| Codium divaricatum is a sort of green algae plant in ocean, and largely grows in middle and low part of the intertidal zone. The genus Codium (Codiaceae, Codiales, Chlorophyta) are widely distributed in the coastal waters of China. In this paper, the sulfated polysac char ides from Codium divaricatum were isolated, and their structural characteristics were investigated by a combination of chemical and spectroscopic methods. The polysaccharides were also submitted to degradation, and a controllable acid degradation method was established to prepare low molecular weight polysaccharides and oligosaccharides. The structures of the low molecular weight polysaccharides and oligosaccharides were studied by nuclear magnetic resonance spectroscopy and mass spectrometry. The polysaccharides and low molecular weight polysaccharides possessed anticoagulant activities. The research results were as follows:1. The green alga, Codium divaricatum from Yantai of China, was extracted by cold water (24 ℃), hot water (100 ℃) and alkali liquid (0.5mol/L NaOH) successively to obtain three polysaccharides. Three kinds of polysaccharides were named as CP, HP and AP, and the yields of the polysaccharides were 10.72%,8.41% and 1.54%, respectively. The chemical compositons of the polysaccharides were analyzed by chemical method, TLC and HPLC. The results showed that the total sugar contents of CP, HP and AP were 66.7%,65.4% and 44.3% respectively, the sulfate contents were 25.6%,11.8% and 28.2%, respectively. The protein content of AP was the highest, and was 20.4%. Three sulfated polysaccharides were mainly composed of glactose with different proportions of arabinose, glucose and mannose.2. Based on the physicochemical analyses, the two polysaccharides CP and HP were first separated by Q-Sepharose Fast F low ion-exchange chromatography giving three factions CP1,CP2 and HP1,and then further purified by Sephacryl S-400/HR size-exclusion chromatography to generate three homo geneous polysaccharides CP1-1,CP2-1 and HP1-1.Chemical composition analyses indicated that CP1-1, CP2-1 and HP 1-1 contained 62.4%,64.8% and 78.1% total sugar,respectively,with 0.5%,0.4% and 3.1% protein,30.4%,23.7% and 12.3% Sulfate,4.5%,3.2% and 6.7% uronic acid.The pyruvic acid content of CP2-1 was about 6.6％.The average molec ular weights of CPl-1,CP2-2 and HP1-1 were estimated by HPGPC to be about 203kDa,37.9kDa and 297kDa,respectively.Monosaccharide composition was tested by PNP pre-column high performance liquid chromatography,the results showed that CP1-1 was mainly composed of arabinose and galactose,CP2-1 was mainly composed of galactose,and HP1-1 was mainly composed of glucose,galactose and arabinose.Determination of the absolute configuration of the main monosaccharides showed that arabinose was present in the L-configuration,galactose and glucose were present in the D-configuration.On the basis of methylation, one-and two-dimensional nuclear magnetic resonance(1D,2D NMR)spectroscopic analyses,the polysaccharides CP1-1,CP2-1 and HP1-1 possessed different structural characteristics.CPl-1 mainly consisted of [→4)-β-L-Arap-(1→]， [→3)-β-D-Galp-(1→], [β-Galp-(1→] and [→3,4)-β-D-Galp-(→], and the sulfate groups were substituted at C一3 of [→4)-β-L-Arap-(1→]and C-4 of[→3)-β-D-Galp-(1→].CP2-1 was galactan,with [→3)-β-D-Galp(1→]as backbone chain having a small degree of branching with single(1→)-linked β-D-galactopyranose units attached to the main chain at C一4 posinions.Sulfate groups were at C-4 of(1→3)-linked β-D-galactopyranose and C一6 of non-reducing terminal galactose residues.In addition,the ketals of pyruvic acid were found to be at 3,4-of non-reducing term.nal galactose residues forming a five.membered ring.HP1-1 was mainly composed with [→4)-α-Glcp-(1→], [→3)-α-Glcp-(1→], [→3,4)-α-Glcp-(1→], [→4)-β-Arap-(1→], [β-D-(3,4Pyr)-Galp-(1→]and[→3)-β-D-Galp-(1→].Sulfate goups were at C-3 of [→4)-α-Glcp-(1→] and C-4 of[→3)-β-D-Galp-(1→].CP1-1,CP2-1 and HP1-1 possessed high anticoagulant activities as assessed by the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) assays. But the anticoagulant activities of the three polysaccharides were lower than that of heparin.3. According to the results of high performance gel permeation chromatography and thin layer chromatography, the hydrolysis condition of the sulfated polysaccharide CP1 was optimized. With the condition of 0.05 mol/L H2SO4 at 40 ℃ for 1.5h and 0.01 mol/L H2SO4 at 60 ℃ for 3h, the polysaccharide CP1 was degraded, and purified by Sephacryl S-400/HR size-exclusion chromatography. Six low molecular weight polysaccharides (L1～L6) were obtained, and their molecular weights were 369 kDa,242 kDa,124 kDa,102 kDa,43 kDa and 22 kDa respectively. The six polysaccharides were composed of arabinose and galactose with different molar ratio, and their sulfate contents were 22.5%～29.9%. Their compositons were similar to those of the parent polysaccharide CP1. The structure of the polysaccharide L5 as representation was studied by methylation analysis and ID,2D NMR spectroscopy, and the results showed that the structure of L5 consisted of [→3)-Galp-(1→], [→4)-Galp-(1→] and [→4)-Arap-(1→] residues. Sulfate groups were at C-2 and C-4 of [→3)-Galp-(1→], C-2 of [→4)-Galp-(1→] and C-3 of [→4)-Arap-(1→], thus L5 was highly sulfated arabingalactan. Base on the anticoagulant activity of APTT, the sulfated polysaccharides with different molecular weights showed different anticoagulant activities. The sulfated polysaccharide fragment with a lower molecular weight had a lower anticoagulant activity. The results suggested that the molecular size has a profound effect on the anticoagulant activity of the sulfated polysaccharides from Codium divaricatum.4. Eight oligosaccharide fractions F1～F8 were obtained after partial depolymerization of the sulfated galactan CP2-1 from Codium divaricatum by TFA and purification by Bio-Gel P4 gel chromatography. The results of ES-MS analyses showed that DP of the oligosaccharides was 1～11, and the oligosaccharides were sulfated and pyruvylated. The structure of the purified fraction F3 as representation was studied by 1D,2D NMR and ES-CID-MS/MS, and the structure of F3 was characterized to be β-D-Galp-(4SO4)-(1→3)-D-Galp. The fragmentation pattern of the homogeneous disaccharide G2S in the product ion spectra was recognized by ES-CID MS/MS, and then the established principles were applied to sequence determination of the other galacto-oligosaccharides. The results showed that the oligosaccharides were linked by 1→3 glycosidic bond, and sulfate groups were at C-4 of [→3)-β-D-Galp-(1→] and C-6 of [β-D-Galp-(1→]. In addition, the ketals ofpyruvic acid were found at 3,4- of non-reducing terminal galactose residues forming a five-membered ring. The established principles of negative-ion ES-CID MS/MS affords an efficient method for the sequence determination of oligosaccharides derived from the sulfated polysaccharides.The research results of this paper deepened the understanding of the structure and activity of the polysaccharides from Codium divaricatum, and laid a foundation for the development and utilization of Codium divaricatum. The research also enriched the data resources of "ocean sugar library", and was of great significance for the development of marine drugs with Chinese characteristics. |
PDF Full Text Request