Identificação por ultrafiltração acoplada à CLUE-EM de ligantes enzimáticos de apirase a partir de fontes naturais e avaliação da sua atividade esquistossomicida in vitro e in vivo

Abstract

The present work describes the identification of natural potato apyrase ligands, which may be potential schistosomicidal substances, through the ultrafiltration (UF) coupled with CLUE-ESI-QTOF-EM. In addition, in vitro and in vivo schistosomicidal activity of selected plant extracts and isolated substances were evaluated. Firstly, cardamonin, a substance with in vitro schistosomicidal and with potential to inhibit the activity of ATP diphosphohydrolases, was evaluated in the in vivo schistosomicidal assay and used to select the incubation conditions for the UF technique. Then some extracts produced were incubated with potato apyrase and submitted to UF coupled with CLUE-ESI-QTOF-EM. The chromatographic peaks of the ligands of apyrase were selected for further fractionation of the plant extracts and isolation and purification of these ligand substances. The isolated substances were evaluated in the potato apyrase inhibition activities assays, as well as in vitro and in vivo schistosomicidal assays. The results this study showed that the cardamonin binds and inhibits the potato apyrase enzymatic activity, with IC50 of 49 M. In addition, in a mouse model of schistosomiasis, cardamonin (400mg/kg) was able to reduce the total number of worms recovered in 46.81% and the number of immature eggs in 62.44%. As a result of the UF coupled with CLUE-ESI-QTOF-EM, the screening of samples for the selection of potential ligands and inhibitors of the potato apyrase enzyme resulted in the selection of 3 plant extracts of the species Ruta graveolens L. (Rutaceae), Acmella oleracea L. (Asteraceae) and Centela erecta L. (Apiaceae). From these extracts we isolate the ligands arborinine, spilanthol and asiaticoside, respectively. Regarding the enzymatic activity, when evaluated at 25 M, arborinine, spilanthol and asiaticoside, were able to inhibit the enzyme by 31.32%, 77.29% and 33.23%, respectively. In the in vitro schistosomicidal assay, the Rg-F4 fraction from R. graveolens extract and the A. oleracea extract caused the death of all parasites after 24h and 48h of incubation. C. erecta extract (100 μg/mL) caused only the death of male worms, while females had only a small reduction in motor activity after 24h of incubation, and all parasites were killed after 48h of incubation. Spilanthol (100 μM) caused the death all parasites after 24h of incubation. Asiaticoside (400mg/kg), when evaluated in the in vivo schistosomicide assay, was able to reduce in 65.43% the number of worms recovered and and 67.87% the number of eggs per gram of feces. The results obtained in this study with cardamonin suggest that apyrase may be used as a target molecule in screening studies to find S. mansoni ATP diphosphohydrolases ligands and possible inhibitors, from herbal extracts. In addition, further studies to investigate are important to investigate the in vivo schistosomicidal activity of arborinine and spilanthol.