| In this paper, the green alga Codium fragile (Suringar) Hariot, was used as starting material, the alga was firstly delipided with 85% ethanol, then extracted with room temperature water,90℃hot water and 2% sidium bicarbonate water solution at 70℃, three types of crude polysaccharides, which named as CFC, CFH and CFA, were acquired. The yields of CFC, CFH and CFA were 7.0%,8.1% and 0.9%, respectively. Their relative molecular weight (Mw), monosaccharide composition and structural features were determined with high performance gel permeation chromatography (HPGPC), high performance ion chromatography (HPIC) and Fourier transform infrared spectroscopy (FTIR), respectively. The results showed that CFC, CFH and CFA were composed of different contents of Gal, Ara, Glc, Man and Xyl, with the molar ratio of 3.65:2.71:1.00:0.85:0.55,1.10:1.01:1.00:0.92:0.21 and 0:51:0.34:1.00:0.61:0.05. Their sulfate contents were different, CFC have the highest sulfate content, then was CFH and CFA, of which were 23.4%,17.5% and 9.7%, respectively.Based on the physicochemical analysis of CFC and CFH, six kinds of polysaccharides (CFCP1～P6) and five kinds of polysaccharides (CFHP1～P5) were respectively separated from CFC and CFH by using Q-Sepharose FF anion-exchange chromatography. Two kinds of molecular uniform pure fractions CFCP4 and CFCP6 were further acquired from CFC with Sepharose 6B Fast Flow gel permeation chromatography. The Mw of CFCP4 and CFCP6 were 29 and 56 kD, separately; the sulfate content were 21.0% and 32.4%, respectively. CFCP4 and CFCP6 was mainly composed by galactose and arabinose, separately. Based on the NMR and methylation analysis to CFCP4 and CFCP6 and their desulfated, counterpart, CFCP4 and CFCP6 were determined as sulfated galactan and sulfated arabinan. The backbone of CFCP4 wasβ(1→3)-linked galactose (60%), and the branched chain wasβ(1→6)-linked galactose residues (40%). The sulfate groups were located at C4-OH and C6-OH, and pyruvic acid was linked at C3-OH and C4-OH of terminal galactose residues. The backbone of the CFCP6 was consists ofβ(1→3)-arabinose residues, and the branch points were found at C2-OH (30%), and the sulfate located at C4-OH and C2-OH.To determine their fine structures, the purified polysaccharide CFCP4 and CFCP6 were degraded with the methods of hydrogen form strong-cation exchange resin and free radical degradation, the oligosaccharides mixture was separated on BioGel-P4 column,14 kinds of oliogosaccharides were acquired. The sequence and structure of eight galactooligosaccharides and six arabinooligosaccharides were identified with electrospray ionization tandem mass spectrometry (ESI-CID-MS/MS). Their structures were as following:Galp6Sβ1→6Galp, Galpβ1→6Galpβ1→3Galp4S, Galp6Sβ1→6Galpβ1→6Galpβ1→6Galp, Galp6Sβ1→3Galpβ1→3 Galpβ1→3Galpβ1→6 Galp, Galp6Sβ1→6Galp4Sβ1→6Galp, Galp6Sβ1→6Galpβ1→6Galp4Sβ1→6 Galp,Galp6Sβ1→6Galpβ1→6Galpβ1→6Galpβ1→6Galp4S,3,4PyrGalp6Sβ1→6Galp; and Arbpβ1→3Arbp4S, Arbp4Sβ1→3Arbpβ1→3Arbp, Arbp4Sβ1→3Arbp4S, Arbp4Sβ1→3Arbp2Sβ1→3)Arbp,Arbp4Sβ1→3Arbp2Sβ1→3Arbpβ1→3Arbp,and Arbp4Sβ1→3Arbpβ1→3Arbp2Sβ1→3Arbpβ1→2Arbp.All these unique oligosaccharides offered useful message for construction of marine carbohydrate library. More importantly, they provide the foundation for the preparation of the oligosaccharide-chip and the investigation on the oligosaccharide-protein Interaction.Based on the structure analysis, the anticoagulant activities of three crude polysaccharides (CFC, CFH and CFA) and three purified polysaccharides (CFCP1, CFCP4 and CFCP6) were evaluated by assays of the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT). The results showed that CFC and CFCP1 showed high anticoagulant activities with heparin as positive control, CFCP6, which have similar physicochemical properties to CFCP1, showed lower anticoagulant activities. Apart from the structural features, the major differences of anticoagulant activities between these polysaccharides were associated with relative molecular weight, the higher Mw showed the stronger anticoagulant. The mechanism of anticoagulant of CFCP1 was due to intrinsic and common pathway, but not extrinsic pathway. The results provided theoretical foundation to marine drugs development.
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