摘要
宽礁膜是我国重要的经济海藻之一,隶属于绿藻门,绿藻纲,石莼目,礁膜科,礁膜属。目前,宽礁膜在我国人工育苗和栽培已获成功。本论文以我国人工养殖宽礁膜为研究对象,采用各种色谱分离技术,获得高抗凝血活性的宽礁膜多糖,采用化学和现代波谱方法对其结构进行研究,并对结构新颖的抗凝血多糖进行降解研究,建立可控酸降解法制备宽礁膜低分子量多糖片段及其寡糖的方法,通过核磁共振波谱和质谱技术,对获得的低分子量的糖基片段和寡糖的结构进行研究,并研究各种低分子量糖基片段对抗凝血活性的影响。研究结果如下: 1人工栽培宽礁膜藻体经冷、热水提取得到两种多糖冷水提多糖和和热水提多糖,相对于干燥藻体的得率分别为9.56%和39.1%。采用离子交换色谱和凝胶渗透色谱对热水提多糖进一步纯化,主要得到三种多糖MAS、MBS和MCS。
通过化学方法对理化性质进行测定,结果表明,MAS、MBS和MCS总糖含量分别为74.1%、68.1%和65.3%;硫酸基含量为26.1%、28.5%和31.8%;糖醛酸含量为4.44%、3.94%和3.27%;蛋白质含量较低。高效凝胶渗透色谱法测得MAS分子量较高为512.5 kDa,而MBS和MCS分子量较低,分别为58.4 kDa和48.5 kDa;气相色谱法测得MAS、MBS和MCS以鼠李糖为主,还含有少量的葡萄糖、木糖、甘露糖和半乳糖等。经红外光谱、脱硫反应、甲基化反应和核磁共振波谱(1D,2D NMR)分析表明,MAS、MBS和MCS结构比较相似,主要由[→3)-α-L-Rhap-(1→]、[→2)-α-L-Rhap-(1→]和[→2,3)-α-L-Rhap-(1→]组成,但摩尔比例不同,硫酸基团主要位于[→3)-α-L-Rhap-(1→]的C-2位和[→2)-α-L-Rhap-(1→]的C-3位。多糖MAS、MBS和MCS中主要含有四种硫酸鼠李二糖结构单元: [→3)-α-L-Rhap(2SO_4)-(1→3)-α-L-Rhap→]、[→3)-α-L-Rhap(2SO_4)-(1→2)-α-L-Rhap→]、[→3)-α-L-Rhap-(1→2)-α-L-Rhap(3SO_4)→]和[→3)-α-L-Rhap(2SO_4)-(1→2,3)-α-L-Rhap→]。通过APTT、PT和TT对多糖MAS、MBS和MCS的抗凝血活性进行评价,得到抗凝血活性较好结构新颖的硫酸鼠李聚糖。
2采用硫酸降解的方法,将多糖MAS降解为不同分子量的多糖。通过相对黏度法、高效薄层色谱法、聚丙烯酰胺凝胶电泳法和高效凝胶渗透色谱法对降解条件进行优化。采用0.1 mol/L H_2SO_4在40℃水解75 min和80℃水解60 min对多糖MAS进行降解,制备得到分子量在405.0~5.1 kDa的七种多糖(MAS1~MAS7)。研究表明,七种多糖的理化性质、单糖组成和红外光谱特征均与母多糖MAS相似。以抗凝血活性高且温和的低分子量多糖MAS5为代表,通过甲基化反应和1D,2D NMR分析表明,MAS5由[→3)-α-L-Rhap-(1→]、[→2)-α-L-Rhap-(1→]和[→2,3)-α-L-Rhap-(1→]组成,三者摩尔比为4:1:1,硫酸基团位于[→3)-α-L-Rhap-(1→]的C-2位和[→2)-α-L-Rhap-(1→]的C-3位。MAS5结构与母多糖MAS相似,即酸降解后多糖主体结构没有发生改变。通过活化部分凝血酶时间(APTT),对多糖MAS及其降解产物MAS1~MAS7的抗凝血活性进行了评价。结果表明,多糖随着分子量的减小,抗凝血活性减弱。分子量对宽礁膜多糖抗凝血活性具有较大的影响,其抗凝血活性依赖于一定长度的糖链。
3多糖MAS通过0.1 mol/L H_2SO_4于80℃水解3 h,Bio-Gel P4凝胶渗透色谱分离纯化,得到寡糖组分O1~O8。通过ES-MS对寡糖组分的聚合度、单糖组成和纯度进行了分析。结果表明,硫酸鼠李寡糖聚合度(DP)在2~9之间。采用1D,2D NMR对纯度较高的硫酸鼠李二糖结构进行研究,确定其结构为α-L-Rhap(2SO_4)-(1→3)-α-L-Rhap。通过对硫酸鼠李二糖ES-CID-MS/MS断裂碎片的分析和归属,建立了硫酸鼠李寡糖负离子模式下二级质谱序列分析方法,并将此方法用于聚合度为3~9的硫酸鼠李寡糖的结构研究。结果表明,制备得到的硫酸鼠李寡糖均为[→3)-α-L-Rhap-(1→]连接的直链结构。这些硫酸鼠李寡糖为具有新型结构的海洋特征寡糖,目前国内外尚未有关硫酸鼠李寡糖的报道。
本论文的研究成果,对宽礁膜多糖的研究和开发、对“海洋糖库”的建设以及发展具有我国特色的海洋药物具有重要的学术价值和经济意义。
Marine green algae often contained bioactive substances with novel functions and structures because of their special living conditions, and the polysaccharides from marine green algae are of interest to new drug discovery. The sulfated polysaccharides from green algae Monostromaceae species show potent anticoagulant activity, and represent potential source to be explored. Until recently, few structural studies have been done on the polysaccharides from Monostromaceae.
