Estudo in vitro da atividade tripanocida de polissacarídeos vegetais

Abstract

Chagas disease caused by the parasite Trypanosoma cruzi is considered a neglected tropical disease. In Brazil, only benzonidazole is used in treatment with limited efficacy and relevant adverse effects. Natural plant products are a promising source of drugs for the fight against T. cruzi infections. In the search for therapeutic alternatives, the objective was to evaluate the effect of plant polysaccharides on the evolutionary forms of T. cruzi and to investigate its mechanism of action. The powder obtained from the vegetable parts (5 g) was suspended in methanol, homogenized and filtered. The residue was suspended in 0.1 M NaOH (1:50, w/v), homogenized and centrifuged (1445 x g, 15 min, r.t). Supernatants were pooled, neutralized, precipitated and centrifuged. The precipitate was dialyzed, centrifuged and the lyophilized supernatant (total polysaccharides-TP). The TP were dissolved in distilled water (2: 1, w/v), applied in ion exchange chromatography (DEAE-cellulose) and acidic fractions eluted with NaCl (0.1-1.0 M). The majority fractions (FI and FII) were dialysed and lyophilized. TP and fractions were analyzed for carbohydrate, uronic acid and protein contents.The polysaccharides were evaluated on epimastigotes (24, 48, 72 h), trypomastigotes (24 h), amastigote (24 and 48h). Cytotoxicity on LLC-MK2 cells (MTT) was also evaluated. Flow cytometry assays (7AAD/annexin V, Rho123, H2DCFH-DA and Acridine Orange) were performed for the evaluation of the mechanism of cell death. Scanning electron microscopy (SEM) was used to observe possible ultrastructural changes. TP of all species inhibited the growth of epimastigote and trypomastigote forms, among these the TP of the leaves of G. americana were distinguished by their lower IC50 compared to the trypanocidal assays, in addition to low cytotoxicity on LLC-MK2. TP [epimastigote (IC50/24h = 740 ± 0.07 μg / mL; IC50/48h = 710±0.05 μg / mL; IC50/72h = 870±0.05 μg/mL); trypomastigote (LC50 = 470±0.08 μg/ml)]; LLC-MK2 (CC50 = 2250±0.07 μg/mL)]. As for fractions (FI and FII), only FI presented IC50 for epimastigote forms [IC50/24 h = 580±0.17 μg/mL; IC50/48 h = 530±0.13 μg/mL; IC50/72 h = 500±0.14 μg mL)]. In trypomastigotes, the following LC50 were present: FI (LC50 = 100±0.09 μg/mL) and FII (LC50 = 23±0.06 μg/mL). FI and FII did not cause toxicity on the host cells, with a selectivity index higher than 15 (FI) and 65 (FII). TP (235 and 470 μg/mL) and fractions [FI (50 and 100 μg/mL); FII (11.5 and 23 μg/mL)] also showed antimastigote effect, with a reduction in the percentage of infected cells, number of amastigotes/cells and survival index. After 7AAD/annexin V, flow cytometry assays demonstrated a higher population of 7-AAD-labeled parasites at the concentrations tested [TP (IC50 -740 μg/mL) and FI (IC50 – 580 μg/mL)] suggesting that death induced by the polysaccharides is predominantly by necrosis. Then, Rho 123, H2DCFH-DA and Acridine Orange tests were performed to investigate the changes of the mitochondrial transmembrane potential, the involvement of ROS and detection of acidic vacuoles. The results showed that there was alteration of transmembrane potential and increase of ROs. The cell death pathway of T.cruzi by a necrotic mechanism was confirmed by MEV, which demonstrated changes in the typical format, rounding and shortening of the parasite and degradation of the cell membrane with pores. In conclusion, the polysaccharides of all plant species inhibited the growth of the epimastigotes and trypomastigote forms, among which the polysaccharides of G. americana leaves inhibited all evolutionary forms of Y strain of T. cruzi, with high selectivity index (FII), with induction of necrosis and oxidative stress.