ABSTRACT
A great number of pharmacological compounds is found in the Brazilian marine diversity. This study evaluated the anticoagulant potential of glycosaminoglycans (GAGs) isolated from the skin of palombeta Chloroscombrus chrysurus and guaiúba Ocyurus chrysurus. GAGs were extracted with crude papain in 0.1 M sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by ion exchange chromatography on DEAE-cellulose column. The chemical composition (contaminant proteins and total carbohydrates) and the analysis by 0.5% agarose gel electrophoresis of fractions were also determined. Anticoagulant assays were performed by activated partial thromboplastin time (APTT) using normal human plasma and standard heparin (193.00 IU mg-1). The obtaining and fractionation procedures of GAGs were effective and similar chromatographic profiles were verified between the species. A similar mobility to dermatan sulfate was revealed for C. chrysurus. This GAG also showed a low activity of 3.30 IU mg-1.
O Brasil abriga uma das maiores biodiversidades marinhas do mundo, favorecendo a descoberta de fontes alternativas de compostos farmacológicos. Desta forma, objetivou-se avaliar o potencial anticoagulante de glicosaminoglicanos (GAGs) isolados das peles da palombeta (Chloroscombrus chrysurus) e guaiúba (Ocyurus chrysurus). Os GAGs foram extraídos com papaína bruta em tampão acetato de sódio 0,1 M (pH 5,0) contendo cisteína 5 mM e EDTA 5 mM, seguido por cromatografia de troca iônica do extrato total em coluna de DEAE-celulose. As frações obtidas foram analisadas quanto à composição química (proteínas contaminantes e carboidratos totais) e os GAGs identificados por eletroforese em gel de agarose a 0,5%. Os ensaios de atividade anticoagulante foram realizados por meio do tempo de tromboplastina parcial ativada (TTPA) usando plasma humano normal e heparina-padrão (193,00 UI mg-1). O procedimento de obtenção e fracionamento dos GAGs mostrou-se eficiente, indicando semelhantes perfis cromatográficos entre as espécies avaliadas e, revelando para C. chrysurus, bandas com mobilidades semelhantes ao dermatam sulfato e com atividade de apenas 3,30 UI mg-1.
ABSTRACT
O Brasil abriga uma das maiores biodiversidades marinhas do mundo, favorecendo a descoberta de fontes alternativas de compostos farmacológicos. Desta forma, objetivou-se avaliar o potencial anticoagulante de glicosaminoglicanos (GAGs) isolados das peles da palombeta (Chloroscombrus chrysurus) e guaiúba (Ocyurus chrysurus). Os GAGs foram extraídos com papaína bruta em tampão acetato de sódio 0,1 M (pH 5,0) contendo cisteína 5 mM e EDTA 5 mM, seguido por cromatografia de troca iônica do extrato total em coluna de DEAE-celulose. As frações obtidas foram analisadas quanto à composição química (proteínas contaminantes e carboidratos totais) e os GAGs identificados por eletroforese em gel de agarose a 0,5%. Os ensaios de atividade anticoagulante foram realizados por meio do tempo de tromboplastina parcial ativada (TTPA) usando plasma humano normal e heparina-padrão (193,00 UI mg-1). O procedimento de obtenção e fracionamento dos GAGs mostrou-se eficiente, indicando semelhantes perfis cromatográficos entre as espécies avaliadas e, revelando para C. chrysurus, bandas com mobilidades semelhantes ao dermatam sulfato e com atividade de apenas 3,30 UI mg-1.
A great number of pharmacological compounds is found in the Brazilian marine diversity. This study evaluated the anticoagulant potential of glycosaminoglycans (GAGs) isolated from the skin of 'palombeta' Chloroscombrus chrysurus and 'guaiúba' Ocyurus chrysurus. GAGs were extracted with crude papain in 0.1 M sodium acetate buffer (pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by ion exchange chromatography on DEAE-cellulose column. The chemical composition (contaminant proteins and total carbohydrates) and the analysis by 0.5% agarose gel electrophoresis of fractions were also determined. Anticoagulant assays were performed by activated partial thromboplastin time (APTT) using normal human plasma and standard heparin (193.00 IU mg-1). The obtaining and fractionation procedures of GAGs were effective and similar chromatographic profiles were verified between the species. A similar mobility to dermatan sulfate was revealed for C. chrysurus. This GAG also showed a low activity of 3.30 IU mg-1.
Subject(s)
Animals , Pharmacology , Blood Coagulation , Marine Environment , Tissue Plasminogen Activator , Biodiversity , GlycosaminoglycansABSTRACT
Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 µM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 µg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.