In vitro viability assay against epimastigotes

The effects of the extract and fractions on the viability of the epimastigote forms of the parasite were determined by a colorimetric assay using MTS/PMS. New cultures, containing 10⁶ parasites/mL, were added to the wells and incubated with the extract and fractions (at 50 µg mL^-1) for 72 h, and the extract and fractions that showed activity were evaluated in a subsequent assay at six different concentrations (50.0-1.06 µg mL^-1) to calculate the IC₅₀. All assays were performed in technical triplicate.

After incubation for 72 h, MTS/PMS solution was added (containing 400 μg mL^-1 and 9.2 μg mL^-1, respectively), and the plates were incubated at 28 °C for 4 h. Then, the optical density was read at 490 nm in a microplate reader (Asys Expert Plus; Biochrom).

Parallel tests were performed using 1 % dimethylsulfoxide (DMSO, negative control) and a replicate of each concentration, and then fixed with 4 % paraformaldehyde prior to the addition of MTS/PMS solution (basal absorbance control).