Studies On Isolation, Structure And Anticoagulant Activity Of The Sulfated Polysaccharide From Monostroma Latissimum

Monostroma latissium is a solt of green algae plant in ocean. It grows widely in the brackish water area in the upper part of the intertidal zone. Monostroma latissium exhibits many biological activities such as anticoagulant, antiviral, antioxidant activities and radioprotective effect. It has been confirmed that the main active substance in Monostroma latissium is soluble sulfated polysaccharide.In this paper, the polysaccharide from Monostroma latissium was extracted by cool water, boiled water and alkali liquid successively. Three kinds of polysaccharides were obtained and named as CE, HE and AE, respectively. The chemical characteristics of the polysaccharides were analyzed by chemical method, GC and HPLC. The results showed that the total yield of the polysaccharides was 40.41% and the three polysaccharides were soluble sulfated polysaccharides with the different sulfate content. The sulfate contents of polysaccharides are 21.33%, 24.92% and 25.34%, respectively. Three sulfated polysaccharides were found to contain mainly rhamnose with small amounts of glucose and xylose and trace amounts of glactose and mannose. The uronic acid is consisted of glucuronic in the sulfated polysaccharide.Based on the physicochemical analysis, three pure fractions HEA, HEB and HEC were obtained from HE using ion-exchange chromatography and low-pressure gel permeation chromatography. The chemical characteristics and primary structures of the pure fractions were investigated. The result showed that HEA was a polysaccharide with high molecular size (725.4kD). The molecular weights of HEB and HEC (54.8 kD, 68.8 kD) are similar. The monosaccharide compositions of the three polysaccharides are similar. The backbone of HEA and HEB were consisted of 1,3-Rha, 1,2-Rha and the sulfate groups were substituted at C-4 or at C-3 in 1,2-Rha. While HEB was a kind of sulfated rhamnan consisting of 1,2-Rha with little branch, HEA was a complicated sulfated rhamnan consisting of 1,2-Rha, 1,3- Rha, 1,2,4-Rha and 1,2,3,4-Rha.The sulfated polysaccharide from Monostroma latissimum was hydrolyzed into smaller fragments with different molecular weights by H2O2 or H2SO4 degradation. The polysaccharide fragments with different molecular weights were further fractionated by gel permeation chromatography. Molecular weight of the polysaccharide fragments prepared by H2O2 were 216.4kDa, 123.7kDa, 61.9kDa, 26.0kDa and 10.6kDa, respectively; the molecular weight of polysaccharide fragments prepared by H2SO4 degradation were assayed as follows: 156.8 kDa, 61.7 kDa, 46.1 kDa, and 15.7 kDa. Moreover, chemical and instrumental analysis indicated that chemical components and structure of the sulfated polysaccharide fragments are similar to that of the parent sulfated polysaccharide. The results showed that the basic structures of polysaccharides had not been destroyed by H2O2. H2SO4 degradation had little effect on the basic structure of the sulfated polysaccharide. A series polysaccharide fragments with different molecular weights can be prepared effectively by the two ways.Anticoagulant activities of the parent sulfated polysaccharide and its fragments were investigated by studying the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) using human plasma. The study on anticoagulant activity showed that the sulfated polysaccharide isolated from Monostroma latissimum inhibited both the intrinsic and or common pathways of coagulation and thrombin activity or conversion of fibrinogen to fibrin. The sulfated polysaccharide fragments with higher molecular weight had slightly higher anticoagulant activities than their parent sulfated polysaccharide at the same concentration. The sulfated polysaccharide fragment with a lower molecular weight had a lower anticoagulant activity. These 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 anticoagulant action. The sulfated polysaccharides from Monostroma latissimum require longer chains to achieve complete thrombin inhibition.In the last part of the paper, the oligosaccharides from the parent sulfated polysaccharide were prepared by H2SO4 method. Seven oligosaccharide products were obtained by gel chromatography. The structures of the oligosaccharides were characterized by methylation analysis, IR, ESI-MS and ESI-CID-MS/MS. The results showed that the oligosaccharide products are mainly consist of disaccharide to heptasaccharide. The oligosaccharide was straight line consisting of 1,3-Rha and the sulfate groups were substituted at C-2 or C-4 in 1,3-Rha.The active substance in Monostroma latissimum is polysaccharide. It is important and neccssary to research the structure and activity of the polysaccharides. This research provided the theory information for the sulfated polysaccharide, settled the foundation for utilizing the sulfated polysaccharide from Monostroma latissimum and develop the establishing the"saccharide-bank".