Monostroma latissimum is widely distributed in China, and has been used as fundamental source of food and drug in traditional Chinese medicine for thousands of years. Recently, the cultivation of marine green seaweed, Monostroma latissimum, was successfully obtained in China. Thus, the analyses of this species are very important in industrial application. In the present work, the heparinoid-active sulfated polysaccharides from the cultivation of Monostroma latissimum were isolated, and their structural characteristics were investigated by a combination of chemical and spectroscopic methods.
The green alga, Monostroma latissimum from Yuhuan County in China, was extracted by cold (24°C) and hot (100°C) water to obtain two polysaccharides. The hot water extracted polysaccharide with higher yield and anticoagulant-active was further purified on Q-Sepharose Fast Flow ion-exchange and Sephacryl S-400/HR size-exclusion chromatography to generate three polysaccharides MAS, MBS and MCS of uniform size and charge. Chemical composition analysis indicated that MAS, MBS and MCS contained 74.1%, 68.1% and 65.3% total polysaccharides, with 26.1%, 28.5% and 31.8% sulfate, 4.44%, 3.94% and 3.27% glucuronic acid, and with a minor amount of protein. MAS, MBS and MCS were high rhamnose-containing sulfated polysaccharides with an average molecular weight of about 512.5 kDa, 58.4 kDa and 48.5 kDa, respectively. On the basis of methylation, one- and two-dimensional nuclear magnetic resonance (1D, 2D NMR) spectroscopic analyses, the polysaccharide chains of MAS, MBS and MCS were characterized to consist of [→3)-α-L-Rhap-(1→], [→2)-α-L-Rhap-(1→] and [→2,3)-α-L-Rhap-(1→] with different molar ratio, and the sulfate groups were substituted at C-2 of [→3)-α-L-Rhap-(1→] and C-3 of the [→2)-α-L-Rhap-(1→]. The disaccharide units in the structure of three polysaccharides were [→3)-α-L-Rhap(2SO_4)-(1→3)-α-L-Rhap→], [→3)-α-L-Rhap(2SO_4)-(1→2)-α- L-Rhap→], [→3)-α-L-Rhap-(1→2)-α-L-Rhap(3SO_4)→] and [→3)-α-L-Rhap(2SO_4)- (1→2,3)-α-L-Rhap→]. The polysaccharides had a high anticoagulant activity as evaluated by assays of the activated partial thromboplastin time and thrombin time. The investigation demonstrated that the polysaccharides appeared to be a sulfated rhamnan with different structural characteristics from other sulfated polysaccharides from Monostromaceae species, and could be a potential source of anticoagulant.
