Extraction, Isolation, Structural Characterization And Anticoagulation Studies Of Polysaccharides From Schizochytrium Limacinum And Two Diatom Species

In this paper, the characterization and structural features of polysaccharides and their oligomers from marine fungi Schizochytrium limacinum were studied. Extracted with70℃hot water and4%NaOH water solution at50℃, respectively, two types of crude polysaccharides, which named as SLW and SLA, were acquired from the marine fungi. The yields of SLW and SLA were4.18%and1.10%, respectively. Their chemical properties, monosaccharide compositions, average molecular weights and structures were determined by high performance gel permeation chromatography (HPGPC), high performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR). The experimental results showed that SLW and SLA mainly consisted of galactose (Gal,>75%) with a small amount of mannose (Man), glucose (Glc), and glucosamine (GlcN). Their sulfate contents were9.38%and14.58%, respectively; molecular weight were633.2kD and48.1kD, respectively.Two polysaccharides, SLW1and SLW2, were separated from SLW on Q-Sepharose Fast Flow anion exchange chromatography. The general analysis results showed that the two polysaccharides were mainly conposed of Gal (>75%) with a small amount of Man, Glc and GlcN. Their relative molecular weight were39.79kD and103.83kD, respectively. Their sugar contents were54.2%and68.4%, respectively. Their sulfate contents were8.16%and12.84%, respectively. Their protein contents were10.55%and9.39%, respectively. The existence of sulfate esters located at C6hydroxyl groups of Gal residues was comfirmed on FTIR spectrum.The structure characterization of SLW2was further studied by methylation and2D NMR spectroscopy. Our data suggested that SLW2consisted of→-6)Galf(1→, →5)Galf(1→,→3,4)Galp(1→,→3)Galp(1→,→4,6)Galp(1→,→3)Galp(1→,→6)Galp(1→and→6)Galp2S(1→residues.To determine the fine structure of SLW2, its oligosaccharides was prepared by TFA acid hydrolysis, and separated on Superdex Peptide chromatography. Seven galacto-oligosaccharides were acquired. Their sequences and structures were identified with negative-ion electrospray tandem mass spectrometry (ESI-CID-MS/MS). Results showed that they were all composed of1→6linkage and their sulfate groups were located at C6of non-reducing ends or C2of reducing ends of the residues. More importantly, it provided samples with multiply structural properties for preparation and carbohydrate-protein interaction studies of carbohydrate macroarray.By methylation、2D NMR spectroscopy and ESI-MS/MS technology, the results indicated that SLW2was composed of→3,4)-α-Galp(1→6)-β-Galf(1→3)-β-Galp(1→3)-α-Galp2S(1→4,6)-β-Galp-(1→4as its backbone chain, with→5)-β-Galf-(1→and β-D-Galp6S-(1→[6)-β-D-Galp-(1]n→6)-β-D-Galp2S-(1→(n=0～3) in side chains.Besides, we have also studied the general characters of polysaccharides extracted from Cylindrotheca fusiformis and Chaetoceros muelleri. Results showed that polysaccharides form C. fusiformis, CF1and CF2, mainly contained Man, Rha, Gal and Xyl, with little amount of GlcA and GalA, The polysaccharides form C. muelleri, CM1and CM2, were mainly composed of Rha, Gal and Xyl, but no uronic acid existed.The sulfate contents of both CF1and CF2were about2.7%, respectively, compared that of CM1and CM2about1.5%, respectively. Their FT-IR spectra analysis indicated that they were sulfated polysaccharides but with low sulfation levels.The anticoagulant activities of polysaccharides, SLW, SLA, SLW1, SLW2, CF1, CF2, CM1and CM2, were evaluated by assays of the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT). The results showed that all the eight polysaccharides with different resources presented anticoagulant activities to some extent. But the effect was much weaker than that of Low-Molecular-Weight Heparin. Some structure modification could be furtherly done to promote their anticoagulant activities.