摘要
浒苔是浒苔属绿藻的总称，在分类学上属于绿藻石莼目、石莼科，是我国沿海常见海藻，其作为食用和药用藻类有着悠久的历史。本文以青岛沿海春季和秋季的缘管浒苔、青岛沿海绿潮浒苔以及厦门沿海的条浒苔为原料，采用冷水提取和热水提取方法，分别对四种不同浒苔中的多糖进行了提取，并采用各种色谱分离技术对多糖进行分离纯化，运用化学和现代波谱方法对多糖的结构进行研究；采用酸降解法对多糖进行降解得到不同聚合度的寡糖，并对寡糖的结构进行了研究。主要研究结果如下：
1.青岛沿海两种缘管浒苔(Enteromorpha linza)藻体粉末分别经冷水和热水提取得到四种多糖MC、MH、SC和SH，其得率分别为12.4%、15.0%、14.0和12.3%。采用离子交换色谱法和凝聚渗透色谱法对多糖进一步分离纯化，主要得到五个多糖组分MCS、MHS、SCS、SH1S和SH2S。通过化学方法对理化性质进行测定，结果表明MCS、MHS、SCS、SH1S和SH2S的总糖含量分别为51.7%、52.8%、45.7%、50.4%和47.6%；糖醛酸含量为20.7%、17.2%、16.8%、19.4%和15.3%；硫酸基含量为16.9%、17.5%、14.3%、10.5%和13.0%；蛋白含量均低于4%。高效凝胶渗透色谱法分析表明，MCS、MHS、SCS、SH1S和SH2S的分子量分别为412.7kDa、535.0kDa、136.2kDa、502.1kDa和213.5kDa。气相色谱法和PMP柱前衍生高效液相色谱法分析表明，MCS和MHS主要由鼠李糖组成，含有少量木糖和葡萄糖醛酸；SCS、SH1S和SH2S主要由鼠李糖组成，含有少量木糖、葡萄糖醛酸和半乳糖。红外光谱法、甲基化及核磁共振波谱法分析表明，MCS和MHS主要由[→4)-α-L-Rhap-(1→]、[→2,4)-α-L-Rhap-(1→]、[→4)-β-D-Xylp-(1→]和[→4)-β-D-GlcUAp-(1→]组成，含有少量[→3)-α-L-Rhap-(1→]、[→2)-α-L-Rhap-(1→]，硫酸基主要位于[→4)-α-L-Rhap-(1→]的C-3位；SCS主要由[→3)-α-L-Rhap-(1→]、[→2)-α-L-Rhap-(1→]、[→4)-β-D-Xylp-(1→]、[→4)-α-L-Rhap-(1→]和[→2,4)-α-L-Rhap-(1→]组成，硫酸基位于[→3)-α-L-Rhap-(1→]的C-2位或C-4位、[→2)-α-L-Rhap-(1→]的C-3位或者C-4位以及[→4)-α-L-Rhap-(1→]的C-2位；SH1S主要由[→4)-α-L-Rhap-(1→]、[→3)-α-L-Rhap-(1→]、[→2,4)-α-L-Rhap-(1→]、[→4)-β-D-Xylp-(1→]和[→4)-β-D-GlcUAp-(1→]组成，硫酸基位于[→4)-α-L-Rhap-(1→]的C-3位；SH2S主要由[→4)-α-L-Rhap-(1→]和[→4)-β-D-Xylp-(1→]组成，含有少量[→2)-α-L-Rhap-(1→]、[→3)-α-L-Rhap-(1→]、[→3,4)-α-L-Rhap-(1→]、[→2,3)-α-L-Rhap-(1→]和[→2,4)-α-L-Rhap-(1→]，硫酸基主要位于[→4)-α-L-Rhap-(1→]的C-3位。
2.青岛绿潮浒苔(Enteromorpha prolifera)藻体粉末经冷水和热水提取法得到两种多糖QC和QH，得率分别为10.6%和22.6%。采用离子交换色谱法和凝聚渗透色谱法对粗多糖进一步分离纯化，主要得到四个多糖组分QC1S、QCQ2、QCQ3和QHS。分析表明QC1S、QCQ2、QCQ3和QHS的总糖含量分别为52.2%、42.4%、51.0%和55.6%；糖醛酸含量为10.9%、4.3%、15.3%和17.3%；硫酸基含量为17.5%、13.2%、20.4%和17.4%；蛋白含量为2.1%、13.6%、2.4%和1.0%。高效凝胶渗透色谱法分析表明，QC1S、QCQ2、QCQ3和QHS的分子量分别为116.3kDa、546.7kDa、541.0kDa和538.4kDa。