According to the relative viscosity, high performance gel permeation chromatography, thin layer chromatography, polyacrylamide gel electrophoresis, the hydrolysis condition of the sulfated polysaccharide MAS was optimized. With the condition of 0.1 mol/L H2SO_4 at 40°C for 75 min and 80°C for 60 min, the polysaccharide MAS was degraded, and isolated to generate seven low molecular weight polysaccharides (MAS1~MAS7), their molecular weights ranged from 405.0 kDa to 5.1 kDa. The physiochemical properties, monosaccharide composition and infra-red spectrum characteristics of the polysaccharides were all similar to those of the parent polysaccharide MAS. The structure of the polysaccharide MAS5 as representation was studied by methylation and 1D, 2D NMR spectroscopy, and the results showed that the structure consisted of [→3)-α-L-Rhap-(1→], [→2)-α-L-Rhap-(1→] and [→2,3)-α-L-Rhap-(1→] in the molar ratio of 4:1:1. The sulfate groups were substituted at C-2 of the [→3)-α-L-Rhap-(1→] and C-3 of the [→2)-α-L-Rhap-(1→], which is consistent with the parent polysaccharide MAS. The results indicated that the structure of the low molecular weight polysaccharides after mild acid hydrolysis was not destroyed. Base on the anticoagulant activity of APTT, the sulfated polysaccharides with different molecular weights showed different anticoagulant activities. The 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 the anticoagulant action.
The oligosaccharide fragments of the sulfated rhamnan MAS from Monostroma latissimum were obtained from partial depolymerization of the polysaccharide by mild acid hydrolysis, and their sequences were investigated with ES-MS. On the basis of negative-ion electrospray tandem mass spectrometry with collision-induced dissociation (ES-CID-MS/MS) and 1D, 2D NMR spectroscopy, the oligosaccharide R2S was characterized to be a monosulfated rhamnobiose and consisted ofα-L-Rhap-(2SO_4)-(1→3)-α-L-Rhap. The fragmentation pattern of the homogeneous disaccharide compositions in the product ion spectra was established, and was then applied to sequence determination of the other oligosaccharides. The results demonstrated that it was possible to derive the sequence of the sulfated rhamno-oligosaccharide directly from the glycosidic cleavage fragmentation in the product ion spectra, and negative-ion ES-CID MS/MS affords an efficient method for the sequence determination of oligosaccharides derived from the sulfated rhamnan. The results indicated that the linkage pattern of the sulfated rhamno-oligosaccharides are all [→3)-α-L-Rhap-(1→]. The structure of the sulfated rhamno-oligosaccharide was the first time to report.
引文
[1]李八方.海洋生物活性物质.山东:中国海洋大学出版社.2007,177-180.
[2]甘建红,周培根.海洋天然物质化学.上海:上海大学出版社.2006,43-44.
[3] Lee J B, Yamagaki T, Maeda M, & Nakanishi H. Rhamnan sulfate from cell walls of Monostroma latissimum. Phytochemistry, 1998, 48, 921-925.
[4] Cassolato J E F, Noseda M D, Pujol C A, Pellizzari F M, Damonte E B, & Duarte M E R. Chemical structure and antiviral activity of the sulfated heterorhamnan isolated from the green seaweed Gayralia oxysperma. Carbohydrate Research, 2008, 343, 3085-3095.
[5] Harada N, & Maeda M. Chemical structure of antithrombin-active rhamnan sulfate from Monostroma nitidum. Bioscience Biotechnology Biochemistry, 1998, 62, 1647-1652.
