Preparation And Structure Of Anticoagulant-active Sulfated Polysaccharides And Oligosaccharides From Green Alga Monostroma Latissimum

Marine green algae often contained bioactive substances with novel functions and structures because of their special living conditions, and the polysaccharides from marine green algae are of interest to new drug discovery. The sulfated polysaccharides from green algae Monostromaceae species show potent anticoagulant activity, and represent potential source to be explored. Until recently, few structural studies have been done on the polysaccharides from Monostromaceae.Monostroma latissimum is widely distributed in China, and has been used as fundamental source of food and drug in traditional Chinese medicine for thousands of years. Recently, the cultivation of marine green seaweed, Monostroma latissimum, was successfully obtained in China. Thus, the analyses of this species are very important in industrial application. In the present work, the heparinoid-active sulfated polysaccharides from the cultivation of Monostroma latissimum were isolated, and their structural characteristics were investigated by a combination of chemical and spectroscopic methods.The green alga, Monostroma latissimum from Yuhuan County in China, was extracted by cold （24°C） and hot （100°C） water to obtain two polysaccharides. The hot water extracted polysaccharide with higher yield and anticoagulant-active was further purified on Q-Sepharose Fast Flow ion-exchange and Sephacryl S-400/HR size-exclusion chromatography to generate three polysaccharides MAS, MBS and MCS of uniform size and charge. Chemical composition analysis indicated that MAS, MBS and MCS contained 74.1%, 68.1% and 65.3% total polysaccharides, with 26.1%, 28.5% and 31.8% sulfate, 4.44%, 3.94% and 3.27% glucuronic acid, and with a minor amount of protein. MAS, MBS and MCS were high rhamnose-containing sulfated polysaccharides with an average molecular weight of about 512.5 kDa, 58.4 kDa and 48.5 kDa, respectively. On the basis of methylation, one- and two-dimensional nuclear magnetic resonance （1D, 2D NMR） spectroscopic analyses, the polysaccharide chains of MAS, MBS and MCS were characterized to consist of [→3)-α-L-Rhap-（1→], [→2）-α-L-Rhap-（1→] and [→2,3）-α-L-Rhap-（1→] with different molar ratio, and the sulfate groups were substituted at C-2 of [→3）-α-L-Rhap-（1→] and C-3 of the [→2）-α-L-Rhap-（1→]. The disaccharide units in the structure of three polysaccharides were [→3）-α-L-Rhap（2SO₄）-（1→3）-α-L-Rhap→], [→3)-α-L-Rhap（2SO₄）-（1→2）-α- L-Rhap→], [→3)-α-L-Rhap-（1→2）-α-L-Rhap（3SO₄）→] and [→3)-α-L-Rhap（2SO₄）- （1→2,3）-α-L-Rhap→]. The polysaccharides had a high anticoagulant activity as evaluated by assays of the activated partial thromboplastin time and thrombin time. The investigation demonstrated that the polysaccharides appeared to be a sulfated rhamnan with different structural characteristics from other sulfated polysaccharides from Monostromaceae species, and could be a potential source of anticoagulant.According to the relative viscosity, high performance gel permeation chromatography, thin layer chromatography, polyacrylamide gel electrophoresis, the hydrolysis condition of the sulfated polysaccharide MAS was optimized. With the condition of 0.1 mol/L H2SO₄ at 40°C for 75 min and 80°C for 60 min, the polysaccharide MAS was degraded, and isolated to generate seven low molecular weight polysaccharides （MAS1^MAS7）, their molecular weights ranged from 405.0 kDa to 5.1 kDa. The physiochemical properties, monosaccharide composition and infra-red spectrum characteristics of the polysaccharides were all similar to those of the parent polysaccharide MAS. The structure of the polysaccharide MAS5 as representation was studied by methylation and 1D, 2D NMR spectroscopy, and the results showed that the structure consisted of [→3)-α-L-Rhap-（1→], [→2）-α-L-Rhap-（1→] and [→2,3）-α-L-Rhap-（1→] in the molar ratio of 4:1:1. The sulfate groups were substituted at C-2 of the [→3）-α-L-Rhap-（1→] and C-3 of the [→2）-α-L-Rhap-（1→], which is consistent with the parent polysaccharide MAS. The results indicated that the structure of the low molecular weight polysaccharides after mild acid hydrolysis was not destroyed. Base on the anticoagulant activity of APTT, the sulfated polysaccharides with different molecular weights showed different anticoagulant activities. The results suggest that the molecular size has a profound effect on the anticoagulant activity of the sulfated polysaccharides from Monostroma latissimum, and plays an important role in the anticoagulant action.The oligosaccharide fragments of the sulfated rhamnan MAS from Monostroma latissimum were obtained from partial depolymerization of the polysaccharide by mild acid hydrolysis, and their sequences were investigated with ES-MS. On the basis of negative-ion electrospray tandem mass spectrometry with collision-induced dissociation （ES-CID-MS/MS） and 1D, 2D NMR spectroscopy, the oligosaccharide R2S was characterized to be a monosulfated rhamnobiose and consisted ofα-L-Rhap-（2SO₄）-（1→3）-α-L-Rhap. The fragmentation pattern of the homogeneous disaccharide compositions in the product ion spectra was established, and was then applied to sequence determination of the other oligosaccharides. The results demonstrated that it was possible to derive the sequence of the sulfated rhamno-oligosaccharide directly from the glycosidic cleavage fragmentation in the product ion spectra, and negative-ion ES-CID MS/MS affords an efficient method for the sequence determination of oligosaccharides derived from the sulfated rhamnan. The results indicated that the linkage pattern of the sulfated rhamno-oligosaccharides are all [→3）-α-L-Rhap-(1→]. The structure of the sulfated rhamno-oligosaccharide was the first time to report.