Diospyros perigrena bark extract induced apoptosis in filarial parasite Setaria cervi through generation of reactive oxygen species.

BACKGROUND

Lymphatic filariasis is a major neglected tropical disease. Diospyros perigrena Gurke (Ebenaceae) was selected for antifilarial chemotherapy because of unavailability of proper medicine. In India, different parts of this plant were used for the treatment of diabetes, diarrhea, dysentery, cholera, mouth ulcers, and wounds.

OBJECTIVE

The present study was undertaken to access antifilarial potential and mechanism of action of n-butanol extract (NBE) of D. perigrena stem bark on Setaria cervi Rudolphi (Onchocercidae).

METHODS

In vitro efficacy and apoptotic mechanism were evaluated by Hoechst, TUNEL, DNA fragmentation assay, pro- and anti-apoptotic gene expression in NBE (250, 125, 62.5, 31.25, and 15.6 µg/ml)-treated S. cervi after 24 h of incubation. Reactive oxygen species (ROS) up-regulation was also determined by GSH, GST, SOD assays, and super oxide anion level.

RESULTS

Significant in vitro antifilarial activity of NBE was found 50% inhibitory concentration (IC50): adult = 57.6 μg/ml, microfilariae (mf) = 56.1 μg/ml, and lethal dose (LD100) in mf is 187.17 μg/ml) after 24 h of treatment. NBF-induced apoptosis was proved by Hoechst, TUNEL, RT-PCR, and Western blot method. NBF (250 µg/ml) decreased the level of GSH (17.8%) and GST (65.4%), increased SOD activity (1.42-fold) and super oxide anion production (1.32-fold) in the treated parasite which culminated into ROS up-regulation.

CONCLUSIONS

NBE induced apoptosis in different life cycle stages of S. cervi. In future, a detailed study of NBF will give us a novel antifilarial compound which will be used for antifilarial chemotherapy.