[6] Lee J B, Koizumi S, Hayashi K, & Hayashi T. Structure of rhamnan sulfate from the green alga Mnonostroma nitidum and its anti-herpetic effect. Carbohydrate Polymers, 2010, 81, 572-577.
[7] Lahaye M, Inizan F, & Vigouroux J. NMR analysis of the chemical structure of ulvan and of ulvan-boron complex formation. Carbohydrate Polymers, 1998, 36, 239-249.
[8] Bilan M I, Vinogradova E V, Shashkov A S, & Usov A. Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense ( Bryopsidales, Chlorophyta ). Carbohydrate Research, 2007, 342, 586-596.
[9] Chattopadhyay K, Adhikari U, Lerouge P, & Ray B. Polysaccharides from Caulerpa racemosa: Purification and structural features. Carbohydrate Research, 2007, 68, 407-415.
[10] Ray B. Polysaccharides from Entermorpha compressa: Isolation, purification and structural features. Carbohydrate Polymers, 2006, 66, 408-416.
[11] Chattopadhyay K, Mandal P, Lerouge P, Driouich A, Ghosal P, & Ray B. Sulphated polysaccharides from Indian samples of Entermorpha compressa (Ulvales, Chlorophyta): Isolation and structural features. Food Chemistry, 2007, 104, 928-935.
[12]张会娟,毛文君,房芳,李红燕,齐晓辉.绿藻多糖结构和生物活性研究进展.海洋科学,2009,33(4):90-93.
[13] Zhang H J, Mao W J, Fang F, Li H Y, Sun H H, Chen Y, & Qi X H. Chemical characteristics and anticoagulant activities of a sulfated polysaccharide and its fragments from Monostroma latissimum. Carbohydrate Polymers, 2008, 71, 428-434.
[14] Mao W J, Fang F, Li H Y, Qi X H, Sun H H, Chen Y, Guo S D. Heparinoid-active two sulfated polysaccharides isolated from marine green algae Monostroma nitidum. Carbohydrate Polymers, 2008, 74, 834-839.
[15] Mao W J, Zang X X, Li Y, & Zhang H J. Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity. Journal of Applied Phycology, 2006, 18, 9–14.
[16] Matsubara K, Matsuura Y, Bacic A, Liao M L, Hori K, & Miyazawa K. Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum. International Journal of Biological Macromolecules, 2001, 28, 395–399.
[17] Athukorala Y, Lee K W, Kim S K, & Jeon Y J. Anticoagulant activity of marine green and brown algae collected from Jeju Island in Korea. Bioresource Technology, 2007, 98, 1711-1716.
[18] Siddhanta A K, Shanmugam M, Mody K H, Goswami A M, & Ramavat B K. Sulphated polysacchardies of Codium dwarkense Boergs. from the west coast of India: chemical composition and blood anticoagulant activity. International Journal of Biological Macromolecules, 1999, 26,151-154.
[19] Matsubara K, Matsuura Y, Hori K, & Miyazawa K. An anticoagulant proteoglycan from the marine green alga, Codium pugniformis. Journal of Applied Phycology, 2000, 12, 9-14.
[20] Witvrouw M & De Clercq E. Sulfated polysaccharides extracted from sea algae as potential antiviral drugs. General Pharmacology, 1997, 29, 497-511.
[21] Lee J B, Hayashi K, Hayashi T, Sankawa U & Maeda M. Antiviral activities against HSV-1, HCMV, and HIV-1 of rhamnan sulfate from Monostroma latissimum. Planta Medica, 1999,65 (5), 439-441.
[22] Lee J B, Hayashi K, Maeda M & Hayashi T. Antiherpetic activities of sulfated polysaccharides from green algae. Planta Medica, 2004, 70, 813-817.
[23] Ivanova V, Rouseva R,Kolarova M, Serkedjieva J, Rachev R & Manolova N. Isolation of a polysaccharide with antiviral effect from Ulva lactuca. Preparative Biochemistry, 1994, 24, 83-97.
