In vitro effect on Leishmania amazonensis amastigote stage.

Activity assay against intracellular amastigotes was performed according to Jain et al. (56). Macrophages (J774A.1 cell line) were placed in a 96-well flat-bottom plate at a density of 2 × 10⁵/ml in RPMI 1640 medium supplemented with 10% FBS and incubated for 1 h at 37°C in a 5% CO₂ environment. Additionally, 100 μl of stationary-phase promastigotes (7-day-old culture) was added in a 10:1 ratio, and plates were reincubated at 37°C overnight to allow a maximum infection. After incubation, free promastigotes were washed off with the culture medium at least 3 times. We added 50 μl of culture medium into each well. Subsequently, a serial dilution of test compounds was made in a 96-deep-well plate with the culture medium, and then 50 μl of this serially diluted standard were added to each well. The plates were incubated at 37°C, 5% CO₂, for 24 h. After incubation, the medium was removed, and 30 μl of Schneider’s medium (with 0.05% sodium dodecyl sulfate) was added to each well. Plates were shacked for 30 s, and 170 μl of medium were added to each well. alamarBlue at 10% was added into each well of the 96-well plates and incubated at 26°C for 72 h to allow transformation of rescued amastigotes to promastigotes. After incubation, the emitted fluorescence was measured in a PerkinElmer EnSpire spectrofluorometer at 585 nm.