Roots of Raphanus sativus were collected from a local market in Dhaka city, Bangladesh during November, 2013. The plant was taxonomically identified at the Bangladesh National Herbarium at Dhaka (Accession Number 38,620). The roots were air-dried in the shade, grounded into a fine powder and 150g of the powder was extracted with methanol (1:5, w/v) for 48 hours. The extract was evaporated to dryness at 40oC. The final weight of the extract was 24.3g.
Chemicals: Glacial acetic acid was obtained from Sigma Chemicals, USA; aspirin was obtained from Square Pharmaceuticals Ltd., Bangladesh.
Animals: In the present study, Swiss albino mice (male), which weighed between 13-15g were used. The animals were obtained from International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B). All animals were kept under ambient temperature with 12h light followed by a 12h dark cycle. The animals were acclimatized for three days prior to actual experiments. The study was conducted following approval by the Institutional Animal Ethical Committee of University of Development Alternative, Dhaka, Bangladesh.
Antinociceptive activity: Antinociceptive activity of the methanol extract of Raphanus sativus roots (MERSR) was examined using previously described procedures (Shanmugasundaram and Venkataraman, 2005). Briefly, mice were divided into seven groups of five mice each. Group 1 served as control and was administered vehicle only. Groups 2 and 3 were orally administered the standard antinociceptive drug aspirin at a dose of 200 and 400 mg per kg body weight, respectively. Groups 4-7 were administered methanol extract of Raphanus sativus roots (MERSR) at doses of 50, 100, 200 and 400 mg per kg body weight, respectively. Following a period of 60 minutes after oral administration of standard drug or extract, all mice were intraperitoneally injected with 1% acetic acid at a dose of 10 ml per kg body weight. A period of 15 minutes was given to each animal to ensure bio-availability of acetic acid, following which period, the number of writhings was counted for 10 min. The following formula was used for calculation of percent inhibition of the number of writhings in aspirin and MERSR administered animals compared to control mice, Percent inhibition = (1 – We /Wc) X 100 where We and Wc represent the number of writhings in aspirin or MERSR administered mice (Groups 2-7), and control mice (Group 1), respectively.
Preliminary phytochemical screening: Preliminary phytochemical analysis of MERSR for the presence of saponins, tannins, alkaloids, and flavonoids were conducted as described before.
Acute toxicity test: Acute toxicity test was conducted as previously described (Ganapaty et al., 2002). Mice were divided into nine groups, each group consisting of six animals. Group 1 was given 1% Tween 80 in normal saline (2 ml per kg body weight). The other eight groups (Groups 2-9) were administered, respectively, 100, 200, 300, 600, 800, 1000, 2000 and 3000 mg of MERSR per kg body weight. All animals were closely observed for the next 8 hours to notice any behavioral changes or mortality and were kept under close observation for the next two weeks.
Statistical analysis: Experimental values are expressed as mean ± SEM. Independent Sample t-test was carried out for statistical comparison. Statistical significance was considered to be indicated by a p value < 0.05 in all cases.