[24] Pardee K I, Ellis P, Bouthillier M, Tower G H N & French C J. Plant virus inhibitors from marine algae. Canadian Journal of Botany, 2004, 82, 304-309.
[25]赵国华,陈宗道,李志孝,阚建全.活性多糖的研究进展.食品与发酵工业,2001,7(27): 45-48.
[26] Jiao L, Li X, Jiang P, Zhang L, Wu M, & Zhang L. Characterization and anti-tumor activity of alkali-extracted polysaccharide from Enteromorpha intestinalis. International Immunopharmacology, 2009, 9, 324-329.
[27] Tanaka K, Konishi F, Himeno K, Taniguchi K, & Nomoto K. Augmentation of antitumor resistance by a strain of unicellular green algae, Chlorella vulgaris. Cancer Immunology Immunotherapy. 1984, 17, 90-94.
[28] Karnjanapratum S, & You S G. Molecular characteristics of sulfated polysaccharides from Monostroma nitidum and their in vitro anticancer and imonodulatory activities. International Journal of Biological Macromolecules, 2011, 48, 311-318.
[29] Yu P, Zhang Q, Li N, Xu Z, Wang Y, & Li Z. Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity. Journal of Applied Phycology, 2003, 15, 21-27.
[30] Yu P, Li N, Liu X, Zhou G, Zhang Q, & Li P. Antihyperlipidemic effects of different molecular weight sulfated polysaccharides from Ulva pertusa (Chlorophyta). Pharmacological Research, 2003, 48, 543-549.
[31]王艳梅,李智恩,牛锡珍,张虹,张全斌.孔石莼多糖降血脂活性的初步研究。中国海洋药物,2003,2:33-35.
[32]周慧萍,蒋巡天,王淑如,陈琼华.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.
[33] Godard M, Decorde K, Ventura E, Soteras G, Baccou J C, Cridtol J P, & Rouanet J M. Polysaccharides from the green alga Ulva rigida improve the antioxidant status and prevent fatty streak lesions in the high cholesterol fed hamster, an animal model of nutritionally-induced atherosclerosis. Food Chemistry, 2009, 115, 176-180.
[34]李蜀眉.天然植物抗氧化作用的研究进展.内蒙古农业大学学报,2005,26:139-142.
[35] Formanek Z, Kerry J P, Higgins F M, Buckley D J, Morrissey P A, Farkas J. Addition of synthetic and natural antioxidants toα-tocopheryl acetate supplemented beef patties: effects of antioxidants and packaging on lipid oxidation. 2001, 58, 337-341.
[36] Qi H, Zhao T, Zhang Q, Li Z, & Zhao Z. Antioxidant activity of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta). Journal of Applied Phycology, 2005, 17, 527-534.
[37] Qi H, Zhang Q, Zhao T, Chen R, Zhang H, Niu X, & Li Z. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. International Journal of Biological Macromolecules, 2005, 37, 195-199.
[38] Kaplan D, Christiaen D, & Arad S. Chelating properties of extracellular polysaccharides from Chlorella Spp. Applied and Environmental Microbiology, 1987, 53, 2953-2956.
[39] Qi H, Zhang Q, Zhao T, Hu R, Zhang K, & Li Z. In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta). Bioorganic & Medicinal Chemistry Letters, 2006, 16, 2441-2445.
[40] Mao W J, Li Y, Wang H Q, Zhang Y, Zang X X, & Zhang H J. Chemical characterization and radioprotective effect of polysaccharide from Monostroma angicava (Chlorophyta). Journal of Applied Phycology, 2005, 17, 349-354.
[41]李德英,浙江省药用海洋生物资源调查报告.中国海洋药物杂志,1989,45:1.
[42]徐娟华,赵孟辉,谢强明,刘伟.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.
[43]徐大伦,黄晓春,欧昌荣,薛长湖,杨文鸽,王海洪.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.
[44]陈丽萍,王弘.硫酸多糖的结构与生物活性关系研究现状.广州化工,2005,33(5): 21-23.
