Characterization and pharmacological properties of a novel multifunctional kunitz inhibitor from erythrina velutina seeds

• Autor(a) principal: Morais, Ana Heloneida de Araújo
• Data de Publicação: 2013
• Outros Autores: Machado, Richele Janaína Araújo, Monteiro, Norberto de Kássio Vieira, Migliolo, Ludovico, Silva, Osmar Nascimento, Pinto, Michele Flaviane Soares, Oliveira, Adeliana, Franco, Octávio Luiz, Kiyota, Sumika, Bemquerer, Marcelo Porto, Uchôa, Adriana Ferreira, Santos, Elizeu Antunes dos
• Tipo de documento: Artigo
• Idioma: por
• Título da fonte: Repositório Institucional da UFRN
• Texto Completo: https://repositorio.ufrn.br/handle/123456789/58137; http://dx.doi.org/10.1371/journal.pone.0063571
• topic: Erythrina velutina; Multifunctional properties; Anti-inflammatory; Trypsin Inhibition

Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30–60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.261028 mol.L21 and constant inhibition (Ki) of 1.061028 mol.L21 , by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anticoagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to antiinflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-a release and stimulating IFN-a and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation