Biological assay

The insecticidal activity of 1-7, 3a-g, 4a-c, 5a, 5c-h and 6a, 6b was tested as the mortality rate values by using the leaf-dipping method against the pre-third-instar larvae of M. separata using the reported procedure³². For each sample, a total of 24 pre-third-instar larvae (6 larvae per group) were used. Each treatment was performed four times. Acetone solutions of 1-7, 3a-g, 4a-c, 5a, 5c-h, 6a, 6b and toosendanin (positive control) were prepared at 1 mg/mL. Fresh wheat leaf discs (1 × 1 cm) were dipped into the corresponding solution for 3 s, then taken out and dried. Leaf discs treated with acetone alone were used as a blank control group. Several pieces of treated leaf discs were kept in each 6 well plate which was then placed in a conditioned room (25 ± 2 °C, 65–80% relative humidity (RH), 12 h/12 h (light/dark). Once the treated leaves were consumed, the corresponding ones were added to the dish. After 2 days, untreated fresh leaves were added to the all dish until the adult pupae emergence. The corrected mortality rates of the tested compounds against M. separata Walker were calculated in three different periods by the following formula: