This study was aimed at trypanocidal potential of purified fractions of Khayaseneegalensis tree bark against Trypanoamaevansi. The current classes of drugs in use are bedeviled with lots of problems. In quest of new trypanocidal compounds from medicinal plants, K. seneegalensis tree bark was extracted with methanolic solvent. The obtained extract (29.8 g) underwent the processes of column chromatography, thin layer chromatography (TLC) and rechromatography of distinct groups of pooled fractions based on unique and similar characteristics of bioactive constituents displayed on TLC plates. It was further subjected to preparative thin layer chromatography and higher performance liquid chromatography (HPLC) to determine purity nature of the pooled fractions. Each set of pooled fractions at different stages of purification was screened against T. evansiat different concentrations(250-1000 µg ml-1)for bioassay to determine its level of trypanocidal activities. A high parasitemic blood of a mouse was diluted with Alsever solution to obtain a final parasite concentration of 1x106 parasites/ml. The medium consist of Alsever solution and inactivated bovine serum at 58C for 1 h. Suspension (180 µl of medium with trypanosomes) was added to test extract and pooled fractions (20 µl) obtained at different stages of purification of K. Senegalensis(root bark) in the ELISA plates wells, which were incubated at 37C under 5% CO2 for the period of observation. The test was repeated at least thrice. In vitro cytotoxicity was done on Vero cell line (SIGMA) grown in DMEM in 96-wells microculture plates. Each well was seeded with 500,000 cells ml-1 and plates were incubated at 37oC with 5% CO2 for 48 h. After the formation of confluent monolayer, the supernatant was discarded and replaced with fresh medium. Confluent monolayer of Vero cell lines was treated with serial dilutions (1.56-100 µg ml-1) of MPE and partially purified fractions of K. senegalensis tree bark in triplicate and incubated for 72 h consecutively under the same conditions described previously. At 24 h interval, plate was observed under inverted microscope for cytotoxic effects as compared to untreated normal cells that served as control. In each case, after 72 h of incubation, the culture media of the incubated Vero cells was discarded. Adhered cells were stained with a drop of crystal violet in phosphate buffered solution. Plate was then incubated for 24 h at 37oC in ordinary incubator. Plates were later observed under inverted microscope for cytotoxic effects. Stock of MPE of K. Senegalensistreebark was solubilized in 1% dimethylsuphoxide (DMSO). The concentration in the experiment had no deleterious effect by itself on host cells or trypanosomes. 1% DMSO in distilled water was used as control. Results of in vitro trypanocidal activity varied from immobilization, reduction and to the killing of trypanosomes in corresponding ELISA plate wells. At 250 µg ml-1of MPE, there was drastic reduction and complete killing of trypanosomes at 6 h of incubation (40.00±0. and 0.0.±0.0) as observed. Trypanosomes counts decreased in concentration and time –dependent manner with significant difference (P ≤ 0.05 to 0.01). In in vivo test of MPE of K. senegalensisat 12.5, 25, 40, 100 and 200 mg/kg body weight, mice in each group died at day 3, 7, 8 and 19 due to parasitaemia. But for in vivo of partially purified fractions of K. senegalensis at the same concentrations, mice in each group died at day 4, 8, 15 and 38. MPE and partially pooled fractions of K. senegalensis with diminazineaceturate, standard drug, were cytotoxic to Vero cells except at concentrations of 1.56-3.13, 1.56-6.25 and 1.56-6. 25 µg ml-1.K. senegalensis tree bark appeared to be promising candidate for a new trypanocidal drug as trypanosomes disappeared for weeks from peripheral blood and reappeared just as in clinical cases.
Key words:Medicinal plant,Khayasenegalensistree bark, in vitro and in vivotrypanocidal activity of methanolic and partial purified fractions of K. senegalensis,ln vitro cytotoxicity test