Purificação e avaliação do potencial larvicida e antimicrobiano de um inibidor de protease de Dioclea megacarpa Rolfe

Abstract

Vector-borne diseases are one of the major public health problems in the world. In Brazil, the Aedes aegypti mosquito causes great concern, being responsible for transmitting diseases such as Dengue, Zika, and Chikungunya. Prevention methods against this and other insects focus mainly on the use of synthetic chemicals, which have a long and worrying history related to risks against human health, environmental imbalance, and the emergence of resistant vector populations. Therefore, bioinsecticides appear as a sustainable and safe solution to the problem. Within this class, some of the most explored bioinsecticides are bioactive compounds extracted from plants, such as protease inhibitors, commonly found in families such as Fabaceae, Cucurbitaceae, and Solanaceae. Thus, the objectives of the present work focused on the purification and evaluation of the larvicidal and antimicrobial potential of a protease inhibitor derived from Dioclea megacarpa. For initial protein profile determination, the crude extract (EbDM), obtained through extraction with buffer Tris HCl 50 mM pH 7.5, was fractionated with 20% trichloroacetic acid (TCA) and subjected to polyacrylamide gel electrophoresis (SDS-PAGE). After determining the best fraction (DmF5), the isolation of the inhibitor was given through trypsin affinity chromatography, where only the sample retained in the column (RDmF5) was selected for subsequent tests. The trypsin inhibition tests showed very promising values, reaching a specific inhibitory activity measurement of 25,569.80 IU/mg. Furthermore, inhibitory activity tests on the midgut proteases of Aedes aegypti showed 20% inhibition at the lowest concentration (0,234 ug/mL), reaching up to 53% in assays with 1,872 ug/mL. As for antimicrobial activity essays, different concentrations of RDmF5 and NRDmF5 were used against Candida spp. and Staphylococcus spp. strains, and they did not show a satisfactory minimum inhibitory concentration. These data show the efficacy of the proteinase inhibitor isolated from Dioclea megacarpa as a larvicidal agent, suggesting its potential use as a bioinsecticide, and demonstrate its inefficiency as an antimicrobial agent.