Evaluation of anti-trypanosomal activity of extracts of selected meliaceae plant species by in vitro and in vivo assays.
Wanzala-Mahiri, Everlyne Nafuna
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African human trypanosomiasis (HAT) and African animal trypanosomiasis (AAT) are vector-borne parasitic diseases, which are among the most neglected diseases in the world. They cause major health and economic problems in rural sub-Saharan Africa. Chemotherapy, the main means of controlling the disease is limited due to parasite resistance and toxicity of the current anti-trypanosomal drugs. The development of a vaccine has been thwarted by antigenic variation of the parasite. Thus, the use of natural products is one of the strategies being explored to address some of the problems encountered with allopathic chemical drugs. The main objective of the current research was to evaluate the anti-trypanosomal activity of methanol and aqueous extracts of selected Meliaceae species through in vitro and in vivo assays against Trypanosoma brucei rhodesiense, Trypanosoma brucei brucei and Trypanosoma evansi. Methanol and aqueous extracts of five Meliaceae plant species: Trichilia emetica, Toona ciliata, Azadirachta indica, Turraea mombassana and Melia azedarach were tested for in vitro anti-trypanosomal activity, acute toxicity and in vivo efficacy using mice as animal models. Sequential extraction of powdered plant samples in hexane, ethyl acetate, methanol and water afforded the crude extracts. In vitro assays were carried out in 96-well microtitre plates and melarsoprol and suramin were used as the positive controls. The results revealed that the minimum inhibitory concentrations (MICs) of MeOH extracts were significantly lower than those of the aqueous extracts. The MeOH extracts of M. azedarach stem bark, A. indica stem bark and T. emetica root bark (MIC 9.11±3.44, 9.93±2.88 and 9.11±3.44 μg/ml respectively) showed the best anti-trypanosomal activity against T. b. rhodesiense; the MeOH extract of T. emetica root bark (MIC 9.93±2.88 μg/ml) showed the best anti-trypanosomal activity against T. b. brucei. The methanol extracts of M. azedarach root bark, T. emetica root bark and A. indica stem bark (9.11±3.44, 9.11±3.44 and 9.93±2.88 μg/ml respectively) showed the best anti-trypanosomal activity against T. evansi. Based on the strength of in vitro anti-trypanosomal activity, four extracts were selected and subjected to acute toxicity tests in mice. It was established that the MeOH extract of A. indica stem bark and the aqueous extract of T. mombassana leaves were safe in mice at dose levels of 100, 200 and 400 mg/kg body weight. The LD50 values of T. emetica root bark and M. azedarach root bark were 707.95±229.25 and 229.09.54±95.54 mg/kg body weight respectively. Two extracts (A. indica stem bark and the T. mombassana leaves) were subjected to in vivo efficacy tests using T. b. rhodesiense infected mice. Melarsoprol and suramin at doses of 3.6 and 5 mk/kg bwt respectively were used as positive controls. The infected-untreated group served as negative control. The study showed that the MeOH extract of A. indica stem bark at 200 and 400 mg/kg and the aqueous extract of T. mombassana leaf at 400 mg/kg reduced parasitemia levels, prevented body weight loss and reduced decline in packed cell volume (p < 0.05) in mice. Chromatographic fractionation of the methanol extract of A. indica stem bark led to the isolation of nimbin (55) and 1-detigloyl salannin (84). Toona ciliata stem extract yielded bis(2-ehtylhexyl) phthalate (85) and bis(2-methylheptyl) phthalate (86). Nimbin (55) displayed the highest in vitro antiptrypanosomal activity with MIC values of 9.74±0.93, 11.90±0.02 and 11.89±0.01 μg/ml against T. b. rhodesiense, T. b. brucei and T. evansi respectively. Bis(2-ehtylhexyl) phthalate (85) showed mild activity against the three strains (MIC > 146 μg/ml). In conclusion, the study established that A. indica stem bark and T. mombassana leaves have in vitro and in vivo anti-trypanomal activities and can be considered portential sources of new anti-trypanosomal compounds. Based on the findings of the current study, it is recommended that extracts of A. indica and T. mombassana may be used as alternative remedies for African trypanosomiasis.