Omar Ali Mondal
Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
K. A. M. S. H. Mondal
Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh.
Nurul Islam
Department of Zoology, University of Rajshahi, Rajshahi-6205, Bangladesh.
Ataur Rahman Khan
Department of Zoology, University of Rajshahi, Rajshahi-6205, Bangladesh.
Derris indica, Cytotoxicity, Artemia salina nauplii, Phytotoxicity, Mustard seeds.
Institute of Biological Sciences, University of Rajshahi
Pest Management
Preparation of plant materials for extraction: The fresh leaves, fruit shell, root bark, root wood, seeds, stem bark, and stem wood of D. indica were collected from the campus of the University of Rajshahi, Bangladesh. After drying under shade the plant materials were powdered in a grinder machine. Chemical extraction of the collected materials: Chloroform was selected as a solvent to extract seven different parts of D. indica separately. The ground dried materials, viz. leaves, fruit shell, root bark, root wood, seeds, stem bark, and stem-wood were extracted with sufficient amounts of chloroform (500g x 1500m1 x 3 times) for each of the items. Separate extracts were collected by the cool method after 72 hours of plunging for each of the plant parts. Extracts were subjected filtration and evaporation of the solvent. The residues were kept in a refrigerator after proper labeling. Since the lethality test involves the culture of brine shrimp nauplii, i.e., the nauplii should be grown in water with salinity similar to that of sea water, while the seawater contains 3.8% sodium chloride. Accordingly, a 3.8% sodium chloride solution was prepared by dissolving 38 gm sodium chloride in 1000 ml distilled water. The PH of the brine water thus prepared was maintained between 8 and 9 using NaHCO3. Brine water was taken in a small tank and shrimp eggs (1.5 gm/L) were added to one side of the perforated tank with a constant oxygen supply. A constant temperature (37ºC) and sufficient light were maintained. After 48 hours, shrimp nauplii were collected and used for the experiment. For the seed germination test, fresh and healthy mustard seeds were collected from the market. Cytotoxicity test: Preparation and application of doses on A. salina Chloroform extracts of the D. indica samples were applied against the brine shrimp nauplii. For the fruit shell, leaves, root bark, root wood, stem bark and stem wood samples 4 mg were initially dissolved in 200μl of pure dimethylsulfoxide (DMSO) to make them hydrophilic before adding 19.98 ml of water to get a concentration of 200 ppm for each of the samples separately which were used as stock solutions for all the extracts and from these concentrations other successive doses were prepared separately for each of the extracts through the serial dilution method. A series of concentrations, e.g. 200, 100, 50, 25, 12.5 and 6.25 ppm were prepared for the extracts separately. However, for the seed extract 2 mg was initially dissolved in 100 μl of DMSO to make it hydrophilic before adding 19.98ml of water to get a concentration of 100 ppm which was used as the stock solution for the seed extract. The following concentrations were made from the stock solution: 100, 50, 25, 12.5, 6.25, 3.125 and 1.563 ppm. Brine shrimp eggs were hatched in simulated seawater to get nauplii. Test samples were prepared by the addition of the requisite amounts of DMSO for obtaining desired concentrations of the test sample. The nauplii were counted by visual inspection and were taken in vials containing 5ml of brine water. Then samples of different concentrations were added to the pre-marked vials with the help of a micropipette. The vials were left for 24 hours and then the nauplii were counted again to find out the cytotoxicity of the test agents and compared to the results with positive control. Preparation and application of doses on mustard seeds: In this experiment 4 doses from the fruit shell, leaves, root bark, root wood, seed, stem bark and stem wood extracts of D. indica were made as 1 mg, 0.75 mg, 0.50 mg and 0.25 mg/ml freshwater. Because of insolubility of the extract in water it was needed to add 100μl DMSO with the weighed extract before mixing with water. For application of doses a number of petridishes 60mm diam. were used. Filter papers were placed inside the petridishes and doses were applied separately. Five mustard seeds were put in every petridish and three replications were set for each concentration and a control with three replications was also maintained. All the petridishes were kept covered to avoid drying. The humid condition inside the petridishes helped the seeds to germinate. Then the petridishes were placed in a safe place with plenty of light and air. Germination (%) was carefully recorded at various concentrations of different extracts of D. indica. Collection and analysis of data for cytotoxicity The test tubes containing the nauplii with the treated brine water were kept on a rack near the window in the laboratory. The recorded mortality was corrected by the Abbott’s (1925) formula.
Univ. j. zool. Rajshahi Univ. Vol. 31, 2012 pp. 59-64 ISSN 1023-6104
Journal