气相色谱法和PMP柱前衍生高效液相色谱法分析表明，QC1S、QCQ2、QCQ3和QHS主要由鼠李糖组成，其次为木糖；QC1S、QCQ3和QHS中还含有少量葡萄糖醛酸，而QCQ2含有少量甘露糖和氨基葡萄糖。红外光谱法、甲基化和核磁共振波谱法分析表明，QC1S主要由[→4)-α-L-Rhap-(1→]和[→4)-β-D-Xylp-(1→]组成，含有少量[→2)-α-L-Rhap4S-(1→]、[→3)-α-L-Rha4S-(1→]、[→4)-α-L-Rhap2S-(1→]和[→3,4)-α-L-Rhap-(1→]；QHS主要由[→4)-α-L-Rhap-(1→]、[→4)-β-D-Xylp-(1→]和[→4)-β-D-GlcUAp-(1→]组成，含有少量[→3)-α-L-Rhap-(1→]和[→2,4)-α-L-Rhap-(1→]，硫酸基位于[→4)-α-L-Rhap-(1→]的C-3位，其主要含有两种二糖结构单元：[→4)-β-D-ClcUAp-(1→4)-α-L-Rhap3S-(1→]和[→4)-β-D-Xylp-(1→4)-α-L-Rhap3S-(1→]。
3.厦门沿海条浒苔(Enteromorpha clathrata)藻体粉末经冷水和热水提取得到两种多糖XC和XH，得率分别为7.3%和10.3%。利用离子交换色谱和凝胶渗透色谱对粗多糖分离纯化，主要得到三个多糖组分XCS、XH1S和XH2S。分析表明XCS、XH1S和XH2S的总糖含量分别为75.6%、74.1%和64.7%；糖醛酸含量为8.6%、9.2%和7.9%；硫酸基含量为14.4%、18.0%和31.0%；蛋白含量为10.4%、2.6%和2.5%。高效凝胶渗透色谱法分析表明， XCS、XH1S和XH2S的分子量分别为6.0kDa、23.8kDa和511.4kDa，XCS和XH1S的分子量明显小于XH2S。气相色谱法和PMP柱前衍生高效液相色谱法分析表明，XCS主要由阿拉伯糖和半乳糖组成，含有少量鼠李糖和微量岩藻糖、木糖、甘露糖和葡萄糖；XH1S和XH2S主要由阿拉伯糖组成，XH1S还含有少量半乳糖和微量鼠李糖，XH2S含有少量鼠李糖和微量半乳糖。红外光谱法、甲基化和核磁波谱法分析表明，XCS主要由[→2)-β-D-Galp-(1→]、[→3)-β-D-Galp-(1→]、[→4)-β-D-Galp-(1→]和[→6)-β-D-Galp-(1→]组成，含有少量[→4)-β-L-Arap-(1→]、[→2)-α-L-Rhap-(1→]、[→3)-α-L-Rhap-(1→]和[→2)-α-L-Rhap-(1→]，硫酸基主要位于[→4)-β-D-Galp-(1→]的C-6位或者C-2位、[→6)-β-D-Galp-(1→]的C-4位或者C-2位。XH1S主要由[→4)-β-L-Arap-(1→]组成，含有少量[→3)-β-D-Galp-(1→]、[→4)-β-D-Galp-(1→]和[→6)-β-D-Galp-(1→]，硫酸基主要位于[→4)-β-L-Arap-(1→]的C-3位；XH2S主要由[→4)-β-L-Arap3S-(1→]组成，含有微量[→4)-β-L-Arap-(1→]和[→3)-α-L-Rhap4S-(1→]，XH2S是一种结构新颖的硫酸阿拉伯糖聚糖。
4.利用酸降解对多糖QHS和XH2S进行降解，经过Bio Gel P4凝胶渗透色谱柱分离，得到了不同聚合度的寡糖。QHS的降解条件为0.05mol/L HCl/100°C水浴/1.5h，得到寡糖组分P1–P8；XH2S的降解条件为0.05mol/L HCl/100°C水浴/1h，得到寡糖组分F1–F7。利用一级及二级质谱分析表明，P1–P8含有β-D-GlcpA-(1→4)-α-L-Rhap3S和α-L-Rhap3S-(1→4)-β-D-Xylp二糖结构；F1–F7为主要由[→4)-β-L-Arap3S-(1→]组成的聚合度1–5硫酸阿拉伯寡糖。获得的硫酸阿拉伯寡糖是尚未见报道的具有新型结构的海洋特征寡糖。
本论文的研究成果对“海洋糖库”的建设提供了结构新颖的海洋多糖和特征寡糖，对浒苔的研究和开发利用具有重要参考价值。
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→]和[→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.
