Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.