Assessment of apoptosis inducing potential of bioactive fractions. (a). Determination of PS externalization in treated promastigotes. L. donovani promastigotes (2 × 106 ml−1) were treated with 500 μg ml−1 of ALE, ASE and pentamidine for 72 h and dual-stained with Annexin-V-FLUOS and PI. The lower left quadrant represents live cells, upper left quadrant = PI positive, necrotic cells, lower right quadrant = Annexin-V-FLUOS positive, apoptotic cells, upper right quadrant = PI and Annexin positive, late apoptotic cells. The percentage of positive cells is shown in the respective quadrants. (b). Probing of in situ DNA fragmentation by TUNEL assay. L. donovani promastigotes (2 × 106 ml−1) were incubated in the absence or presence of ALE, ASE, and pentamidine (500 μg ml−1) for 72 h. The samples were processed according to instructions provided with ApoDirect kit (Roche), as described in Methods. ***P < 0.001 and **P < 0.01, in comparison to parasite control.