引文
[1]齐继承.海藻的开发应用.中国保健食品,2004,(5):10-12.
[2]吕惠敏，张侃.海藻的利用与开发.食品科技,1998,6:29-30.
[3]阮积惠.海藻主要药用成分的研究和展望.东海海洋,2001,19(2):1-9.
[4]李红燕.宽礁膜抗凝血活性多糖及其寡糖的制备和结构研究:[博士学位论文].青岛:中国海洋大学,2011.
[5]孟春，郭养浩，石贤爱，等.海藻多糖生物活性与分子修饰.中国生物工程杂志,2004,24(3):35-39.
[6] Li Hongyan, Mao Wenjun, Zhang Xiuli, et al. Structural characterization of ananticoagulant-active sulfated polysaccharide isolated from green alga Monostromalatissimum. Carbohydrate Polymers,2011,85(2):394-400.
[7] Kim Jin-Kyung, Cho Myoung Lae, Karnjanapratum Supatra, et al. In vitro and in vivoimmunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera.International Journal of Biological Macromolecules,2011,49(5):1051-1058.
[8] Karnjanapratum Supatra, You Sangguan. Molecular characteristics of sulfatedpolysaccharides from Monostroma nitidum and their in vitro anticancer andimmunomodulatory activities. International Journal of Biological Macromolecules,2011,48(2):311-318.
[9] Zhang Zhongshan, Wang Feng, Wang Xiaomei, et al. Extraction of the polysaccharides fromfive algae and their potential antioxidant activity in vitro. Carbohydrate Polymers,2010,82(1):118-121.
[10] Jiao Lili, Jiang Peng, Zhang Liping, et al. Antitumor and Immunomodulating Activity ofPolysaccharides from Enteromorpha intestinalis. Biotechnology and BioprocessEngineering,2010,15(3):421-428.
[11] Costa L. S., Fidelis G. P., Cordeiro S. L., et al. Biological activities of sulfatedpolysaccharides from tropical seaweeds. Biomedicine& Pharmacotherapy,2010,64(1):21-28.
[12] Cho Myounglae, Yang Chen, Kim Sang, et al. Molecular characterization and biologicalactivities of watersoluble sulfated polysaccharides from Enteromorpha prolifera. FoodScience and Biotechnology,2010,19(2):525-533.
[13] Mao Wenjun, Li Hongyan, Li Yi, et al. Chemical characteristic and anticoagulant activity ofthe sulfated polysaccharide isolated from Monostroma latissimum (Chlorophyta).International Journal of Biological Macromolecules,2009,44(1):70-74.
[14] Jiao Lili, Li Xia, Li Tianbao, et al. Characterization and anti-tumor activity ofalkali-extracted polysaccharide from Enteromorpha intestinalis. InternationalImmunopharmacology,2009,9(3):324-329.
[15] Cassolato Juliana E. F., Noseda Miguel D., Pujol Carlos A., et al. Chemical structure andantiviral activity of the sulfated heterorhamnan isolated from the green seaweed Gayraliaoxysperma. Carbohydrate Research,2008,343(18):3085-3095.
[16]纪明侯.海藻化学.北京:科学出版社,1997,356-366.
[17] Lahaye M., Inizan F., Vigoureux J. NMR analysis of the chemical structure of ulvan and ofulvan-boron complex formation. Carbohydrate Polymers,1998,36(2-3):239-249.
[18] Lahaye Marc, Brunel Magali, Bonnin Estelle. Fine chemical structure analysis ofoligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wallpolysaccharides from Ulva sp.(Ulvales, Chlorophyta). Carbohydrate Research,1997,304(3-4):325-333.
