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
Sulfated polysaccharides (SPs) have attracted growing interest for various biotechnological applications. We evaluated the efficiency of two methods of drying SPs (M I and II) extracted from Halymenia sp Rhodophyceae in order to compare the yield, purification and anticoagulant activity. The total SPs (TSPs) were first extracted with papain in 100 mM sodium acetate (pH 5.0) containing cysteine and EDTA (5 mM). The TSPs obtained were dried in an oven (M I) or lyophilized (M II) and then examined by ion exchange chromatography (DEAE-cellulose) using the NaCl gradient technique. The fractions were analyzed by 0.5% agarose gel electrophoresis and the in vitro anticoagulant activity was evaluated by the activated partial thromboplastin time test using normal human plasma and compared to heparin (HEP) (193.00 IU mg-1). There was a difference in TSP yield of 19.05% and similar chromatographic SP profiles. Electrophoresis revealed fractions with distinct resolutions. The fractions eluted with 0.75 M of salt (M I and II) were the most active, measuring 27.40 and 72.66 IU mg-1, respectively, when compared to HEP. Therefore, obtaining SP with anticoagulant activity from Halymenia sp. is more efficient by freeze-drying.