ATIVIDADES BACTERICIDAS E ANTICANCERÍGENAS DO INIBIDOR DE TRIPSINA PURIFICADO DE SEMENTES DE Enterolobium timbouva

Abstract

One of the problems faced worldwide is microbial resistance. In this context, plant peptidase inhibitors have potential antimicrobial activity and can also serve as a model for the design of new drugs, capable of inducing death. In this work we aimed to isolate and purify a trypsin inhibitor from Enterolobium Timbouva seeds (EtTI), and to verify its bactericidal and antitumor activities. EtTI was purified in two chromatographic steps. Polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS) revealed that EtTI has a molecular weight of 19 kDa. EtTI showed minimum inhibitory concentration (MIC) for Gram-positive bacterial species: Staphylococcus epidermidis (9µM), Staphylococcus haemolyticus (9µM) and Staphylococcus saprophyticus (4.5µM) and Gram-negative: Escherichia coli (9µM). By doing the treatment with MIC and 10 times the MIC value we obtained the results for biofilms: reduction in biofilm formation and in mature biofilm for the S. saprophyticus strain. Representative images of biofilm assay, were obtained by fluorescence microscopy. In the assay with Sytox Green - permeable in cells with damaged membranes - we had a 48% permeation in 2 hours, indicating possible mechanism by membrane damage. Furthermore, EtTI showed synergistic effect with the antibiotic Vancomycin. In the evaluation of anticancer activity, the inhibitor showed an IC50 of 2.69µM in murine melanoma cells. In an in vivo test, using an invertebrate model, EtTI showed no acute toxicity at the concentrations studied. The biological activity of EtTI demonstrates antimicrobial potential and, from the sequence elucidation of EtTI, antimicrobial peptides can be designed in order to obtain potential molecules with clinical application.