[19] Lahaye Marc. NMR spectroscopic characterisation of oligosaccharides from two Ulvarigida ulvan samples (Ulvales, Chlorophyta) degraded by a lyase. Carbohydrate Research,1998,314(1-2):1-12.
[20] Harada Naoki, Maeda Masaakira. Chemical Structure of Antithrombin-active RhamnanSulfate from Monostrom nitidum. Bioscience, Biotechnology, and Biochemistry,1998,62(9):1647-1652.
[21] Lee Jung-Bum, Yamagaki Tohru, Maeda Masaakira, et al. Rhamnan sulfate from cell wallsof monostroma latissimum. Phytochemistry,1998,48(6):921-925.
[22] Matsubara Kiminori, Matsuura Yasushi, Bacic Antony, et al. Anticoagulant properties of asulfated galactan preparation from a marine green alga, Codium cylindricum. InternationalJournal of Biological Macromolecules,2001,28(5):395-399.
[23] Bilan Maria I., Vinogradova Ekaterina V., Shashkov Alexander S., et al. Structure of ahighly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense(Bryopsidales, Chlorophyta). Carbohydrate Research,2007,342(3–4):586-596.
[24] Siddhanta A. K., Shanmugam M., Mody K. H., et al. Sulphated polysaccharides of Codiumdwarkense Boergs. from the west coast of India: chemical composition and bloodanticoagulant activity. International Journal of Biological Macromolecules,1999,26(2–3):151-154.
[25]嵇国利.刺松藻（Codium fragile（Suringar）Hariot）多糖的提取分离、结构表征及其抗凝活性研究:[硕士学位论文].青岛:中国海洋大学,2010.
[26] Chattopadhyay Kausik, Adhikari Utpal, Lerouge Patrice, et al. Polysaccharides fromCaulerpa racemosa: Purification and structural features. Carbohydrate Polymers,2007,68(3):407-415.
[27] Rao N. V. S. A. V. Prasada, Rao E. Venkata. Structural features of the sulphatedpolysaccharide from a green seaweed, Caulerpa taxifolia. Phytochemistry,1986,25(7):1645-1647.
[28] Sri Ramana K., Venkata Rao E. Structural features of the sulphated polysaccharide from agreen seaweed, Cladophora socialis. Phytochemistry,1991,30(1):259-262.
[29] Erick Reyes Suárez, Jaroslav A. Kralovec, Grindley T. Bruce. Isolation of phosphorylatedpolysaccharides from algae: the immunostimulatory principle of Chlorella pyrenoidosa.Carbohydrate Research,2010,345:1190-1204.
[30] Hayakawa Yumiko, Hayashi Toshimitsu, Lee Jung-Bum, et al. Inhibition of thrombin bysulfated polysaccharides isolated from green algae. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology,2000,1543(1):86-94.
[31] Mao Wenjun, Zang Xiaoxue, Li Yi, et al. Sulfated polysaccharides from marine green algaeUlva conglobata and their anticoagulant activity. Journal of Applied Phycology,2006,18:9-14.
[32]王晓宇，邹明明，王蓉，等.海藻多糖抗肿瘤机理研究进展.大连医科大学学报,2007,29(3):318-322.
[33] Ji Hongwu, Shao Haiyan, Zhang Chaohua, et al. Separation of the polysaccharides inCaulerpa racemosa and their chemical composition and antitumor activity. Journal ofApplied Polymer Science,2008,110(3):1435-1440.
[34] Isuru Wijesekaraa, Ratih Pangestutia, Se-Kwon Kima. Biological activities and potentialhealth benefits of sulfated polysaccharides derived from marine algae. CarbohydratePolymers,2011,84:14-21.
[35] Ghosh Tuhin, Chattopadhyay Kausik, Marschall Manfred, et al. Focus on antivirally activesulfated polysaccharides: From structure–activity analysis to clinical evaluation.Glycobiology,2009,19(1):2-15.
[36] Lee Jung-Bum, Koizumi Seiji, Hayashi Kyoko, et al. Structure of rhamnan sulfate from thegreen alga Monostroma nitidum and its anti-herpetic effect. Carbohydrate Polymers,2010,81(3):572-577.
