Characterization and Pharmacological Properties of a Novel Multifunctional Kunitz Inhibitor from Erythrina velutina Seeds

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.

Extraction, purification and characterization of inhibitor of trypsin from Chenopodium quinoa seeds

“…The quantity of histidine, lysine and leucine demonstrated better similarity with legumes species (Silva et al, 2001;Machado et al, 2013). The concentration of Glu, Gli and Arg was better than the concentration in cereal and legume (Tamir et al, 1996;Silva et al, 2001).…”

“…The BBI are small cysteine-rich proteins in size (8-20kDa), with seven disulfide linkages, high cysteine content, and two independent reactive sites for trypsin and chymotrypsin, soluble between pH 1.5 and 12 and in dry state or in 0.02% aqueous solution is stable at 100 °C or at 105 °C for 10 min, respectively (Losso, 2008). These classes of proteins has been studied due to its application in the treatment of different pathologies as cancer (Zhang et al, 2011), antifungal activity (Kim et al, 2005), inflammation and coagulation (Machado et al, 2013) or as bioinsecticides (Rai et al, 2010), demonstrating its importance as new therapeutic agents for the pharmaceutical industry. Despite there are abundantly studies of PIs in leguminous plants, little is known in cereals, such as amaranth (Tamir et al, 1996); buckwheat (Park et al, 1997) and buckwheat Seeds (Tsybina et al, 2001).…”

Effect of the Erythrina sp. leaves powder on biochemical profile, biological parameters and liver histopathology of Cavia porcellus

“…Sin embargo, la Erythrina ha mostrado menor nivel de degradabilidad que otros forrajes tropicales lo cual se podría asociar a la presencia de sustancias anti nutricionales en su composición química (Joubert y Sharon, 1985;Birk, 2003;Machado et al, 2013;Pesoti et al, 2014;Pompeu et al, 2014) y alcaloides como la hypaphroina y la erythroidina (Paterson, 1994). Estos estudios preliminares muestran por un lado a la Erythrina como un forraje proteico que se acerca aquellos reportados para la alfalfa, pero no muestra una mejora de los parámetros productivos de cuyes (De la Cruz, 2012;.…”

“…Considerando que, en los animales en crecimiento, un incremento en el consumo de proteína conduce a un incremento lineal de la deposición de proteína en músculos (Titgemeyer, 2003) y por tanto un incremento de ganancia de peso (Brosh et al, 2000), la harina de hojas Erythrina (Erytrhina fusca) por su considerable contenido de proteína total (Rios, 1981;Kongmanila y Ledin, 2009) podría llegar a ser una alternativa de forraje para la alimentación de cuyes en la región de Selva Alta. Sin embargo, el uso de hojas de esta planta hasta en un 24% en la ración, no ha influenciado en los índices zootécnicos de cuyes (De la Cruz, 2012; lo que podría estar relacionado con el contenido de inhibidores de proteasas encontrados en extractos de esta y otras plantas (Machado et al, 2013;Pesoti et al, 2014;Pompeu et al, 2014) por lo cual sería de necesidad desarrollar investí-gaciones orientadas a determinar el efecto biológico de esta planta en el organismo animal. El objetivo del presente trabajo fue determinar el efecto de la harina de Erytrhina en el bienestar de Cavia porcellus mediante la medición de los perfiles bioquímicos, parámetros biológicos e histología del hígado.…”

Atividade Antitríptica Em Semente E Produto Alimentício De Chia (Salvia Hispanica L.)

“…18 Porém, quanto aos inibidores enzimáticos, tem-se observado um potencial terapêutico em sua utilização heteróloga para o tratamento de alguns estados de doença. [19][20][21][22][23][24] Embora os inibidores de tripsina estejam presentes naturalmente no reino vegetal, nas sementes de chia não existe relato na literatura sobre tal presença. Além disso, é importante investigar se esses inibidores comuns em sementes, também se apresentam e se mantêm nos seus produtos, como na farinha.…”