Identifying new potential antimalarial compound in toad venoms through a bio-guided fractionation process

Aim
This work aims to develop a fractionation process that allows to isolate and identify compounds displaying a potential antiplasmodial activity from toad venom extracts.
Methods
The strategy combines analytical and separative techniques with biological evaluations of the obtained samples. Three species were considered: Rhinella marina (RM), Bufo bufo (BB), Incillius alvarius (IA). Crude extracts were obtained through sonication-assisted solvent extractions of dried venoms. The different crude extracts were characterized by TLC and LC-MS and fractionated using flash chromatography and preparative TLC.
Each crude extract and the subsequently obtained fractions were tested for their antiplasmodial activity on two Plasmodium falciparum strains (3D7 and W2) using the SYBR-Green I assay. Their cytotoxicities were also assessed on FHs74int and HUVEC cell lines using the MTT and Crystal Violet assays. The hemolytic activity was evaluated.
Results
Among the tested samples, several displayed an interesting antiplasmodial activity, mainly those originating from the venoms of RM and IA. One of the fractions obtained from the dichloromethane extract of RM displayed an activity against in vitro cultures of Plasmodium falciparum . The main component of this sample was identified as being resibufogenin. The antiplasmodial activity of this isolated compound seems promising. No hemolytic activity was highlighted. Cytotoxicity assays on human cells are still ongoing but have already pointed out the toxicity of several samples including resibufogenin.
Main Conclusions
This work has demonstrated that toad venoms could constitute an interesting source for new antimalarial drug candidates. Resibufogenin, a cardiotonic steroid, was identified as being the main protagonist. This compound being exempt of hemolytic activity still, however, displays a certain level of toxicity against human cells. However, many perspectives are possible to reduce toxicity while maintaining antiplasmodial activity (chemical modifications through hemi-synthesis, nanoencapsulation).