Mosquito larvicidal assay

Mosquito larvicidal assay was carried out following standard procedure described by WHO [18] and adopted by Ndung’u et al. [19–21] with an exception of the solvents used, dimethyl sulfoxide (DMSO, 99.9%, Sigma-Aldrich), instead of acetone. Briefly, 1 ml standard w/v of each test material or treatment in DMSO was made up to 20 ml with distilled water in 100 ml beakers in three replicates. Azadirachtin, a potent anti-insect naturally occurring limonoid [22], previously isolated and characterized in our laboratory from neem Azadirachta indica [20] and used as a positive control was similarly prepared in DMSO (1 ml). Twenty late third-fourthinstar larvae each were transferred into the test and control solutions, and larval mortality was monitored and recorded for up to 96 h. Dead larvae were removed from each treatment daily (after 24 h). The room temperature was maintained at 25–27 °C and larvae in each treatment were fed daily with approximately 1 mg of Tetramin fish food (Melle, Germany).

For the assays, we prepared a stock solution of 10 mg/ml by dissolving 40 mg of crude extract, fractions 3 and 4, and compound 1 in 4 ml of dimethyl sulfoxide (DMSO) and 2.5 mg of compound 4 in 0.25 ml DMSO (due to limited material available). From the stock solution, five concentrations of 0.1, 0.01, 0.005, 0.0025 and 0.001 mg/l (corresponding to 100, 10, 5, 2.5 and 1 ppm, respectively) for the crude extract, fractions 3 and 4 and compound 1, and only three concentrations of 0.01, 0.0025 and 0.001 mg/ml (corresponding to 5, 2.5 and 1 ppm, respectively) for compound 4.