Caracterização química e espectroscópica de polissacarídeos sulfatados extraídos de Spirulina sp.

Abstract

Spirulina sp are microalgae of the group of cyanobacteria rich in compounds with biological activity, being the antiviral, antioxidant and anticoagulant activity already attributed to sulphated polysaccharides present in these microalgae. However, the relationship between structure and biological activity of sulphated polysaccharides are not yet clearly established. In this work, polysaccharides from the commercial biomass Spirulina sp were extracted, characterized and analyzed for anticoagulant and antioxidant activities in vitro. The biomass was submeted to different conditions of polysaccharides extraction, those were analyzed by electrophoresis in 0.5% agarose gel in 0.05M buffer of 1,3-diaminopropane acetate (PDA) pH 9.0, dosages of protein, total sugar, uronic acid, sulfate and phenolic compounds. Subsequently, the fractions of sulfated polysaccharides were subjected to paper, ion exchange and gel filtration chromatographies, electrophoresis in 1.0% agarose gel in tris-borate-EDTA (TBE) buffer pH 8.3, Fluorophore Assisted Carbohydrate Electrophoresis (FACE), β-elimination and desulfation. In addition, were performed Raman, IR and NMR C13 solid state spectroscopy and analysis of the anticoagulant activity by the USP method and antioxidant activity by sequestration of the DPPH radical and β-carotene / linoleic acid system. The total sugar yields of the polysaccharides ranged from 2.27% to 20.23%. The DPB fraction obtained from the cell lysis process and papain proteolysis it presented one of the best yields and the most intense metachromasia in the electrophoresis in gel 0.5% agarose in PDA buffer, characteristic of the presence of sulphated polysaccharides. Thus, this fraction was chosen to follow the work. The analysis of the chemical composition revealed the presence of sulfate, rhamnose, glucuronic acid and galactose, being the glucose the major monosaccharide. Beside those, the presence of aldobiuronic acid in the structure of the sulfated polysaccharides is suggested. The estimated molecular weight for those polysaccharides was near to 130 and 40 kDa. When the depigmentation process was performed (DPBdespig fraction) it was observed that the chemical composition and molecular weight remained similar to the DPB fraction as well as the DPB 2.0M and DPB 3.0M fractions obtained after ion exchange chromatography. However, DPB 3.0M showed higher sulphate content. Analyzing the product of the fractions DPB and DPBdespig submeted to β-elimination, was observed a single estimated molecular weight, close to 100KDa. The spectroscopic analysis revealed characteristic bands of sulphated polysaccharides in all analyzed fractions, but the fraction DPB 3,0 M showed bands attributed to sulfate groups intense and well defined, while the fractions submitted to desulphation not. No analyzed fraction presented anticoagulant activity by the method used. The fraction DPB and DPBdespig showed good antioxidant activity, around 45% by DPPH sequestration method and 55% by β-carotene / linoleic acid system. The kinetics of the antioxidant activity suggested that the fractions act on the primary mechanisms of oxidation. Those same fractions when subjected to β-elimination and desulphation had their antioxidant activity reduced. The results suggest that the cell lysis method followed by proteolysis provided the best yield of sulfated polysaccharides with antioxidant activity from Spirulina sp, possibly related to the structure those polysaccharides.