Arabinogalactan-Glycoconjugate Fractions from Genipa americana Leaves as a Source of Antichagasic Natural Products

Abstract

The extract from leaves of Genipa americana L., Rubiaceae, has antichagasic effect. The major components of this extract, arabinogalactan-glycoconjugate fractions (PFI and PFII), have shown similar biological effects of the crude extract, becoming interesting to evaluate the antichagasic effect of PFI and PFII. This work aimed to access the trypanocidal effect of PFI and PFII against the benznidazole-resistant Trypanosoma cruzi Y strain and to investigate the mechanism of action. PFI and PFII were evaluated on epimastigote (24, 48, 72 h), trypomastigote (24 h), and amastigote (24 and 48 h) forms. The cytotoxicity on LLC-MK2 cells was made by MTT assay. Flow cytometry analysis with PE-annexin V (Ax), 7-AAD, and DCFH-DA staining and scanning electron microscopy (SEM) were performed in epimastigote forms for evaluation of the cell death pathway. PFI showed effect against epimastigote forms (EC50/24 h = 580 ± 0.17 μg/ml; EC50/48 h = 530 ± 0.13 μg/ml; EC50/72 h = 500 ± 0.14 μg/ml), while PFII did not show effect on any tested concentrations. In trypomastigotes, the PFI and PFII showed effect with EC50 values of 100 ± 0.09 and 23 ± 0.06 μg/ml respectively. PFI and PFII were also able to decrease amastigotes/100 cells parameter. PFI and PFII were not cytotoxic in LLC-MK2 cells at the highest tested concentration, resulting in selectivity index (SI) higher than 15 for PFI and higher than 65 for PFII. The increase in labeling of 7-AAD and DCFH-DA fluorescence on cytometry flow assays suggested necrosis as the cell death pathway of PFI in epimastigotes of T. cruzi, being confirmed by pores in the membrane observed on SEM. PFI and PFII are partially responsible by the trypanocidal effect of the crude extract of G. americana, with PFI showing the same action mechanism of crude extract and PFII being the most selective and potent fraction, presenting the biotechnological potential for antichagasic natural products.