Estudo químico da alga Lithothamnion calcareum e avaliação da atividade inibitória do rolamento de leucócitos

Abstract

Lithothamnion calcareum (Hapaladiceae) is a marine alga found in deep waters, commercialized in Brazil and abroad as food supplement of calcium and other minerals. Polysaccharides have been reported as constituents of marine algae, possessing severalbiological activities, including anti-inflammatory effect. The species was obtained from a commercial source (Phoster Algamar, Belo Horizonte, Brazil). Different conditions for polysaccharide extraction were evaluated, including aqueous medium under heating (40, 60,70, 80 and 100°C) and alkaline extraction with 1% and 2% w/v Na2CO3 solution, at 60 °C. Ethanol was added to the extracts (4:1) for polysaccharide precipitation, following dialysation(cut off 10,000 Da) and extract concentration. Extractives ranged from 0.008 to 0.050%, being the highest yield found for alkaline extraction (2% w/v Na2CO3, 60 °C). The polysaccharide mixture was fractionated by size exclusion chromatography (Sephadex G-100 and G-25) andion exchange chromatography (Sepharose Fast Flow). The obtained compounds were characterized by UV and IR spectrometry, in addition to colorimetric reaction with phenolsulphuric acid. Four polysaccharides were isolated and had their chemical compositioncharacterized by quantification of sulfate content, carried out by turbidimetry, along with uronic acid, proteins and total sugar contents, determined by colorimetric methods. The molecular weight (MW) was estimated by high performance size exclusion chromatography(HPSEC), by comparison with pullulan standards of different MW. The polysaccharides presented MW in the range 38 ± 2 to 270 ± 20 KDa and uronic acid contents from 1.7 ± 0.1 to 9.0 ± 1.0 %. The protein proportion varied from 3.0 ± 1.0 to 32.0 ± 1.0%, whereas total sugarcontent was found between 6.0 ± 1.0 and 51.0 ± 2.0%. On its turn, sulfate contents reached up to 37 ± 5% in some polysaccharides and showed minimum value of 3 ± 3%. The antiinflammatory activity was assayed in mice, using a model of leukocyte rolling inhibition(n=3) employing intravital microscopy. Fucoidin and saline were employed as positive and negative controls, respectively. The intravenous injection of sulfated polyssaccharides B1P 1- 19 (100 mg/Kg) and B1P 22-37 (100 mg/Kg) in mice reduced leukocyte rolling byapproximately 90%, whereas fucoidin (10 mg/Kg) induced a similar response. Analysis of chemical composition and biological activity induced by the polysaccharides indicate that high level of sulfatation and high MW favour inhibition of leukocyte rolling. The isolationand chemical characterization of polysaccharides from L. calcareum and the evaluation of their potential anti-inflammatory activity are here reported for the first time.