Extraction, Isolation, Structural Characterization And Anticoagulation Of Polysaccharides From Four Kinds Of Green Seaweeds

Four kinds of green seaweeds Codium fragile, Haematococcus pluvialis andCaulerpa peltata and an unidentified green alga, as starting material, were firstlydelipided, then extracted with water and alkaline solution, and15types of crudepolysaccharides were acquired at last. The relative molecular weight (Mw) andmonosaccharide composition were determined with high performance gel permeationchromatography (HPGPC) and high performance liquid chromatography (HPLC),respectively. Combined with the general analysis, the results showed thatpolysaccharides from Codium fragile (CFC, CFH, CFA, CFS and CFT) were sulfatedarabinogalactan and mannan, the ones from Caulerpa peltat (CPC, CPH, CPA andCPA2) were xylogalactan with slight sulfation, mannan, galactan and glucan,especially hot water extracted polysaccharide (CPH) with the high uronic acid content,the ones from an unidentified green alga (C-C, C-H and C-A2) were arabinogalactanand mannan, the ones from Haematococcus pluvialis (HPC, HPH and HPA) werecomplex, including common monosaccharides, and with high content of crude protein.Due to the broad range of average molecular mass of the polysaccharides from greenalgae, the polysaccharides should be purified at next step of the study.The crude water extract from Haematococcus pluvialis was fractionated byion-exchange chromatography on Q-Sepharose Fast Flow column. The purifiedextracts were designated HPC1, HPC2and HPC3. The monosaccharide composition,average molecular mass and structural feature of the polysaccharides were comparedby HPLC and Fourier Transform Infrared spectroscopy (FTIR), respectively. Theresults showed that the monosaccharide composition was complex and the maincontents were galactose, mannose, arabinose, glucose, glucuronic acid, galacturonicacid, xylose, rhamnose, fucose, glucosamine and galactosamine, especially the content of Galactose was higher than20%. The relative molecular masses ofpolysaccharides HPC1, HPC2and HPC3were502.6,373.2,577.5and300.5ku,respectively.To determine their fine structures, the alkaline extracts from Codium fragile CFSand CFT was degraded in the acid solution, then the oligosaccharides mixture wasseparated on Superdex peptide10/300GL column,6kinds of mannoseoliogosaccharides and10types of sulfated mannose oliogosaccharides were acquired.The sequences of these oligosaccharides were identified with electrospray ionizationtandem mass spectrometry (ESI-MS). The main structures of CFS were mannan andCFT were sulfated mannan. Besides, OxiCFS and CFSS were prepared by TEMPOoxidation and chemical sulfation of CFS respectively, in order to improve its watersolubility. The comparison of results of general analysis, monosaccharide composition,FTIR and NMR demonstrated OxiCFS contained typical β-D-1,4-polymannuronate,and furthermore to confirm that β-D-1,4-mannan was the predominant structure ofCFS.Based on the structure analysis, the anticoagulant activities of11kinds ofwater-soluble polysaccharides from green seaweeds and2types of polysaccharideswith chemically modified were evaluated by assays of the activated partialthromboplastin time (APTT), thrombin time (TT), prothrombin time (PT). The resultsrevealed that C-H and CFC exhibited high anticoagulant activities with heparin aspositive control. Their mechanisms of anticoagulant were due to intrinsic pathwaywith the association of monosaccharide composition and degree of sulfation.Meanwhile, we determined the tissue plasminogen activator (t-PA), plasminogenactivator, urokinase (u-PA) and plasminogen activator inhibitor-1(PAI-1), anddiscussed the effect of these polysaccharides on the fibrinolytic system. Onlypolysaccharides from an unidentified green alga C-C, C-H and C-A2could enhancethe activity of u-PA. All polysaccharides could effectively enhance the activity of t-PAand raised the proportionality of t-PA/PAI-1, i.e. these polysaccharides had significantfibrinolytic activity, especially CPH, CPA and C-H. The polysaccharides werelabelled with FITC by reductive animation reaction, and all these fluorescent labelled polysaccharides were prepared for glycoarray, and all of them offered bases for thepolysaccharide-protein interaction study in the future.