Abstract
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 2.2×10−8 mol.L−1 and constant inhibition (Ki) of 1.0×10−8 mol.L−1, 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 anti-coagulant 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 anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α 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.
Highlights
The coral tree, Erythrina velutina, is a widely known species belonging to the Erythrina genus, Fabaceae family, Papillionoideae subfamily and known popularly as mulungu
The retained proteins (TR) was further purified by HPLC in a Shimadzu analytical column and two protein fractions were observed (P1 and Peak 2 (P2)) (Figure 1B)
ESI (Q-Electrospray microTOF) was further utilized for purity degree analyses demonstrating various ions charged with the presence of M20+ (962.44 Da), M19+ (1,013.04 Da), M18+ (1,069.27 Da), M17+ (1,132.11 Da), M16+ (1,202.78 Da), M15+ (1,282.91 Da), M14+ (1,374.46 Da), M13+ (1,480.13 Da), M12+ (1,603.38 Da) and M11+ (1,749.04 Da) charges for Erythrina velutina (EvTI) protein (Figure 1H)
Summary
The coral tree, Erythrina velutina, is a widely known species belonging to the Erythrina genus, Fabaceae family, Papillionoideae subfamily and known popularly as mulungu. This plant is commonly found in tropical and sub-tropical regions of the World [1]. Many studies have attributed some of these activities to proteinaceous proteinase inhibitors [5,6,7,8,9,10,11,12,13,14]. A Kunitz-type chymotrypsin inhibitor from Erythrina velutina seeds (EvCI) was purified and showed potential for combating inflammation related disorders (sepsis) and disseminated intravascular coagulation (DIC) [18]
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