| In this paper,3kinds of algae (Nemacystus decipiens, Gracilaria asiatica andGelidium amansii) were used as starting materials. The algae were extracted withwater and alkaline solution after delipided, thus，12types of crude polysaccharideswere acquired. Their relative molecular weight (Mw) and composition ofmonosaccharides were determined with high performance gel permeationchromatography (HPGPC) and high performance liquid chromatography (HPLC),respectively. The results showed that most of these crude polysaccharides containeddifferent content of fucose. Polysaccharides from Nemacystus decipiens have thehighset fucose content, and the hot water extract fraction, named NW2, wascomposed of fucose and glucuronic acid, with the molar ratio of7:2. Andpolysaccharides from Gracilaria asiatica and Gelidium amansii were mainlycomposed of galactose, glucose and fucose; the cold water extract of Gracilariaasiatica has a relative higher content of fucose (18%). Their sulfate contents weredifferent, NW2has the highest sulfate content (20.3%), but polysaccharides fromGracilaria asiatica have a lower sulfate content (no more than10%), andpolysaccharides from Gelidium amansii were neutral. Based on the analysis above,4kinds of polysaccharides (named NP1-4) and5kinds of polysaccharides (GP1-3)were respectively separated from NW2and GVC by using Q-Sepharose FFanion-exchange chromatography. The main component were NP2and GP2, their Mwwere487kD and1173kD, separately; the sulfate content were19.4%and5.77%,respectively, and the content of fucose were77%and20%, respectively. Based onthe FTIR, NMR and methylation analysis, NP2was determined as sulfated fucan. Thebackbone of NP2was (1â†'3)-linked fucose (80%), and the branched residue was (1â†'2)-linked glucuronic acid (20%). To determine their fine structures, the purifiedpolysaccharide NP2was degraded in the acid solution, then the oligosaccharidesmixture was separated on BioGel-P4column,8kinds of oliogosaccharides were acquired. The sequence and structure of these fucoseoligosaccharides were identifiedwith electrospray ionization tandem mass spectrometry (ESI-CID-MS/MS). Theirstructures were listed as following: Fucp2S4Ac1â†'3Fucp, Fucp4S1â†'3Fucp4S,Fucp1â†'3(GlcUAp1â†'2) Fucp, Fucp2S4Ac1â†'3Fucp1â†'3Fucp，Fucp4S1â†'3(GlcUAp1â†'2) Fucp4Ac1â†'3Fucp， GluUAp1â†'2Fucp1â†'3(GlcUAp1â†'2) Fucp1â†'3Fucp， FucpSAc1â†'3Fucp1â†'3(GlcUAp1â†'2)Fucp1â†'3(GlcUAp1â†'2) Fucp1â†'3Fucp andFucpS1â†'3FucpAc1â†'3Fucp1â†'3(GlcUAp1â†'2) Fucp1â†'3FucpS1â†'3(GlcUAp1â†'2) Fucp1â†'3FucpS. The sulfate groups were located at C2-OH andC4-OH, and acetyl groups were linked at fucose residues. All these uniqueoligosaccharides offered useful message for construction of marine carbohydratelibrary. More importantly, they provide the foundation for the preparation of theoligosaccharide-chip and the investigation on the oligosaccharide-protein Interaction.Based on the structure analysis, the anticoagulant activities of purifiedpolysaccharides NP1-4and GP1-3were evaluated by assays of the activated partialthromboplastin time (APTT), thrombin time (TT), prothrombin time (PT). At thesame time, we determined the tissue plasminogen activator (t-PA), plasminogenactivator, urokinase (u-PA) and plasminogen activator inhibitor-1(PAI-1), anddiscussed the polysaccharides’ effect on the fibrinolytic system. The results showedthat NP2, NP4and GP2exhibited slightly anticoagulant activities with heparin aspositive control. These polysaccharides could effectively enhance the activity of t-PAand the proportionality of t-PA/PAI-1was raised, it accounts that thesepolysaccharides had high fibrinolytic activity. For the purpose of studying on thesaccharide-protein interaction, the purified polysaccharides were marked by FITC andthe oligosaccharides were prepared as neoglycolipids (NGLs), glycochip wereprepared at last. The results provided theoretical foundation to marine oligosaccharidedrugs development. |
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