Application of flow cytometry and microscopical methods to characterize the effect of herbal drugs on Leishmania Spp

The World Health Organization has identified leishmania sis as a major public health problem, particularly in Africa, Asia, and Latin America. About 1.5 to 2 million people are affected annually by this parasitic infection. As there is no vaccine, there is still a strong need for sufficient drugs. In a preliminary screening, extracts of 50 different plants were evaluated for their possible leishmanicidal activity against the promastigote form of Leishmania mexicana amazonensis. Eighteen extracts showed at least 50% inhibition at 100 microg/ml. The ethanolic extract from Yucca filamentosa L. showed the strongest leishmanicidal activity (100% inhibition at 5 microg/ml). The bioactivity-guided fractionation of this extract led to the isolation of three main components (Yuccasaponins MC 1--3). In further experiments, the effect of Yuccasaponin MC 3 on the promastigote form of L. mexicana amazonensis was quantified and characterized using flow cytometry and specific fluorescent dyes [propidium iodide, Syto 9, and DiBAC(4)(3)]. The data revealed that the membrane of the promastigote is attacked. The effect of Yuccasaponin MC 3 on intracellular forms (amastigote) was also characterized; green fluorescent protein-transfected Leishmania major were used. By this method, an inhibition of intracellular growth of L. major was demonstrated. This paper shows that, together, flow cytometry and microscopy are quick, sensitive, and easily reproducible methods to describe the effects of drugs on parasites.