New method for screening anti-Leishmania compounds in plants extracts by HPTLC-bioautography.

ABSTRACT

Leishmania genus is responsible for leishmaniasis, a group of diseases affecting 12 million people in the tropical and subtropical zone. Currently, the few drugs that are available to treat this disease are expensive and cause many side effects. Searching for new therapeutics from plant species seems to be a promising path. This work proposes an original HPTLC test against parasites, in particular on Leishmania infantum, to screen new molecules from plant extracts. The technique uses protozoa transformed to express the luciferase gene to observe the bioautogram in bioluminescence. We have developed two different test protocols based on the two dimorphic stages of the parasite. The free promastigote stage, and an intracellular stage parasitizing macrophage cells called the amastigote stage. These two stages only survive under extremely different conditions which required the development of two very different test protocols. For the promastigote free stage of the protozoa, the direct bioautography technique was chosen while for the intracellular amastigote stage, bioautography by immersion (agar overlay) was required. Amphotericine B was chosen as the reference compound for this assay. The development of each of these two tests made it possible to clearly detect areas of activity on the bioautogram, allowing a rapid and inexpensive screening of the antiparasitic properties of molecules in natural extracts.