Therapeutic Activity of Partially Purified Fractions of Emblica officinalis (Syn. Phyllanthus emblica) Dried Fruits against Trypanosoma evansi

Abstract

Emblica officinalis (E. officinalis) dried fruits were evaluated for its antitrypanosomal activity and cytotoxic effects. Vero cell line maintained in DMEM (Dubecco's Modified Eagle Medium) and incubated with Trypanosoma evansi for more than 12 h. MPE was added to the Vero cell culture medium at different concentrations (250~1,000 µg/mL) with trypanosomes concentration (1 × 10 6 trypanosomes/mL in each ELISA plate well) and incubated at appropriate conditions for 72 h. In-vitro cytotoxicity of MPE of E. officinalis was determined on Vero cells at concentrations ((1.56~100 µg/mL). Acute toxicity and in-vivo infectivity tests were done in mice. Obtained MPE of E. officinalis underwent process of purification via column chromatography, preparative chromatography and HPLC (higher performance liquid chromatography) with bioassay at different strata on Alsever's medium. In-vivo assay for trypanocidal activity, MPE and PPFs (partially purified fractions) of E. officinalis with two sets of mice, each mouse was inoculated with 1 × 10 4 /mL of trypanosomes and treated (48 h post inoculation) at concentrations (12.5, 25, 50, 100 and 200 mg/kg body weight)were administered at dose rate of 100 µL per mouse via intraperitoneal route (in treating parassitemic mice) to different groups of mice, 6 mice per concentration. HPLC of partially purified fractions of E. officinalis was carried out with mobile phase of acetonitrile: water (40:60) in gradient mode. In vitro, MPE induced immobilization and killing of the parasites in concentration-time dependent manner. Significant reduction of trypanosomes counts from concentration of 250 µg/mL and complete killing of trypanosomes at 5th hour of observation, which was statistically equivalent to 4th hour of Diminazine Aceturate (Berenil), standard reference drug used. HPLC of the partially purified fractions revealed two major prominent peaks at retention time of 1~4 min. In vivo, both MPE and PPFs of test material did prolong lives of mice by 6~9 days but could not cure them. At concentration of 2,000 kg/kg body weight of MPE in acute test, all mice survived. For in-vivo infectivity test, mice injected with immobilized trypanosomes developed parasitemia and died while, the other group survived. MPE, PPFs and Diminazine Aceturate were toxic to Vero cells at all concentrations exception of 1.56, 1.56~3.13 and 1.56~6.25 µg/mL, respectively. From this report, PPFs of E. officinalis dried fruits demonstrated potential pathway for a new development of trypanocide in near future if additional investigations are put in place.Key words: Emblica officinalis dried fruits, in vitro and in vivo partially purified fractions trypanocidal activity, Trypanosoma evansi, in vitro cytotoxicity.

Medicinal values of the genus Phyllanthus - A review

“…This is statistically equivalent to the 4th hour of diminazine aceturate (Berenil) treatment, the standard reference drug used. E. officinalis dried fruits demonstrated a potential pathway for the development of new trypanocides if in-depth investigations were to be put in place (Peter et al, 2016). ii) Cardiovascular protection A large number of people across the globe suffer from heart/stroke attacks, mainly due to poor life styles and increased sugar/carbohydrate intakes.…”