Cytotoxicity assay

The extracts were also tested for cytotoxicity against transformed human monocytic (THP1) cells. A 4 days’ old culture of THP1 cells in the experimental phase was diluted with RPMI medium to 2.5X 10⁵ cells/mL. Phorbol 12-myristate 13-acetate (PMA) was added to the culture at 25 ng/mL concentration for transformation of the cells to adherent macrophages [13]. The PMA treated THP1 cell culture was dispensed in 96 well plates with 200 μl culture (2.5X 10⁵ cells/mL) in each well and plates were incubated at 37 °C in 5 % CO₂ incubator for overnight. Extracts were diluted in separate plates (Daughter plates) in RPMI medium. The medium in plates with THP1 cells was replaced with fresh medium. The diluted plant extracts were added to these plates. The plates were placed again in CO₂ incubator at 37 °C, 5 % CO₂ for 48 h. After 48 h 10 μl of alamar blue solution was added to each well and the plates were incubated further for overnight. Standard fluorescence was measured on a fluorometer at 544 nm ex, 590 nm em. Cytotoxicity screening was done for active extracts, which have shown more than 90 % inhibition in primary T. brucei screening. None of the T. brucei active plant extracts have shown more than 50 % inhibition on differentiated THP1 cells at 10 μg/mL concentration.