[37] Ghosh Partha, Adhikari Utpal, Ghosal Prodyot K., et al. In vitro anti-herpetic activity ofsulfated polysaccharide fractions from Caulerpa racemosa. Phytochemistry,2004,65(23):3151-3157.
[38]周杰，陈安国.海藻多糖的生物活性研究进展.饲料工业,2005,26(18):12-15.
[39]石学连,张晶晶,王晶,等.浒苔多糖的分级纯化及体外抗氧化活性研究.中国海洋药物杂志,2009,28(3):44-49.
[40] Qi Huimin, Zhang Quanbin, Zhao Tingting, et al. Antioxidant activity of different sulfatecontent derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro.International Journal of Biological Macromolecules,2005,37(4):195-199.
[41] Guzmán S., Gato A., Lamela M., et al. Anti-inflammatory and immunomodulatory activitiesof polysaccharide from Chlorella stigmatophora and Phaeodactylum tricornutum.Phytotherapy Research,2003,17(6):665-670.
[42] Yu Pengzhan, Zhang Quanbin, Li Ning, et al. Polysaccharides from Ulva pertusa(Chlorophyta) and preliminary studies on their antihyperlipidemia activity. Journal ofApplied Phycology,2003,15(1):21-27.
[43]周慧萍，蒋巡天，王淑如，等.浒苔多糖的降血脂及其对SOD活力和LPO含量的影响.生物化学杂志,1995,11(2):161-165.
[44] Mao Wenjun, Li Yi, Wu Ligen, et al. Chemical characterization and radioprotective effect ofpolysaccharide from Monostroma angicava (Chlorophyta). Journal of Applied Phycology,2005,17(4):349-354.
[45]徐娟华,赵孟辉,谢强明,等.海藻石莼降血糖作用的实验研究.中国中医药科技,2002,9(3):168-169.
[46]石学连,张晶晶,宋厚芳,等.浒苔多糖的分级纯化及保湿活性研究.海洋科学,2010,34(7):81-99.
[47]周鹏，谢明勇，傅博强.多糖的结构研究.南昌大学学报（理科版）,2001,25(2):197-204.
[48]阳佛送,李雪华.多糖结构研究的方法和进展.食品科技,2008,(3):200-203.
[49]张惟杰.糖复合物生化研究技术.杭州:浙江大学出版社,1999,193-253.
[50]陈志强,金杨,王昌禄.多糖结构分析方法的研究.饲料工业,2008,29(6):59-61.
[51]高小荣，刘培勋.多糖结构分析研究进展.天津药学,2003,15(6):67-70.
[52] Yang Liqun, Zhang Liming. Chemical structural and chain conformational characterizationof some bioactive polysaccharides isolated from natural sources. Carbohydrate Polymers,2009,76:349-361.
[53]李波,陈海华,许时婴.二维核磁共振波谱在多糖结构研究中的应用.天然产物研究与开发,2005,17(4):523-536.
[54]刘玉红,王凤山.核磁共振波谱法在多糖结构分析中的应用.食品与药品,2007,9(8):39-42.
[55]刘明，李春霞，辛现良.核磁共振技术在糖类化合物化学结构研究的应用.中国药学杂志,2009,44(5):324-327.
[56] Rief Matthias, Gautel Mathias, Oesterhelt Filipp, et al. Reversible Unfolding of IndividualTitin Immunoglobulin Domains by AFM. Science,1997,276(5315):1109-1112.
[57] Abu-Lail N. I., Camesano T. A. Polysaccharide properties probed with atomic forcemicroscopy. Journal of Microscopy,2003,212(3):217-238.
[58] Rief Matthias, Oesterhelt Filipp, Heymann Berthold, et al. Single Molecule ForceSpectroscopy on Polysaccharides by Atomic Force Microscopy. Science,1997,275(5304):1295-1297.
[59]朱劲华,张威,卢薇.寡糖的结构、生物学功能及其应用.化工纵横,1999,(6):15-17.
[60]董权锋,于荣敏.寡糖研究新进展.食品与药品,2009,11(7):63-66.
[61]郭丽民,张汝学,贾正平.寡糖的药理作用和机制研究进展.中成药,2006,28(9):1353-1356.
[62]曹朝晖,李邦良,乔新惠,等.甲壳低聚糖对NOD鼠糖尿病的预防作用.医学理论与实践,2004,17(1):1-3.
