ascorbic acid was obtained from SD Fine chem. Ltd., Biosar, India. Naphthyl ethylene diamine dihydrochloride was purchased from Roch-light Ltd., Suffolk, England. Sodium nitro prusside was obtained from Ranbaxy Lab., Mohali, India and potassium ferricyanide from May and Backer, Dagenham, UK. Diclofenac-Na was collected from Square Pharmaceuticals Ltd., Bangladesh and Nalbuphine was from Incepta Pharmaceuticals Ltd., Bangladesh.
Plant material: The whole plant with leaves, stems and roots was collected from Savar, Dhaka in February 2008 and was identified by Prof. Dr. Abdul Ghani, Stamford University Bangladesh. The plant was thoroughly washed with water; roots and stems were discarded and the leaves were dried in hot air woven at 55°C for 3 days and at 40°C for the next 4 days.
Extraction: The dried leaves were coarsely powdered and extracted with a mixture of methanol: water (7:3, v/v) by a Soxhlet apparatus at 50°C. The solvent was completely removed and obtained dried crude extract which was used for investigation.
Animal: For the experiment Swiss albino mice of either sex, 3-4 weeks of age, weighing between 20-25 g, were collected from the animal research branch of the International Center for Diarrheal Disease and Research, Bangladesh (ICDDRB). Animals were maintained under standard environmental conditions (temperature: (24.0±1.0°), relative humidity: 55-65% and 12 h light/12 h dark cycle) and had free access to feed and water adlibitum. The animals were acclimatized to laboratory condition for one week prior to experiments. All protocols for animal experiment were approved by the institutional animal ethical committee.
Phyto-chemical screening: The freshly prepared crude extract was qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extract was performed using the following reagents and chemicals: Alkaloids with Dragendorff’s reagent, flavonoids with the use of Mg and HCl; tannins with ferric chloride and potassium dichromate solutions and saponins with ability to produce stable foam and steroids with Libermann- Burchard reagent. Gum was tested using Molish reagent and concentrated sulfuric acid; reducing sugars with Benedict’s reagent. These were identified by characteristic color changes using standard procedures.
Analgesic screening:
Hot plate method: The animals were divided into four groups with five mice in each group. Group I animals received vehicle (1% Tween 80 in water, 10 mL kg-1 body weight), animals of Group II received Nalbuphine at 10 mg kg-1 body weight while animals of Group III and Group IV were treated with 250 and 500 mg kg-1 body weight (p.o.) of the crude extract of M. scandens. The animals were placed on Eddy’s hot plate kept at a temperature of 55±0.5°C. A cut off period of 15 s, 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.
Tail immersion test: The procedure is based on the observation that morphine like drugs selectively prolongs the reaction time of the typical tail withdrawal reflex in mice. The animals were treated as discussed above. From 1-2 cm of the tail of mice was immersed in warm water kept constant at 55°C. The reaction time was the time taken by the mice to deflect their tails. The first reading was discarded and the reaction time was recorded as a mean of the next three readings. A latency period of 20 s was defined as complete analgesia and