| The green algae belonging to Enteromorpha species are widely distributed inChina and have been used as drug in traditional Chinese medicine for hundreds ofyears. In the present work, four kinds of green algae Enteromorpha species werechosen, including Enteromorpha linza collected in spring and autumn in the coast ofQingdao, Enteromorpha prolifera from the green tide in the coast of Qingdao andEnteromorpha clathrata collected in the coast of Xiamen. The sulfatedpolysaccharides from the green algae Enteromorpha sp. were isolated and theirstructural characteristics were investigated by a combination of chemical andspectroscopic methods. Moreover, the sulfated oligosaccharides with different degreepolymerization were prepared by mild acid hydrolysis of the parent polysaccharidesand their structures were studied. The results were as follows:1. The green algae, Enteromorpha linza collected in spring and autumn, wereextracted by cold and hot water, and four polysaccharides MC, MH, SC and SH wereobtained. The yields of MC, MH, SC and SH were12.4%,15.0%,14.0%and12.3%,respectively. The four crude polysaccharides were further purified by anion exchangechromatography and gel-permeation chromatography to obtain five polysaccharidesMCS, MHS, SCS, SH1S and SH2S. Chemical composition analysis indicated thatMCS, MHS, SCS, SH1S and SH2S contained51.7%,52.8%,45.7%,50.4%and47.6%total polysaccharides, with20.7%,17.2%,16.8%,19.4%and15.3%uronicacid,16.9%,17.5%,14.3%,10.5%and13.0%sulfate ester, and with less than4%protein. GC and reversed-phase HPLC analyses indicated that MCS and MHS weremainly composed of rhamnose with minor amounts of xylose and glucuronic acid. SCS, SH1S and SH2S were mainly composed of rhamnose with minor amounts ofxylose, glucuronic acid and galactose. HPGPC analysis indicated that the averagemolecular weights of MCS、MHS, SCS, SH1S and SH2S were412.7kDa,535.0kDa,136.2kDa,502.1kDa and213.5kDa, respectively. On the basis of IR, methylationand NMR spectroscopic analyses, MCS and MHS were characterized to be mainlycomposed of ['4)-α-L-Rhap-(1'],['2,4)-α-L-Rhap-(1'],['4)-β-D-Xylp-(1'] and['4)-β-D-GlcUAp-(1'] with a minor amount of ['3)-α-L-Rhap-(1'] and['2)-α-L-Rhap-(1'], and the sulfate groups were substituted at C-3of['4)-α-L-Rhap-(1']. SCS was mainly composed of ['3)-α-L-Rhap-(1'],['2)-α-L-Rhap-(1'],['4)-β-D-Xylp-(1'],['4)-α-L-Rhap-(1'] and ['2,4)-α-L-Rhap-(1'],with sulfate groups substituted at C-2or C-4of ['3)-α-L-Rhap-(1'], C-3or C-4of['2)-α-L-Rhap-(1'],and C-2of ['4)-α-L-Rhap-(1']. SH1S was mainly composed of['4)-α-L-Rhap-(1'],['3)-α-L-Rhap-(1'],['2,4)-α-L-Rhap-(1'],['4)-β-D-Xylp-(1'] and['4)-β-D-GlcUAp-(1'], with sulfate groups substituted at C-3of ['4)-α-L-Rhap-(1'].SH2S was mainly composed of ['4)-α-L-Rhap-(1'] and ['4)-β-D-Xylp-(1'] withminor amounts of ['2)-α-L-Rhap-(1'],['3)-α-L-Rhap-(1'],['3,4)-α-L-Rhap-(1'],['2,3)-α-L-Rhap-(1'] and ['2,4)-α-L-Rhap-(1'], and the sulfate groups were mainlysubstituted at C-3of ['4)-α-L-Rhap-(1'].2. The green alga Enteromorpha prolifera was extracted by cold and hot waterand two polysaccharides QC and QH were obtained. The yields of QC and QH were10.6%and22.6%, respectively. QC and QH were further purified by anion-exchangechromatography and gel-permeation chromatography to obtain four polysaccharidesQC1S, QCQ2, QCQ3and QHS. Chemical composition analysis indicated that QC1S,QCQ2, QCQ3and QHS contained52.2%,42.4%,51.0%, and55.6%totalpolysaccharides, with10.9%,4.3%,15.3%and17.3%uronic acid,17.5%,13.2%,20.4%and17.4%sulfate ester, and with2.1%,13.6%,2.4%and1.0%protein.HPGPC analysis indicated that the average molecular weights of QC1S, QCQ2,QCQ3and QHS were116.3kDa,546.7kDa,541.0kDa and538.4kDa, respectively.GC and reversed-phase HPLC analyses indicated that QC1S, QCQ3and QHS weremainly composed of rhamnose with minor amounts of xylose and glucuronic acid. QCQ2were mainly composed of rhamnose with minor amounts of xylose, mannoseand glucosamine. On the basis of IR, methylation and NMR spectroscopic analyses,QC1S was characterized to be mainly composed of ['4)-α-L-Rhap-(1'] and['4)-β-D-Xylp-(1'] with a minor amount of ['2)-α-L-Rhap4S-(1'],['3)-α-L-Rha4S-(1'],['4)-α-L-Rhap2S-(1']'['3,4)-α-L-Rhap-(1']. QHS was mainlycomposed of ['4)-α-L-Rhap-(1'],['4)-β-D-Xylp-(1'] and ['4)-β-D-GlcUAp-(1'] witha minor amount of ['3)-α-L-Rhap-(1'] and ['2,4)-α-L-Rhap-(1'], and the sulfategroups were substituted at C-3of ['4)-α-L-Rhap-(1']. The major disaccharide units ofQHS were ['4)-β-D-ClcUAp-(1'4)-α-L-Rhap3S-(1'] and['4)-β-D-Xylp-(1'4)-α-L-Rhap3S-(1'].3. The green alga Enteromorpha clathrata was extracted by cold and hot waterand two polysaccharides FC and FH were obtained. The yields of FC and FH were7.3%and10.3%, respectively. FC and FH were further purified by anion-exchangechromatography and gel-permeation chromatography to obtain three polysaccharidesFCS, FH1S and FH2S. Chemical composition analysis indicated that FCS, FH1S andFH2S contained75.6%,74.1%and64.7%total polysaccharides, with8.6%,9.2%and7.9%uronic acid,14.4%,18.0%and31.0%sulfate ester, and with10.4%,2.6%and2.5%protein. HPGPC analysis indicated the average molecular weights of FCS,FH1S and FH2S were6.0kDa,23.8kDa and511.4kDa, respectively. GC andreversed-phase HPLC analyses indicated that FCS was mainly composed of galactoseand arabinose, with minor amounts of rhamnose and trace amounts of fucose, xylose,mannose and glucose. FH1S was mainly composed of arabinose with minor amountsof galactose and trace amounts of rhamnose. FH2S was mainly composed ofarabinose with minor amounts rhamnose and trace amounts of galactose. On the basisof IR, methylation and NMR spectroscopic analyses, FCS was characterized to bemainly composed of ['2)-β-D-Galp-(1'],['3)-β-D-Galp-(1'],['4)-β-D-Galp-(1'] and['6)-β-D-Galp-(1'], with minor amounts of ['4)-β-L-Arap-(1'],['2)-α-L-Rhap-(1'],['3)-α-L-Rhap-(1'] and ['2)-α-L-Rhap-(1'], and the sulfate groups were substitutedat C-6or C-2of ['4)-β-D-Galp-(1'], and C-4or C-2of ['6)-β-D-Galp-(1']. FH1S wasmainly composed of ['4)-β-L-Arap-(1'] with minor amounts of ['3)-β-D-Galp-(1'], ['4)-β-D-Galp-(1'] and ['6)-β-D-Galp-(1'], and the sulfate groups were mainlysubstituted at C-3of ['4)-β-L-Arap-(1']. FH2S was mainly composed of['4)-β-L-Arap3S-(1'], with tiny amounts of ['4)-β-L-Arap-(1'] and['3)-α-L-Rhap4S-(1'].4. The oligosaccharide fragments P1–P8were prepared by mild acid hydrolysisof QHS and purified by gel-permeation chromatography. The oligosaccharidefragments F1–F7were prepared by mild acid hydrolysis of FH2S and purified bygel-permeation chromatography. The sequences of the oligosaccharides wereanalyzed by ES-CID MS/MS and NMR spectroscopy analyses. The results showedthat P1–P8consisted of ['4)-β-D-GlcpA-(1'4)-α-L-Rhap3S-(1'] and['4)-α-L-Rhap3S-(1'4)-β-D-Xylp-(1']. F1–F7were sulfated arabino-oligosaccharideswhich were mainly composed of ['4)-β-L-Arap-(1']with the sulfate groups substitutedat C-3. The sulfated arabino-oligosaccharides were novel sulfated oligosaccharideswith different structural characteristics from other marine oligosaccharides.The results provided novel marine polysaccharides and special oligosaccharidesfor “marine polysaccharides database”. Moreover, the study had potential value andimportant significant for further development of sulfated polysaccharides from greenalga Enteromorpha sp. |
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