[45]戴晋军,周小辉.常见多糖的研究进展.中国牧业通讯,2009,23:19-20.
[46] Mazumder S, Ghosal PK, Pujol CA, Carlucci, MJ, Damonte AB, & Ray B. Isolation, chemical investigation and antiviral activity of polysaccharides from Gracilaria corticata (Gracilariaceae, Rhodophyta). International Journal of Biological Macromolecules, 2002, 31, 87-95.
[47] Alban S, & Franz G. Characterization of the anticoagulant actions of a semisynthetic curdlan sulfate. Thrombosis Research, 2000, 99, 377-388.
[48] Koyanagi S, Tanigawa N, Nakagawa H, Soeda S, & Shimeno H. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochemical Pharmacology, 2003, 65, 173-179.
[49] Zvyagintseva T, Tatiana N, Shevchenko NM, Nazarova IV, Scobun AS, Lukyanov PA, & Elyakova LA. Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds. Comparative Biochemistry and Physiology, Part C: Toxicology & Pharmacology, 2000, 126C, 209-215.
[50] Mizumoto K, Sugawara I, Ito W, Kodama T, Hayami M, & Mori S. Sulfated homopolysaccharides with immunomodulating activities are more potent anti-HTLV-III agents than sulfated heteropolysaccharides. Japanese Journal of Experimental Medicine, 1988, 58, 145-151.
[51] Chaidedgumjorn A, Toyoda H, Woo E R, Lee K B, Yeong S, Tioda T, & Imanari T. Effect of (1→3)- and (1→4)-linkages of fully sulfated polysaccharides on their anticoagulant activity. Carbohydrate Research, 2002, 337, 925-933.
[52]戴伟娟,仲伟法,索金良,林志彬.4-硒硫酸酯多糖对人肝癌细胞Bel/7402 DNA RNA及蛋白质合成的影响.济宁医学院学报,2001,24(1):18-19.
[53]赵峡,于广利,李少平,吕志华,徐家敏.几种海藻多糖硫酸酯碱式铝盐的制备.青岛海洋大学学报(自然科学版),2002,32(3):385-390.
[54]肖锡湘,上官新晨.多糖的应用研究[J].中国食物与营养.2006,5:21-23.
[55]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,2006,8(9):1353-1356.
[56] Lahaye M. NMR spectroscopic characterization of oligosaccharides from two Ulva rigida ulvan samples (Ulvales, Chlorophyta) degraded by a lyase. Carbohydrate Research. 1998, 314, 1-12.
[57] Ogawa K, Ikeda Y, & Kondo S. A new trisaccharide,α-D-glucopyranuronosyl -(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranose from Chlorella vulgaris. Carbohydrate Research, 1999, 321, 128-131.
[58] Zheng Y. & Mort A. Isolation and structural characterization of a novel oligosaccharide from the rhamnogalacturonan of Gossypium hirsutum L.. Carbohydrate Research, 2008, 343, 1041-1049.
[59] Zhang Z Q, Yu G L, Guan H S, Zhao X, Du Y G, Jiang X L. Preparation and structure elucidation of alginate oligosaccharides degraded by alginate lyase from Vibro sp. 510. Carbohydrate Researchm, 339, 1475-1481.
[60] Zhang Z Q, Yu G L, Zhao X, Guan H S, Lawson A M, Chai W G. Sequence analysis of Alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry. American Society for Mass Spectrometry, 2006, 17, 621-630.
[61] Yu G L, Zhao X, Yang B, Ren S M, Guan H S, Zhang Y B, Lawson A M, Chai W G. Sequence determination of sulfated carrageenan-derived oligosaccharides by high-sensitivity negative-ion electrospray tandem mass spectrometry. Analytical Chemistry, 2006, 78, 8499-8505.
[62]毛文君,林洪,管华诗.琼胶寡糖的ESI-MS分析研究.中国水产科学,2001,8(3): 69-72.