[63]李翊,王海清.卡拉胶寡糖对放射损伤的防护作用.中国放射医学与防护杂志,2005,25(2):116-117.
[64]李翊,王海青.卡拉胶寡糖对放射损伤小鼠T细胞功能和亚型的影响.中华放射医学与防护杂志,2005,25(1):41-42.
[65]许青松,白雪芳,杜昱光.壳寡糖及其衍生物抗肿瘤作用的研究进展.食品与药品,2008,10(5):60-62.
[66]官杰,罗晓庆,王琪,等.壳寡糖抑制肿瘤作用的实验研究.中国免疫学杂志,2007,23(5):421-425.
[67]曹秀明,徐海娇,尚明,等.壳寡糖抗肿瘤和免疫调节作用的实验研究.哈尔滨商业大学学报(自然科学版),2006,22(4):8-10.
[68] Wang Jingxue, Jiang Xiaolu, Mou Haijin, et al. Anti-oxidation of agar oligosaccharidesproduced by agarase from a marine bacterium. Journal of Applied Phycology,2004,16(5):333-340.
[69]陈筱春,文质君,屈菊兰.甲壳寡糖对运动小鼠红细胞畸变及脂质过氧化的保护.中国体育科技,2005,41(2):115-116.
[70]曹红,张世玲,姬胜利,等.硫酸寡糖对豚鼠气管平滑肌的作用.中国生化药物杂志,2003,24(6):283-285.
[71]赵智辉,褚健,陈元,等.几种寡糖对白细胞-内皮细胞粘附影响及抗休克作用探讨.中国病理生理杂志,2001,17(11):1125.
[72] Lahaye Marc, Ray Bimalendu. Cell-wall polysaccharides from the marine green alga Ulva"rigida"(Ulvales, Chlorophyta)--NMR analysis of ulvan oligosaccharides. CarbohydrateResearch,1996,283:161-173.
[73] Zheng Yun, Mort Andrew. Isolation and structural characterization of a noveloligosaccharide from the rhamnogalacturonan of Gossypium hirsutum L. CarbohydrateResearch,2008,343(6):1041-1049.
[74]汪建明,赵征,王勇志.低聚糖的分离与鉴定.食品研究与开发,2006,21(3):3-6.
[75]林常青.质谱在多糖结构分析中的应用.分析测试技术与仪器,2005,11(3):221-227.
[76]刘晗,白雪芳,杜昱光.寡糖类物质分离及结构研究现状.精细与专用化学品,2005,13(9):1-5.
[77]王文娟,赵宏,米锴,等.大型绿藻浒苔属植物研究进展.湖南农业科学,2009,(8):1-4.
[78]中国预防医学科学院营养食品卫生研究所.食物成分表.北京:人民卫生出版社,1998,
[79]钟礼云.浒苔系列产品生理功能活性研究:[硕士学位论文]福建医科大学,2006.
[80] Hiqashi-Okaj Kiyoka, Otani Shuzo, Okai Yasuji. Potent suppressive effect of a Japaneseedible seaweed, Enteromorpha prolifera (Sujiao-nori) on initiation and promotion phases ofchemically induced mouse skin tumorigenesis. Cancer Letters,1999,140(1-2):21-25.
[81] Cho Myounglae, Lee Hyi-Seung, Kang Il-Jun, et al. Antioxidant properties of extract andfractions from Enteromorpha prolifera, a type of green seaweed. Food Chemistry,2011,127(3):999-1006.
[82] Hudson J., Kim J., Lee M., et al. Antiviral compounds in extracts of Korean seaweeds:Evidence for multiple activities. Journal of Applied Phycology,1998,10(5):427-434.
[83] Okai Yasuji, Higashi-Okai Kiyoka. Potent anti-inflammatory activity of pheophytin aderived from edible green alga, Enteromorpha prolifera (Sujiao-nori). International Journalof Immunopharmacology,1997,19(6):355-358.
[84]徐大伦,黄晓春,欧昌荣,等.浒苔多糖对非特异性免疫功能的体外实验研究.食品科学,2005,26(10):232-235.
[85]关燕清，郭宝江，徐和德.干苔多糖的分离纯化及抗肿瘤活性研究.功能高分子学报,2002,15(1):19-23.
