In Vitro Trypanocidal Effect of the Extract and Isolated Compound of Corymbia torelliana Stem Bark against Trypanosoma brucei brucei (Federe Strain)

Abstract

African animal trypanosomosis (AAT) is a severe debilitating protozoan disease of livestock and some wild animal species. It is caused by the pathogenic trypanosome species, and transmitted by Tsetse flies. It affects about 60 million cattle in Africa, with about US$1.5 billion in annual losses. Eradication of the disease through chemotherapy and vaccine development has been hindered by parasite antigenic variation and its resistance to drugs. In this study, the crude ethanol stem bark extract of Corymbia torelliana was prepared by cold maceration in 70% Ethanol. It was further purified using the modified Kupchan partition method into n-hexane, Ethyl acetate, N-butanol and Aqueous pure fractions.  A pure compound was isolated from the ethyl acetate purified fraction using the column and thin layer chromatographic techniques. The isolated compound was named Makoshi_Mak1. The in vitro antitrypanosomal effects of the crude extract, the purified fractions and the isolated compound were then tested against the blood stream forms of Trypanosoma brucei brucei (Federe strain). 0.2 ml of prepared trypanosome suspension containing about 2x104 parasites was dispensed into each of seven clean test tubes using micropipettes. 0.2ml of prepared Ringer’s solution was also added to stabilize the parasites. Five varying concentrations of the crude extract, the extract fractions and the isolated compound (Makoshi_Mak1) were added to test tubes 1-5. Test tube 6 and 7 served as the negative and positive controls respectively. Aliquots of the contents of each test tube were examined under the microscope at x400 magnification for parasites motility at different time interval for 2 hours. The crude extract was effective at the concentrations of 12 mg/ml, 1.2 mg/ml,0.12 mg/ml and 0.012 mg/ml. the n-hexane fraction had efficacy at 0.12mg/ml and 0.096mg/ml. The Eethyl acetate fractions were effective at  0.12mg/ml, 0.096 mg/ml, 0.048mg/ml and 0.024mg/ml, the N-butanol fraction had efficacy at 0.12mg/ml and 0.096mg/ml, while the Aqueous fraction was effective at 0.2mg/ml, 0.16mg/ml and 0.08mg/ml. Makoshi_Mak1 was effective in vitro against T. brucei beucei at all the tested concentrations of 0.219mg/ml, 0.109mg/ml, 0.055mg/ml and 0.027mg/ml. Efficacy of the extracts and isolated compound was determined by parasite mortality, and was seen to be concentration dependent. Dead parasites appeared to float freely on the fluid environment, while the lysed parasites were seen as floating debris.  The results of the study showed that both the crude extract, the purified extract and the pure isolated compound of Corymbia torelliana had in vitro activity against Trypanosoma brucei brucei in a concentration dependent manner. Corymbia torelliana can then be considered as a potential herbal antitrypanosomal drug candidate.