Mode of action of the natural sesquiterpene lactones estafietin and eupatoriopicrin on Trypanosoma cruzi

Abstract

Chagas disease is an endemic disease in Latin America. The drugs available for treatment have limitations because of their adverse effects, toxicity and lack of efficacy in chronic chagasic patients. Sesquieterpene lactones (STLs) arise as promising compounds because of their anti-trypanosomal activity. Knowing that the main mechanism of action of these compounds is the alkylation of the thiol groups of biological molecules, the aim of this study was to evaluate the in vitro interaction of two STLs, estafietin (EF) and eupatoriopicrin (EP), with low molecular weight thiols and the induction of oxidative stress on T. cruzi epimastigotes treated with both STLs. ES and EP have been isolated from Stevia alpina Griseb.and Stevia maimarensis Hieron. (both Asteraceae), respectively. The purity of both compounds was confirmed by HPLC analysis. The content of free thiol groups was determined by measuring the change in absorbance at 410 nm occurring when SH-groups reduce DTNB. The intracellular oxidative stress was assessed by flow cytometry using the oxidant-sensitive fluorescent probe H2DCFDA.Both STLs showed a high interaction with sulphydryl reagents such as cysteine, beta-mercaptoethanol and glutathione. The affinity of EP for three compounds was higher than that of ES. The fact that EP possesses two potentially active sites at which Michael-type additions could occur, might explain the difference in affinity with ES, which only has one site. Similar behavior was observed when epimastigotes of T.cruzi were treated with EP or ES during 2-7 hours. Between 2 and 4 hours, EP showed a higher reduction (40 ? 60%) in the free thiols levels than that ES (30 ? 50%), reaching similar values at 7 hours of treatment (about 65%). This reduction on levels of thiols groups could lead to an oxidative stress within the parasite, which it showed after 7 hour of treatment. Both drugs could exert it antiparasitic effect by interaction with intracellular low weight thiols.