[63]毛文君,林洪,管华诗.琼胶寡糖的制备及其13C-NMR研究.水产科学,2001,25(6): 582-584.
[64]李翊,王海青.卡拉胶寡糖对放射损伤的防护作用.中华放射医学与防护杂志,2005,25(2):116-117.
[65]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.
[66] Mou H J, Jiang X L, & Guan H S. A ?-carrageenan derived oligosaccharide prepared by enzymatic degradation containing anti-tumor activity. Journal of Applied Phycology, 2003, 15, 297-303.
[67]杜昱光,白雪芳,金宗濂,燕秋,朱正美.壳寡糖抑制肿瘤作用的研究.中国海洋药物,2002,21 (2):18-21.
[68]柳红,崔涛,刘莹.壳寡糖作用后人结肠癌LoVo细胞株bcl-2、bax表达改变.徐州医学院学报,2005,25(1):1-3.
[69] Yoshio O, Makiko K, Shigeo S, & Masuko S. Comparative study of protective effects of chitin, chitosan, and N-acetyl chitohexaose against Pseudomonas aeruginosa and Listeria monocytogenes infection in mice. Biological & Pharmaceutical Bulletin, 2003, 26, 902-904.
[70]郭晓农,张汝学,贾正平.治疗胰岛素抵抗的中药及活性成分研究进展.中药材,2005,28(4), 350-353.
[71]曹朝晖,李邦良,乔新惠,周娣先,谭洁琼.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.
[72]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.
[73] Wang J X, Jiang X L, Mou H J, & Guan H S. Antioxidation of agar oligosaccharides produced by agarase from a marine bacterium. Journal of Applied Phycology, 2004, 16, 333-340.
[74] Chen A S, Taguchi T, Sakai K, Kikuchi K, Wang M W, & Miwa L. Antioxidant activities of chitobiose and chitotriose. Biological & Pharmaceutical Bulletin, 2003, 26, 1326-1330.
[75]赵智辉,褚健,陈元,高巍,楚燕灵.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):11-15.
[76]曹红,张世玲,姬胜利,程艳娜.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.
[77]杨悦,石森林,葛卫红.多糖的化学研究概况.中国药师,2008,11(1):93-95.
[78]杨云,冯卫生,雷高明.大枣渣多糖精制纯化工艺研究.中药材,2006,29(1):78-80.
[79]王卫国,赵永亮.一种在多糖分离纯化过程中新的脱蛋白方法.中草药,2003,34(10):891-895.
[80]霍贤,梁忠岩,张雅君,张旭,张丽霞.红花水溶性多糖的分离、纯化及初步研究.中国药学杂志,2005,40(8):620-622.
[81]刘锐.多糖类物质的研究进展.安徽农业科学,2005,33(9):1722-1725.
[82] Sugiwa M, Ohno H, Kunihisa M, Hirata F, & Ito H. Studies on antitumor polysaccharides, especially D-II, from mycelium of Coriolus versicolor. Japanese Journal of Pharmacology, 1980, 30, 503-513.
[83] Lindberg B. High-aoltage electrophoresis for polysaccharides. Methods in Carbohydrate Chemistry, 1963, 7, 116 -145.
[84]李林,罗琼,张声华.海带多糖的分类提取、鉴定及理化特性研究.食品科学,2000,21(4):28-32.
[85]陈玉香,张丽萍,梁忠岩,苗春艳,张翼伸.沙棘果皮水溶性多糖Hn的结构研究.分子科学学报,2000,16(1):19-22.
[86] Golovchenko V V, Ovodova R G, Shashkov A S, & Ovodov Y S. Structural studies of the pectic polysaccharide from duckweed Lemna minor L.. Phytochemistry, 2002, 60:89-97.
[87] Hakomori S. A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. Journal of biochemistry, 1964, 55:205-208.
[88]陶乐平,丁在富,张部昌.气相色谱在多糖结构测定中的应用.色谱,1994,12(5):351-354.
