Leishmanicidal and cytotoxicity assays

Leishmania parasites were resuspended with the corresponding drug concentration at 2 × 10⁶ parasites/mL (final concentration) in their respective growth media in 96 microwell plates (200 µL/well). The parasites were allowed to growth for 72 h at 26 °C for promastigotes and 96 h at 32 °C for axenic amastigotes. Afterwards, inhibition of proliferation was measured by the inhibition of MTT reduction by the parasites. To this end, MTT (3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well (0.5 mg/mL, final concentration). MTT reduction was allowed to proceed for 2 h at the respective temperature, and the resulting formazan solubilised by addition of 50 µL/well of 10% SDS. Afterwards, the plate was read at 595 nm in a BioRad Microplate-reader, model 680.

For peritoneal macrophages, the cells were seeded in 96 microwell plates at 1 × 10⁵ cells/well and incubated (37 °C, 5% CO₂) with the drug for 48 h. The toxicity was evaluated by the inhibition of MTT reduction as above.

Samples were made by triplicate and assays were repeated at least twice. EC₅₀ (effective concentration capable that inhibits parasite growth by 50%) was calculated using the statistical module of SigmaPlot v11.0 software.