Enyne acetogenins from Porcelia macrocarpa fruit peels displayed anti-Trypanosoma cruzi activity in vitro and cause a reduction in the intracellular calcium level in the parasites

Abstract Natural products are a promising source of new compounds with a wide spectrum of pharmacological properties, including antiprotozoal activities. Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is one of several neglected tropical diseases with reduced options for treatment, which presents limitations such as toxicity and ineffectiveness in the chronic stage of the disease. Aiming to investigate the Brazilian flora for the discovery of new anti-T. cruzi compounds, the MeOH extract from Porcelia macrocarpa R.E. Fries (Annonaceae) fruit peels displayed potent activity against trypomastigotes and intracellular amastigotes and was subjected to bioactivity-guided fractionation. Using different chromatographic steps, it was obtained a fraction composed of a mixture of four new chemically related acetogenins which were characterized as (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-octadeca-13’,17’-dien-11’-inil)butanolide (1), (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-eicosa-13’,19’-dien-11’-inil)butanolide (2), (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-octadec-13’-en-11’-inil)butanolide (3), and (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-eicosa-13’-en-11’-inil)butanolide (4) by analysis of NMR and UHPLC/ESI-HRMS data. The fraction composed of the mixture of compounds 1–4, displayed an EC50 of 4.9 and 2.5 mg/mL against trypomastigote and amastigote forms of T. cruzi, respectively, similar to standard drug benznidazole (EC50 of 4.8 and 1.4 mg/mL). Additionally, the fraction composed of 1–4 displayed no mammalian toxicity for murine fibroblasts (CC50 > 200 mg/mL), resulting in a SI > 40.8 and > 83.3 against trypomastigotes and amastigotes, respectively. Based on these results, the mechanism of action of this fraction was investigated. After a short-time incubation with the trypomastigotes, no alterations in the cell membrane permeability were observed. However, it was verified a decrease in the intracellular calcium of the parasites, without significant pH variations of the acidocalcisomes. The intracellular damages were followed by an upregulation of the reactive oxygen species (ROS) and ATP, but no depolarization effects were observed in the mitochondrial membrane potential. These data suggest that the fraction composed of 1–4 caused an irreversible oxidative stress in the parasites, leading to death. If adequately studied, these acetogenins can open new insights for discovery of new routes of death in T. cruzi.