Polissacarídeos sulfatados da alga verde Ulva lactuca L.: caracterização química parcial e análise das atividades anticoagulante, antitrombótica e anti-inflamatória

Abstract

Cardiovascular diseases, including thrombotic disorders, are among the leading causes of mortality in the world. Heparin is a sulfated polysaccharide of animal origin used worldwide as a therapeutic agent (anticoagulant and antithrombotic). Sulfated polysaccharides (SP) of seaweed have complex structures, which have become interesting studies of their possible biological activities. In this study, SP from Ulva lactuca L. (Ul) and Fucus vesiculosus L. (Fv) were extracted by enzymatic proteolysis, followed by fractionation with different volumes of acetone. The fractions obtained were initially analyzed by agarose gel electrophoresis. The amounts of proteins, total sugars, uronic acids, and sulfate were determined by chemical dosages, and molecular weight was determined by agarose gel electrophoresis. Monosaccharide composition was determined by Fluorophore-assisted Carbohydrate Electrophoresis (FACE) and by paper chromatography. Also, analyses were made by FTIR/ATR and Raman spectroscopy. Anticoagulant activity was investigated in vitro by TTPa, TP, TT and by chromogenic assays (anti-factor Xa and IIa). Antithrombotic activity was evaluated in vivo by cava vein ligation model in rats and in vitro by measurement of NO production and metalloproteinase activity dosage in rabbit aorta cells culture (RAEC). Anti-inflammatory activity was investigated by measurement of NO, cytokines (IL-6 and TNF-α) and metalloproteinases in macrophage culture (RAW 264.7). F50Ul, F70Ul, F50Fv and F70Fv fractions presented bands with metachromasia and a migration pattern in agarose gel electrophoresis characteristic of the presence of sulfated polysaccharides led to the selection of fractions for further analyses. Chemical composition analysis revealed the presence of polysaccharides rich in sulfate and carboxyl, which was confirmed by spectroscopic analyses. SP had a high molecular weight, and U. lactuca L. fractions present rhamnose as major monosaccharide constituent, and glicose, galactose and uronic acids were also present., while F. vesiculosus L. fractions presented fucose, glicose and uronic acids. F50Ul, F50Fv and F70Fv fractions prolonged the TTPa (0.1-1.0 μg/mL) and inhibited Xa and IIa factors activity (1μg/ml). Also, these fractions (20 μg/g) showed in vivo antithrombotic effect after 24 hours of action. F50Ul showed higher activity at concentrations (5-10 μg/g) and showed a time-dependent effect, with a complete inhibition of thrombus formation after 12 hours of action (10 μg/g). In vitro evaluation, F50Ul (25-100 μg/mL) increased NO production by rabbit aortic endothelial cells and after 12 hours of incubation, this increase was more significantly. In RAW 264.7 cells culture, SP algae were able to interfere with NO production, interleukins, and metalloproteinases, and F50Fv and F70Fv inhibited inflammatory responses, while F50Ul stimulated the increase of these mediators. The results suggest important activities for U. lactuca L. SP and allowing propose further study to understand better the mechanisms involved, as well as their structures.