Studies On Extraction, Isolation, Structural Characterization And Anticoagulant Activity Of The Sulfated Polysaccharides From Green Alga Monostroma Arcticum

The green algae Monostroma arcticum is widely distributed in China andhas been used as drug in traditional Chinese medicine for hundreds of years. Thepolysaccharide is its important bioactive substance. In the present work, thesulfated polysaccharides from the green algae Monostroma arcticum wereisolated, and their structural characteristics and anticoagulant activity wereinvestigated. The results were as follows:The green algae Monostroma arcticum was extracted by cool water, boilingwater and alkali liquid successively. Four kinds of polysaccharides were obtainedand named as CN, HN, AN1and AN2, respectively. The physicochemical propertieswere analyzed by phenol-sulfuric acid method, Folin-phenol reagent method, bariumsulfate turbidimetry method, GC and HPLC. The results showed that the yields ofCN, HN, AN1and AN2were8.65%、25.85%、0.55%and4.25%, respectively. Thecontents of the sulfate group were21.7%,28.6%,32.8%and30.4%, respectively.The protein contents were7.35%、6.30%、13.50%and2.85%, respectively. CN werecomposed of rhamnose, xylose and glucose. HN was mainly composed ofrhamnose with minor amounts of xylose and glucose.AN1consisted of glucosewith minor amount of rhamnose.AN2was mainly composed of rhamnose, withsmall amounts of galactose and glucose and minor amount of xylose.Moreover, the four kind polysaccharides contained minor amounts ofglucuronic acid.The crude polysaccharide HN was further purified by anion exchangechromatography and gel-permeation chromatography to obtain threepolysaccharides HF0, HAF0and HF3. The physicochemical properties andmonosaccharide compositions of HF0, HAF0and HF3were analyzed by phenol-sulfuric acid method, Folin-phenol reagent method, barium sulfateturbidimetry method, IR, GC, and HPLC. The results showed that HF0, HAF0andHF3were highly pure, and the average molecular weights of HF0, HAF0andHF3were940.85kD,9.36kD and73.96kD, respectively. The molecular weight ofHF0was higher than that of HF3. The three sulfated polysaccharides were mainlycomposed of rhamnose with minor amounts of xylose and glucose. In IRspectra of HF0, HAF0and HF3,1256cm-1showed strong absorption peaks of S=Ostretching vibration.The structural characterization of HAF0was further studied by desulfatereaction, methylation, IR and NMR spectroscopic analyses. The results showedthat HAF0was mainly composed of�'3)--L-Rhap(1�'and�'2)--L-Rhap(1�'.The sulfate groups were mainly substituted at C-2of�'3)--L-Rhap(1�'and C-3of�'2)--L-Rhap(1�', with minor amount of sulfate groups substitution at C-4of�'3)--L-Rhap(1�'.The anticoagulant activity of HAF0was studied by APTT, TT and PT. APTT andTT were effectively prolonged by HAF0, while lack of prolongation effect on PTwas observed. The prolongation of APTT indicates inhibition of the intrinsic and/orcommon pathway. TT assay is helpful to investigate the sample effect in thrombinaccelerated clot formation in platelet poor plasma, whereas prolongation of TTsuggests inhibition of thrombin activity or fibrin polymerization. Thus, HAF0inhibited both the intrinsic and/or common pathways of coagulation and thrombinactivity or conversion of fibrinogen to fibrin. HAF0did not inhibit extrinsic pathwayof coagulation.The results provided novel marine polysaccharides for “marine polysaccharidesdatabase”. Moreover, the study had potential value and important significant forfurther development of sulfated polysaccharides from green alga Monostromaarcticum.