[86] Mckinnell J. P., Percival Elizabeth.606. Structural investigations on the water-solublepolysaccharide of the green seaweed Enteromorpha compressa. Journal of the ChemicalSociety (Resumed),1962:3141-3148.
[87] Bimalendu Ray. Polysaccharides from Enteromorpha compressa: Isolation, purification andstructural features. Carbohydrate Polymers,2006,66(3):408-416.
[88] Chattopadhyay Kausik, Mandal Pinaki, Lerouge Patrice, el al. Sulphated polysaccharidesfrom Indian samples of Enteromorpha compressa (Ulvales, Chlorophyta): Isolation andstructural features. Food Chemistry,2007,104(3):928-935.
[89] Lahaye Marc, Robic Audrey. Structure and Functional Properties of Ulvan, aPolysaccharide from Green Seaweeds. Biomacromolecules,2007,8(6):1765-1774.
[90] Marinho-Soriano E., Fonseca P. C., Carneiro M. A. A., et al. Seasonal variation in thechemical composition of two tropical seaweeds. Bioresource Technology,2006,97(18):2402-2406.
[91]徐大伦，黄晓春，欧昌荣，等.浒苔水溶性多糖提取的工艺研究.浙江海洋学院学报（自然科学版）,2005,24(1):67-69.
[92] Michel Dubois K. A. Gilles, J. K. Hamilton, P. A. Rebers, Fred Smith. Colorimetric Methodfor Determination of Sugars and Related Substances. Analytical Chemistry,1956,28(3):6.
[93] Marion M Bradford. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry,1976,72(1–2):248-254.
[94] Bitter T., Muir H. M. A modified uronic acid carbazole reaction. Analytical Biochemistry,1962,4(4):330-334.
[95]从建波,王长振,李妍.褐藻硫酸多糖硫酸基含量测定-硫酸钡比浊法研究.解放军药学学报,2003,19(3):181-183.
[96] Mao Wen-Jun, Fang Fang, Li Hong-Yan, et al. Heparinoid-active two sulfatedpolysaccharides isolated from marine green algae Monostroma nitidum. CarbohydratePolymers,2008,74(4):834-839.
[97]房芳.礁膜（Monostroma nitidum）多糖的提取分离、结构和抗凝血活性研究:[硕士学位论文].青岛:中国海洋大学,2008.
[98] Sun Hai-Hong, Mao Wen-Jun, Chen Yin, et al. Isolation, chemical characteristics andantioxidant properties of the polysaccharides from marine fungus Penicillium sp. F23-2.Carbohydrate Polymers,2009,78(1):117-124.
[99] Falshaw Ruth, Furneaux Richard H. Structural analysis of carrageenans from the tetrasporicstages of the red algae, Gigartina lanceata and Gigartina chapmanii (Gigartinaceae,Rhodophyta). Carbohydrate Research,1998,307(3–4):325-331.
[100] Hakomori Sen-Itiroh. A Rapid Permethylation of Glycolipid, and PolysaccharideCatalyzed by Methylsulfinyl Carbanion in Dimethyl Sulfoxide. Journal of Biochemistry,1964,55(2):205-208.
[101]董群,方积年.寡糖及多糖甲基化方法的发展及现状.天然产物研究与开发,1995,7(2):60-65.
[102]石学连.浒苔多糖的化学组分及生物学活性的基本研究:[硕士学位论文]中国科学院海洋研究所,2009.
[103]何清，胡晓波，周峙苗，等.东海绿藻缘管浒苔营养成分分析及评价.海洋科学,2006,30(1):34-38.
[104] Ray B., Lahaye M. Cell-wall polysaccharides from the marine green alga Ulva "rigida"(Ulvales, Chlorophyta). Chemical structure of ulvan. Carbohydrate Research,1995,274:313-318.
[105] Lahaye Marc, Alvarez-Cabal Cimadevilla Enrique, Kuhlenkamp Ralph, et al. Chemicalcomposition and13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales,Chlorophyta). Journal of Applied Phycology,1999,11(1):1-7.
[106]周慧萍，朱海燕，陈琼华.浒苔多糖的分离、纯化和分析.生物化学杂志,1995,11(1):91-94.
[107] Milton H. Fischer Nanxiong Yu, Gary R. Gray, John Ralph, Laurens Anderson, Judith A.Marlett. The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk).Carbohydrate Research,2004,339:2009-2017.