Antimicrobial effect of a crude sulfated polysaccharide from the red seaweed Gracilaria ornata

The aim of this study was to determine the yield, chemical composition, specific rotation (SR), infrared (IR) spectroscopy and the effect on bacterial growth of a crude sulfated polysaccharide (SP) from the red marine alga G. ornata (Go). Go-1 (25°C), Go-2 (80°C), and Go-3 (80°C) were sequentially extracted and yielded 9.2%. The contents of sulfate (5.88-10.3%) and proteins (0.1-3.7%) were small. The values of SR were [µ]D20°f -19.0, -51.0, and -56.5, respectively. IR spectrums showed the presence of galactose-4 sulfate and absence of 3,6-anydrogalactose-2 sulfate, galactose-6 sulfate and galactose-2 sulfate. SR and IR techniques confirmed SPs. Go-3 was tested on the growth of bacteria (Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Pseudomonas aeruginosa, Salmonela choleraesuis and Salmonela typhi), but only E. coli was inhibited.

Analysis of two drying methods on the yield and activity of sulfated polysaccharides extracted from Halymenia sp (Rhodophyceae) / Análise de dois métodos de secagem sobre o rendimento e atividade de polissacarídeos sulfatados extraídos da rodofícea Halymenia sp

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.

Os polissacarídeos sulfatados (PS) têm despertado interesse crescente para diversas aplicações em biotecnologia. Avaliou-se a eficiência de duas metodologias de secagem de PSs (M I e II) extraídos da rodofícea Halymenia sp. a fim de se comparar o rendimento, a purificação e a atividade anticoagulante. Inicialmente, os PS totais (PSTs) foram extraídos com papaína em tampão acetato de sódio 100 mM (pH 5,0) contendo cisteína e EDTA (5 mM). Os PSTs foram secados em estufa (M I) ou liofilizados (M II), seguido por cromatografia de troca iônica (DEAE-celulose) utilizando um gradiente de NaCl. As frações foram analisadas por eletroforese em gel de agarose a 0,5% e a atividade anticoagulante "in vitro" avaliada por meio do teste do tempo de tromboplastina parcial ativada usando plasma humano normal e comparadas à heparina (HEP) (193.00 UI mg-1). Verificaram-se diferença no rendimento de PSTs (19,05%) e semelhantes perfis cromatográficos de PS, revelando, por eletroforese, frações de PS distintas em grau de resolução entre os métodos. As frações eluídas com 0,75 M de sal (M I e II) foram as mais ativas, com valores de atividade da ordem de 27,40 e 72,66 UI mg-1, respectivamente, em relação à HEP. Portanto, a secagem por liofilização seria uma forma mais eficaz na obtenção de PSs com atividade anticoagulante de Halymenia sp.

Anticoagulant activity of a sulfated polysaccharide isolated from the green seaweed Caulerpa cupressoides

The aim of this study was to evaluate certain molecular characteristics of a sulfated polysaccharide (SPs) with anticoagulant properties, isolated from Caulerpa cupressoides (Chlorophyta). Crude SPs were extracted by proteolytic digestion (papain), followed by ion-exchange chromatography on a DEAE-cellulose column. The fractions obtained were analyzed for molecular mass, 0.5 percent agarose gel electrophoresis and chemical composition. The activated partial thromboplastin time (APTT) test was applied using normal human plasma and standard heparin (HEP) (193 IU mg-1). The yield was ~ 3 percent, and the chromatography procedure separated the material into three different SP fractions (F I, F II and F III), eluted at the concentrations of 0.50, 0.75 and 1.00 M of NaCl, respectively. Only fraction F II was active (24.62 IU mg-1), with high sulfate content (23.79 percent) and number of molecular mass peaks. Therefore, the APTT of a fraction isolated from C. cupressoides was less potent than HEP.

Polissacarídeos sulfatados isolados das clorofíceas Caulerpa racemosae Caulerpa cupressoides – extração, fracionamento e atividadeanticoagulante / Sulfated polysaccharides isolated from Caulerpa racemosa and Caulerpa cupressoides (Chlorophyceaes) – extraction, fractionation and anticoagulant activity

A incidência de doenças cardiovasculares e os efeitos adversos da heparinoterapiatêm motivado a busca por novos agentes terapêuticos e os polissacarídeos sulfatados (PS) de algasmarinhas têm sido reportados como fontes alternativas para tal. Objetivou-se avaliar o potencialanticoagulante dos PS totais (PST) isolados e fracionados das clorofíceas Caulerpa racemosa eCaulerpa cupressoides. Inicialmente, os PST foram extraídos com papaína em tampão acetato desódio 0,1 M (pH 5,0) contendo cisteína 5 mM e EDTA 5 mM, seguidos de fracionamento emcoluna de troca iônica de DEAE-celulose com gradiente de NaCl. As frações obtidas foramanalisadas por eletroforese em gel de agarose a 0,5% e a atividade anticoagulante, mensurada pelotempo de tromboplastina parcial ativada (TTPA), usando-se plasma humano normal ecomparada a uma curva-padrão de heparina (193 UI mg-1). Verificaram-se semelhantes perfiscromatográficos entre os PS de ambas as espécies, porém com padrões de mobilidades distintasquando as frações foram comparadas por eletroforese. Os PS modificaram o TTPA, cujasatividades anticoagulantes foram de apenas 21,23 e 24,36 UI mg-1, quando eluídos com 0,75 M desal para C. racemosa e C. cupressoides, respectivamente. Portanto, PS anticoagulantes isolados dasclorofíceas C. racemosa e C. cupressoides resultaram em efeitos anticoagulantes inferiores aos daheparina. Estudos comparativos dessas moléculas também são sugeridos como ferramentasauxiliares na identificação de algas do mesmo gênero.

The incidence of cardiovascular diseases and adverse effects from heparintherapy have led to asearch for new therapeutic agents, and the sulfated polysaccharides (SP) of seaweeds have beenreported as alternative sources. The aim of this work was to evaluate the anticoagulant potentialof total SP (TSP) isolated and fractionated from Caulerpa racemosa and Caulerpa cupressoides(Chlorophyceaes). Initially, the TSP were extracted with papain in 0.1 M sodium acetate buffer(pH 5.0) containing 5 mM cysteine and 5 mM EDTA, followed by fractionation on ionexchange DEAE-cellulose column with NaCl gradient. The obtained fractions were analyzed by0.5% agarose gel electrophoresis and the anticoagulant activity measured by the activated partialthromboplastin time (APTT) using normal human plasma, and compared to a standard heparincurve (193 IU mg-1). Similar chromatographic profiles of SP were shown on both species, butwith distinct mobility patterns, when the SP fractions were compared by electrophoresis. SPeluted with 0.75 M of NaCl modified the APTT, whose anticoagulant activities were only 21.23and 24.36 IU mg-1 for C. racemosa and C. cupressoides, respectively. Therefore, anticoagulant SPisolated from chlorophyceaes showed effects inferior to heparin, and comparative studies of thesemolecules are also suggested as auxiliary tools in the identification of algae of the same genus.