An in vitro study of apoptotic like death in Leishmania donovani promastigotes by withanolides.

The aim of this study was to isolate and evaluate the withanolides in inducing apoptotic like death in Leishmania donovani in vitro. Withanolides were fractionated and isolated from the leaves of Withania somnifera and LC-MS/MS analysis of two fractions namely, F5 and F6 of ethanolic extracts, obtained through column chromatography with silica gel, was performed. The antileishmanial effect of withanolides on L. donovani promastigotes was assessed in vitro using PI dye exclusion test. The effect of withanolides on promastigote morphology was determined by scanning electron microscopy. To understand their mode of action against L. donovani, DNA fragmentation, quantification of parasites at sub G0/G1 phase, determination of phosphatidylserine externalization, measurement of reactive oxygen species (ROS) and mitochondrial membrane potential (Ψm) were done. Results showed that LC-MS/MS analysis confirmed the presence of withanolides in isolated fractions. Treatment with withanolides resulted in morphological alterations from spindle to round shape and loss of flagella/cell integrity in promastigotes. Moreover, it induced DNA nicks, cell cycle arrest at sub G0/G1 phase and externalization of phosphatidylserine in dose and time dependent manner via increase in ROS and decrease in Ψm. Results of this study indicate that withanolides induce apoptotic like death through the production of ROS from mitochondria and disruption of Ψm in promastigotes of L donovani.