Plant material: The leaves of Cymbidium aloifolium were collected from Dhaka in December 2010 and were identified by the experts of national herbarium, Mirpur, Dhaka, Bangladesh (Accession No. DACB-35414) and a voucher specimen was kept for future reference.
Preparation of the extract: The leaves were thoroughly washed with water. The collected leaves were cut into small pieces and dried in sun for 15 days. The dried small pieces of the plant leaves were ground into small powder by blender machine. Then the powders were preserved in separate airtight container for further use. The plant powders (100 g) were extracted by cold extraction process using ethanol (600 mL) as solvent in a bottom glass container, through occasional shaking and stirring for 7 days. After 7 days the extract filtered through the cotton at first and then through the filter paper. Then the liquid was dried with rotary evaporator to achieve a blackish mass.
Animals: Swiss albino mice (20-30 g) of either sex were purchased from the Animal Research Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). The animals were housed under standard laboratory conditions. The animals were fed with laboratory food and water ad libitum. The experiments were done on an isolated and noiseless condition. The ethical clearance of the experiment was taken from the “Internal Ethical Board For Animal Research” of East West University, Dhaka, Bangladesh.
Drugs and chemicals: Acetic acid and Carrageenan were obtained from Merck, Germany. Tween-80 was obtained from BDH Chemicals, UK. Normal saline solution was purchased from Beximco Infusion Ltd., Bangladesh. Diclofenac was obtained from Square Pharmaceuticals Ltd., Bangladesh.
Experimental design: The animals were randomly divided into four groups and each group consisting of six mice. The test groups received ELECA (ethanolic leaf extract of cymbidium aloifolium) at the doses of 200 and 400 mg kg-1 while positive control was treated with Diclofenac Na (10 mg kg-1) and control with vehicle (1% Tween 80 in water).
Procedure
Acetic acid induced writhing test in mice: To evaluate the analgesic effects of Cymbidium aloifolium, the method described by Dharmasiri was used with slight modifications. Four groups of six mice each received orally normal saline solution (10 mL kg-1) (i.e., control); Diclofenac (10 mg kg-1) and plant extract (200 and 400 mg kg-1). Forty minutes later, the mice were treated with 0.7% acetic acid (10 mL kg-1) solution was injected intraperitoneally. Six minutes after acetic acid injection, mice were placed in individual cage and the number of writhes (abdominal contractions) was counted for each mouse for a period of 10 min and the percentage inhibition of writhing was calculated.
Carrageenan-induced paw oedema: The method described by Winter was slightly modified and adopted to determine anti-inflammatory activity in mice. The mice were divided into four groups each containing six mice each. Acute inflammation was produced by injecting 0.1 μL of carrageenan (1% w/v suspension which contained Tween 80) into plantar surface of mice hind paw. The ethanolic leaf extract (200 and 400 mg kg-1), normal saline (1 mL kg-1) and Diclofenac (10 mg kg-1) as reference agent were given 1 h before carrageenan injection. The paw volume was measured at 30 min before and 3 h after carrageenan injection using a vernier caliper to determine the linear diameter of paw oedema. The difference between the readings at thirty mins before and three hours after the injection of carrageenan was taken as the thickness of oedema. The percent inhibition of the inflammatory reaction was determined for each animal by comparing with control and calculated by the formula.
where, dt is the difference in paw volume of the treated group and dc is the difference in paw volume in the control group.
Statistical analysis: The data were expressed as the Mean±SEM (standard error mean). ANOVA (analysis of variance) followed by Dunnett’s ‘t’ test was performed as a post hoc test to evaluate the statistical significance while taking vehicle treated animals as control; p value of p<0.05 was considered as statistically significant.