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% 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%, 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%) and number of molecular mass peaks. Therefore, the APTT of a fraction isolated from C. cupressoides was less potent than HEP.

Objetivou-se isolar, fracionar e avaliar a toxicidade in vivo dos polissacarídeos sulfatados (PSs) da rodofícea Hypnea musciformis, quando obtidos por três métodos de extração (M I; M II e M III). Os PSs foram extraídos com papaína em tampão acetato de sódio 100mM (pH 5,0), contendo cisteína e EDTA (5mM) (M I) ou água (25-80°C (M II); 80°C (M III)) e, em seguida, determinados sua composição química de carboidratos totais, sulfato livre (SL) e proteínas contaminantes (PCs). Os PSs foram submetidos à cromatografia de troca iônica (DEAE-celulose) usando um gradiente de cloreto de sódio, sendo avaliado o grau de homogeneidade e densidade de carga por eletroforese em gel de agarose das frações obtidas e comparadas à heparina. O ensaio in vivo foi realizado em grupos (n=6) de camundongos Swiss machos e fêmeas (24-33g), os quais receberam: PSs (9mg kg-1; i.p.) isentos do PCs (M I) e salina 0,9% (0,1mL 10g-1; i.p.), durante 14 dias consecutivos. No 15o dia, os animais foram anestesiados e sacrificados para coletas de sangue e órgãos, os quais foram utilizados para dosagens bioquímicas e correlações com suas massas corpóreas, respectivamente. O teor de SL (31,05±0,53%) (P<0,05) e o fracionamento, em DEAE-celulose, indicaram o M I mais eficiente na obtenção de PSs, comparado ao M II e M III. Os animais mostraram-se tolerantes aos PSs do M I e não se observou alteração de ordem hepática ou renal (P>0,05).

This study aimed to isolate, fractionate and evaluate the in vivo toxicity of sulfated polysaccharides (SPs) from Hypnea musciformis (Rhodophyta), when obtained by three extraction methods (M I, M II and M III). SPs were extracted with papain in 100mM sodium acetate (pH 5.0) containing cysteine and EDTA (5mM) (M I) or water (25-80°C (M II), 80°C (M III)), and then their chemical composition of total carbohydrates, free sulfate (FS) and contaminant proteins (CPs) was determined. SPs were submitted to ion-exchange chromatography (DEAE-celulose) using a sodium chloride gradient, being the degree of homogeneity and charge density evaluated by agarose gel electrophoresis of the fractions obtained and compared to heparin. The in vivo assay was performed using groups (n=6) of male and female Swiss mice (24-33g), which received: SPs (9mg kg-1, i.p.) absence of CPs (M I) and 0.9% saline (0.1mL 10g-1, i.p.), for 14 consecutive days. On the 15th day, collect blood and organs for biochemical dosages and corporal mass correlation, respectively, from the animals anesthetized and sacrificed were performed. The sulfate content of FS (31.05±0.53%) (P<0.05) and the fractionation by DEAE-cellulose showed M I more effectiveness in obtaining SPs compared to M II and M III. The animals were tolerable to SPs from M I, and it wasn't observed hepatic or renal alteration (P>0.05)

Este estudo teve como objetivo isolar, fracionar e avaliar o potencial anticoagulante de iota-carragenanas (i-CARs) da rodofícea Solieria filiformis, quando obtidas por dois métodos de extração (M I e M II). As i-CARs foram isoladas com papaína bruta em tampão acetato de sódio 0,1M (pH 5,0), contendo cisteína 5mM e EDTA 5mM (M I) ou água (80°C) (M II) e, em seguida, determinada sua composição química de carboidratos totais, sulfato livre (SL) e proteínas contaminantes. As i-CARs foram submetidas à cromatografia de troca iônica (DEAE-celulose) usando um gradiente de cloreto de sódio, sendo avaliado o tempo de tromboplastina parcial ativada (TTPA) e tempo de protrombina das frações obtidas e comparadas à heparina (193UI mg-1). Uma fração anticoagulante também foi submetida ao procedimento de eletroforese em gel de agarose a 0,5%. A diferença no rendimento de i-CARs entre os métodos foi 10,14%. A composição química de SL (29,40%) e o fracionamento, por DEAE-celulose, indicaram o M I mais eficiente na obtenção de i-CARs, comparado ao M II. O TTPA também foi somente alterado para as i-CARs do M I. Contudo, a atividade anticoagulante in vitro de uma fração rica (8,52UI mg-1) foi inferior à da heparina.

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 anion-exchange 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 (193IU mg-1). A rich fraction of anticoagulant was also submitted to 0.5% agarose gel electrophoresis procedure. The difference of yield between methods was 10.14%. The chemical composition of FS (29.40%) and the fractionation by DEAE-cellulose showed M I more effectiveness in the obtaining of i-CARs compared to M II. The APTT was also modified for i-CARs from M I. However, the in vitro anticoagulant activity of a rich fraction (8.52IU mg-1) was inferior to heparin.