Isolation, Structural Characterization And Anticoagulant Activity Of The Sulfated Polysaccharides From Green Alga Ulva Fasciata

The green alga Ulva fasciata belonging to Ulva species is widely distributed onthe coast of China, and often contains novel bioactive substances because of itsspecial living condition. The polysaccharides from green algae hold a great potentialapplication in new drug discovery and biology. In the present work, the sulfatedpolysaccharides from the green alga Ulva fasciata were isolated and their structuralcharacteristics were investigated by a combination of chemical and spectroscopicmethods. Moreover, the anticoagulant activities of the sulfated polysaccharides wereevaluated by studying the activated partial thromboplastin time (APTT), thrombintime (TT) and prothrombin time (PT) using human plasma. The results were asfollows:1. The green alga Ulva fasciata was extracted with cold water and90℃hot water,and the two crude polysaccharides, named as CP and HP, were obtained. The yields ofCP and HP were9.2%and28.5%, respectively. The chemical analysis demonstratedthat the two polysaccharides contain high sulfate ester, which were19.8%and21.9%,respectively. The sugar contents were56.6%and58.9%, respectively. The uronicacid contents were10.8%and11.6%, respectively. The protein contents were12.7%and7.6%, respectively. The monosaccharide compositions of the polysaccharideswere determined by reversed-phase HPLC, and the results showed that the twopolysaccharides were mainly composed of rhamnose with small amounts of glucoseand glucuronic acid, and minor amounts of mannose, galactose and xylose.2. The two crude polysaccharides CP and HP were further purified by Q-SepharoseFast Flow column and Sephacryl S-400column to obtain seven polysaccharides CP1S,CP2S-1, CP2S-2, CP2S-3, CP2S-4, HP1S and HP2S. HPGPC analyses indicated that CP2S-2, CP2S-3, CP2S-4, HP1S and HP2S were all homogeneous polysaccharideswith the average molecular weight of about100.4kDa,90.3kDa,82.3kDa,871kDaand90.5kDa, respectively. The seven poly-saccharides were highrhamnose-containing polysaccharides with sulfate ester from24.0%to33.5%.3. On the basis of desulfation, methylation, GC-MS and infrared spectroscopyanalyses, HP1S and HP2S were characterized to mainly consist of→3)-α-L-Rhap-(1→,→2)-α-L-Rhap-(1→and→2,3)-α-L-Rhap-(1→with differentmolar ratio, and sulfate group were mainly substituted at C-2of→3)-α-L-Rhap-(1→and C-3of→2)-α-L-Rhap-(1→. Moreover,→3,4)-β-D-Glcp-(1→and→4)-β-D-Glcp-(1→also exited.1D NMR and2D NMR analyses were used for HP1Sto obtain more detailed structure data, and the results showed that→2,3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→、→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→、→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→werethe three main units in the structure of HP1S.4. The anticoagulant activities of the two crude polysaccharides (CP and HP) andthe two purified polysaccharides (HP1S and HP2S) were evaluated by assays of theactivated partial thromboplastin time (APTT), thrombin time (TT) and prothrombintime (PT) with heparin as positive control. The results demonstrated that the foursulfated polysaccharides showed high anticoagulant activities, but their anticoagulantactivities of the four polysaccharides were weaker than that of the heparin. The foursulfated polysaccharides did not affect PT even at the concentration at which APTTand TT were prolonged. Thus, the sulfated polysaccharides inhibited both the intrinsicand or common pathways of coagulation and thrombin activity or conversion offibrinogen to fibrin, and did not inhibit extrinsic pathway of coagulation.The results provided novel marine polysaccharides for “marine polysaccharidesdatabase”. Moreover, the study had potential value and important significance forfurther development of sulfated polysaccharides from green alga Ulva fasciata.