Comparative antitrypanosomal activity of Terminalia chebula dried fruits against Trypanosoma evansi

Trypanosoma evansi, one of the causative agents of trypanosomosis in animals was used to evaluate the trypanocidal potential of Terminalia chebula dried fruits. Powdered T. chebula dried fruits was comparatively extracted with solvents of different polarities (hexane, chloroform, methanol and water) and obtained dried extracts were solubilized in 1% dimethylsulphoxide. Alsever medium supplemented with inactivate bovine serum at 56°C in ratio of 1:1 was incubated at 37°C and under 5% CO₂ for 5h. 20µl of each extract was added to 180µl of Alsever medium with trypanosomes (1×10⁶/ml) at different concentrations of extracts (250–1000µg/ml) and incubated for 5h. In vitro trypanocidal activity was in order of methanol, aqueous, chloroform and hexane. At 250µg/ml, methanolic plant extract (MPE) exhibited strong immobilization and killing of trypanosomes. At same concentration, there was complete killing of trypanosomes at 5h of incubation, which was same as diminazine aceturate (50µg/ml) standard drug at 4h. Trypanocidal activity observed in all extracts was in concentration-time dependent faction. The cytotoxicity test was carried out on Vero cell line maintained in Dubecco's Modified Eagle Medium (DMEM) and seeded in 96-wells flat bottom ELISA plates. Each well contained 500,000 cells/ml. Different concentrations of methanolic plant extract (MPE) (1.56–100µg/ml) were dissolved in DMEM, solubilized in 1% dimethylsulphoxide and incubated at same conditions for 72h. MPE of T. chebula and diminazine aceturate were cytotoxic to Vero cells except at 6.25 and 1.56µg/ml, respectively. In vivo, mice inoculated with 1×10⁴/ml trypanosomes became parasitemic after 48h and were subsequently treated with methanolic extract using concentrations (12.5, 25, 50 100 and 200mg/kg body weight) at dose rate of 100µl per mouse via intraperitoneal route consecutively for 3 days. However, mice treated with MPE of T. chebula at a dose of 200mg/kg body weight showed a survival time of maximum 8 days.