3.5. Biology

Leishmania (Leishmania) donovani (MHOM/ET/1967/HU3) was provided by Oswaldo Cruz Institute Leishmania Collection-Fiocruz-RJ-Brazil. Promastigotes were maintained in vitro at 26 °C in Schneider’s medium, pH 7 (20% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2% of male urine), as previously described [29].

Briefly, promastigotes in the logarithmic growth phase were cultured in 96-well cell culture plates at 1 × 10⁶ parasites per well in 100 mL of Schneider's medium with increasing concentrations of 3.12, 6.25, 12.5, 25, 50, 100, 200, and 400 µg/mL of Morita-Baylis-Hillman adduct. The plates were incubated for 72 h in a biological oxygen demand (B.O.D.) incubator at 26 °C. Cytotoxicity to promastigotes was evaluated by the 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as described by Valadares et al. [30].The absorbance was measured using an ELISA plate reader (ELx800, BIOTEK) at 540 nm. Amphotericin^® was used as a positive control. The viability of promastigotes incubated in the presence of different concentrations of adducts was determined by comparing to a culture control (L. donovani cultivated in Schneider only). The concentration that caused a 50% reduction in cell viability (IC₅₀) was calculated by Probit analysis (SPSS 13.0 for Windows). Each experiment was performed in duplicate and repeated at least three times. Haemolytic assays (HC₅₀ > 400 μg·L^–1) towards human red blood cells were performed as described in the literature [30].