ABSTRACT. Heparin (HEP) is known due to their side effects and the red seaweed Halymenia floresia (Hf) sulfated polysaccharides (SP) are heparinoids. In this study we purified the Hf-SP obtained from an aqueous extract and evaluated their anticoagulant activities. Hf-SP 1 (25°C), Hf-SP 2 (80°C) and Hf-SP 3 (80°C) were sequentially isolated. Hf-SP 3 had the highest sulfate content (37.45%). Hf-SP 3 was fractionated by ion exchange chromatography on a DEAEcellulose column using a NaCl gradient. Fractions were lyophilized and submitted to 0.5% agarose gel electrophoresis. The anticoagulant activity was evaluated by the activated partial thromboplastin time using rabbits plasma and expressed in international units per mg of SP using standard HEP ). The chromatographic procedure separated into four different SP fractions (F I, F II, F III and F IV) eluted at concentrations of 0.50, 0.75, 1.00 and 1.25 M of NaCl, respectively, reveling among them different marked on charge density, when compared by electrophoresis. F III had the highest anticoagulant activity (10.72 IU mg -1 ), suggesting that the sulfate is important in this process. In conclusion, our results suggest that sequential extractions of Hf-SP are an important biotechnological tool for identification of novel anticoagulants and studies of structural characterization are already in progress.Keywords: marine alga, sulfated macromolecules, purification, physical-chemical characterization, blood coagulation.RESUMO. Atividade anticoagulante de frações polissacarídicas sulfatadas de um extrato aquoso obtido da alga marinha vermelha Halymenia floresia (Clemente) C. Agardh. A heparina (HEP) é conhecida devido aos seus efeitos colaterais e os polissacarídeos sulfatados (PS) da alga marinha vermelha Halymenia floresia (Hf) são heparinoides. Objetivou-se purificar os Hf-PS obtidos de um extrato aquoso e avaliar suas atividades anticoagulantes. Foram isolados seqüencialmente Hf-SP 1 (25°C); Hf-SP 2 (80°C) e Hf-SP 3 (80°C). Os Hf-SP 3 apresentaram o maior conteúdo de sulfato (37,45%), sendo fracionados por cromatografia de troca iônica em coluna de DEAE-celulose utilizando um gradiente de NaCl. As frações obtidas foram liofilizadas e submetidas à eletroforese em gel de agarose a 0,5%. A atividade anticoagulante foi avaliada por meio do tempo de tromboplastina parcial ativada usando plasma de coelho e HEP padrão ). O procedimento cromatográfico separou em quatro diferentes frações de PS (F I, F II, F III e F IV), eluídas nas concentrações 0,50; 0,75; 1,00 e 1,25 M de NaCl, respectivamente, revelando diferenças marcantes na densidade de carga entre elas, quando comparadas por eletroforese. A maior atividade anticoagulante foi obtida na F III (10,72 UI mg -1 ), sugerindo que o sulfato é importante nesse processo. Os resultados sugerem que extrações seqüenciais de Hf-SP são uma ferramenta biotecnológica importante para a identificação de novos anticoagulantes. Estudos relacionados à caracterização estrutural já estão em andamento.Palavras-chave: alga marinha, macromolécu...
RESUMO Este estudo teve como objetivo isolar, fracionar e avaliar o potencial anticoagulante de iota-carragenanas (iCARs) da rodofícea Solieria filiformis, quando obtidas por dois métodos de extração (M I e M II). As i-CARs foram isoladas ABSTRACT This study aimed to isolate, fractionate and evaluate the anticoagulant potential of iota-carrageenans (i-CARs) from Solieria filiformis when two extraction methods (M I and M II) were used. i-CARs were isolated with papain in 0.1M sodium acetate (pH 5.0) containing 5mM cystein and 5mM EDTA (M I) or water (80°C) (M II), and then their chemical composition of total carbohydrates, free sulfate (FS) and contaminant proteins were determined. i-CARs were submitted to anionexchange chromatography (DEAE-cellulose) using a sodium chloride gradient,being evaluated the activated partial thromboplastin time (APTT) and prothrombin time of obtained fractions and compared to heparin (
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.Análise de dois métodos de secagem sobre o rendimento e atividade de polissacarídeos sulfatados extraídos da rodofícea Halymenia sp. RESUMO.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. Palavras-chave:Halymeniales, macromoléculas sulfatadas, métodos de secagem, coagulação sanguínea.
RESUMO.O cultivo da rodofícea Solieria filiformis vem sendo desenvolvido na praia de Flecheiras, Estado do Ceará, como perspectiva de utilização sustentável do recurso para biotecnologia, mas o crescimento de epífitas sobre a espécie compromete seu bom crescimento. Desta forma, avaliou-se a eficiência de duas metodologias (M I e M II) no isolamento da Kappa-carragenana (κ-CAR) da alga marinha vermelha Hypnea musciformis. A κ-CAR foi obtida mediante digestão proteolítica (M I) ou água quente (80°C) (M II). Os extratos foram analisados quanto à sua composição química, seguido por cromatografia de troca iônica em coluna de DEAE-celulose usando um gradiente de NaCl. A atividade anticoagulante foi avaliada pelo teste do tempo de tromboplastina parcial ativada (TTPA), usando plasma humano normal e heparina (HEP) padrão (193 UI mg ABSTRACT. Carragennan from Hypnea musciformis epiphyte obtained fromSolieria filiformis experimental culture at Flecheiras, Ceará State, Brazil. Solieria filiformis (Rhodophyceae) culture is being developed at Flecheiras Beach, state of Ceará, Brazil, for its future sustainable use in biotechnology. However, epiphyte growth on the species jeopardizes suitable development. The efficiency of two methods (M I and M II) in the isolation of kappa-carrageenan (κ-CAR) from red marine alga Hypnea musciformis has been evaluated. κ-CAR was obtained by proteolytic digestion (M I) or by hot water (80°C) (M II) and extracts were analyzed with regard to their chemical composition and then by ion-exchange chromatography on DEAE-cellulose column with NaCl gradient. Anticoagulant activity was evaluated by the activated partial thromboplastine time (APTT) test with common human plasma and standard heparin (HEP) ). Yield difference reached 4.28 ± 0.26%% (p < 0.05), and the lowest sulfate content was obtained with M I (17.00%) when compared to that with M II (26.30%). Further, different chromatographic profiles were reported between the two methods. Whereas M I was efficacious in obtaining κ-CAR anticoagulants (2.67 IU mg -1 ), alternative M II was efficient for the food industry. Results suggest that κ-CAR extraction method from H. musciformis depends on the industry's usage criterion.
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