ISOLATION AND CHARACTERIZATION OF PLANT ANTITRYPANOSOMALS FOR AFRICAN TRYPANOSOMES

ABSTRACT African trypanosomiasis is a disease caused by the parasitic protozoans of African trypanosomes. Despite several efforts at chemotherapeutic interventions, the disease poses serious health and economic concerns to humans and animals of various sub-Saharan African countries. Commercially available drugs such as suramin, eflornithine, pentamidine, melarsoprol and nifurtimox have reported cases of undesirable side effects, drug resistance, and difficulty in regimen application. Studies have reported on the antitrypanosomal activities of several medicinal plants although their mechanisms of action remain poorly understood. Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Z. zanthoxyloides) and Bidens pilosa L. (B. pilosa) are plant species of important phytochemical and pharmacological relevance found in several tropical and temperate zones. However, their effects and mechanisms of antitrypanosomal action in African trypanosomes remain largely unknown. The aim of this study was thus to isolate antitrypanosomals from these plants and determine the effects and mechanisms of action of selected antitrypanosomals in Trypanosoma brucei (T. brucei). The Kupchan method of solvent extraction was used to prepare crude extracts and fractions from the air-dried pulverized plant material of Z. zanthoxyloides (root) and B. pilosa (whole), thereby giving rise to methanol (ZRFM, BPFM), dichloromethane (ZRFD, BPFD), hexane (ZRFH, BPFH) and butanol (ZRWB, BPWB) fractions from the respective plants. Effects and mechanisms of action of selected fractions were determined through analysis of cell viability, cell death, cell cycle, fluorescence microscopy and RNA interference target sequencing. The fractions ZRFD, ZRFM, ZRWB, BPFD and BPFM were selectively active against T. brucei with respective half-maximal inhibitory concentrations of 5.70, 3.89, 4.02, 3.29, and 5.86 µg/ml. Fractions ZRFD, ZRFM, ZRWB, BPFD, BPFM and BPFH caused a significant induction of apoptosis-like cell death. The ZRFD, ZRFM, ZRWB, BPFD and BPFM fractions caused significant inhibition and arrest of the G0-G1 and G2-M cell cycle phases respectively, while ZRFD and ZRFM resulted in a significant increase in the S phase of T. brucei. Fractions ZRFD, ZRFM, BPFD, and BPFM also induced distortion and aggregation of the parasites. Antitrypanosomal compounds were isolated from the dichloromethane fraction of both plant species in a bioactivity-guided process using high performance liquid chromatography. Isolated compounds were identified through mass spectrometry, ultraviolet spectroscopy and nuclear magnetic resonance spectroscopy as aromatic nitrogen-containing alkaloids. Promising compounds included new and known alkaloids from Z. zanthoxyloides (root), as well as esters of tryptophan from B. pilosa (whole plant). Compounds exhibited selective activities, significant induction of apoptosis-like cell death and a marked alteration of cell cycle in T. brucei. Through an RNA interference target sequencing, putative forms of leucine-rich repeat proteins, stumpy formation signaling pathway proteins, receptor-type adenylate cyclase and other hypothetically conserved proteins were identified for the most promising compounds, thereby suggesting the potential interference of pathogenesis, growth, development and secretory pathways of T. brucei. Taken together, the results indicate that Z. zanthoxyloides (root) and B. pilosa (whole plant) have promising antitrypanosomal activities with implications for novel therapeutic interventions in African trypanosomiasis.