Evaluating Anthelmintic Activity of Extracts

This was done to eliminate inactive extracts. Adult worm assays were conducted in 24-well plates (NUNC, USA) at 37°C in humidified air containing 5% CO₂ for 5 days (120 hours) without change of medium. Nodular masses (each generally containing a few males and a female worm) were first put in the wells (with 2 mL CCM) without extract overnight to confirm their viability. The crude extracts were then added in quadruplicate wells at a single final concentration of 500 μg/mL by substituting 1 mL of the medium in the well with 1 mL of CCM containing 1000 μg/mL of extract. Six nodular masses each, were used in the negative (2% DMSO only) and in the positive (amocarzine-CGP 6140, 10 μg/mL) control wells in which each well received only one nodular mass. Adult worm viability was assessed by the MTT/formazan assay in which each nodular mass was placed under sterile conditions in a well of a 96-well microtitre plate containing 200 μL/well of 0.5 mg/mL MTT (Sigma, USA) in phosphate buffered saline (PBS), and then incubated under the culture conditions. Adult worm viability was taken as least % inhibition of formazan formation relative to negative control at 120 hours post addition of drug. An extract was considered active if there was a 90% or greater inhibition of formazan formation compared to the negative controls; or moderately active if the inhibition was 50 - 89%. It was considered inactive if the inhibition was less than 50%.