Butenolides from Nectandra oppositifolia (Lauraceae) displayed anti-Trypanosoma cruzi activity via deregulation of mitochondria

Background: From a previous screening of Brazilian biodiversity for antitrypanosomal activity, the n-hexane extract from twigs of Nectandra oppositifolia (Lauraceae) demonstrated in vitro activity against Trypanosoma cruzi. Purpose: To perform the isolation and chemical characterization of bioactive compounds from n-hexane extract from twigs of N. oppositifolia and evaluate their therapeutical potential as well as to elucidate their mechanism of action against T. cruzi. Methods/Study design: Bioactivity-guided fractionation of the n-hexane extract from twigs of N. oppositifolia af-forded three related butenolides: isolinderanolide D (1), isolinderanolide E (2) and secosubamolide A (3). These compounds were evaluated in vitro against T. cruzi (trypomastigote and amastigote forms) and against NCTC (L929) cells for mammalian cytotoxicity. Additionally, phenotypic analyzes of compounds-treated parasites were performed: alterations in the plasma membrane permeability, plasma membrane electric potential (Delta Psi(p)), mitochondrial membrane potential (Delta Psi(m)) and induction of ROS. Results: Compounds 1-3 were effective against T. cruzi, with IC50 values of 12.9, 29.9 and 12.5 mu M for trypomastigotes and 25.3, 10.1 and 12.3 mu M for intracellular amastigotes. Furthermore, it was observed alteration in the mitochondrial membrane potential (Delta Psi(m)) of parasites treated with butenolides 1-3. These compounds caused no alteration to the parasite plasma membrane, and the deregulation of the mitochondria might be an early event to cell death. In addition, in silico studies showed that all butenolides were predicted to be non-mutagenic, non-carcinogenic, non hERG blockers, with acceptable human intestinal absorption, low inhibitory promiscuity with the main five CYP isoforms, and with high metabolic stability. Otherwise, tested butenolides showed unfavorable blood-brain barrier penetration (BBB +). Conclusion: Our results demonstrated the anti-T. cruzi effects of compounds 1-3 isolated from N. oppositifolia and indicated that the lethal effect of these compounds in trypomastigotes of T. cruzi could be associated to the alteration in the mitochondrial membrane potential (Delta Psi(m)).