[89] Sidorczyk Z, Zych K, Toukach F V, Arbatsky N P, Zablotni A, Shashkov A S, & Knirel Y A. Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3. European Journal of Biochemistry, 2002, 269, 1406-1412.
[90] Tikhomicrov M M, Khorlin A Y, Voelter W, & Bauer H. High-performance liquid chromatographic investigation of the amino acid, amino sugar and neutral sugar content in glycoproteins. Journal of chromatography, 1978, 167, 197-203.
[91] Nashabeh W, & El R Z. Capillary zone electrophoresis of pyridylamino derivatives of maltooligosaccharides. Journal of chromatography, 1990, 514, 57-64.
[92] Vorndran A E, Grill E, Huber C, Oefner P J, & Bonn G K. Capillary zone electrophoresis of aldroses, ketoses and uronic acids derivatized with ethyl P-aminobenzoate. Chromatographia, 1992, 34, 109-114.
[93] Goubet F, Jackson P, Deery M J, & Dupree P. Polysaccharide analysis using carbohydrate gel electrophoresis: A method to study plant cell wall polysaccharides and polysaccharide hydrolases. Analytical Biochemistry, 2002, 300, 53-68.
[94]张惟杰.糖复合物生化研究技术.杭州:浙江大学出版社,1999.
[95] Stroop, C. J. M., Xu, Q, W., Retzlaff, M., Abeygunawardana, C., & Bush, C. A. Structural analysis and chemical depolymerization of the capsular polysaccharide of Streptococcus pneumoniae type 1. Carbohydrate Research, 2002, 337, 335-344.
[96]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.
[97]张惟杰.糖复合物生化研究技术.第二版.杭州:浙江大学出版社,1999,133-135.
[98] Ovalle R, Soll C E, Lim F. Systermatic analysis of oxidative degradation of polysaccharides using PAGE and HPLC-MS. Cabohydrate Research, 2001, 330, 131-139.
[99] Chen H M, Zheng L, Lin W, Yan X J. Product monitoring and quantitation of oligosaccharides composition in agar hydrolysates by precolumn labeling HPLC. Talanta, 2004, 64, 773-777.
[100]刘翠平,方积年.质谱技术在糖类结构分析中的应用.分析化学评述与进展,2001,29(6):716-720.
[101]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.
[102] Gamian A, Ulrich J, Defaye J, Mieszala M, Witkoska D, Romanowska E. Structural heterogeneity of the sialic-acid-containing oligosaccharides from the lipopolysaccharides of Hafnia alvei strain 2 as detected by FABMS studies. Carbohydrate Research, 1998, 314, 201-209.
[103] Reinhold V N, Reinhold B B, Chan S. Carbohydrate sequence analysis by electrospray ionization-mass spectrometry. Methods in Enzymology, 1996, 271, 377-402.
[104] Duffin K L, Welply J K, Huang E, Henion J D. Characterization of N-linked oligosaccharides by electrospray and tandem mass spectrometry. Analytical Chemistry, 1992, 64, 1440-1448.
[105] Yu G L, Zhao X, Yang B, Ren S M, Guan H S, Zhang Y B, Lawson A M, Chai W G. Sequence determination of sulfated carrageenan-derived oligosaccharides by high-sensitivity negative-ion electrospray tandem mass spectrometry. Analytical Chemistry, 2006, 78, 8499-8505.
[106]阳佛送,李雪华.多糖结构研究的方法和进展.食品安全与检测,2008,3:200-203.
[107]王顺春,方积年. X-射线纤维衍射在多糖构型分析中应用的研究进展.天然产物研究与开发,2000,12(2):75-80.
[108]鞠建华,周亮,杨峻山.二维核磁共振波谱在阐明一种三萜多糖皂苷结构中的应用.波谱学杂志,2001,18(4):229-341.
[109] Engle A R, Purdie N, & Hyatt J A. Induced circular dichroism study of the aqueous solution complexation of cello-oligosaccharides and related polysaccharides with aromatic dyes. Carbohydrate Research, 1994, 265, 181-195.
[110]沈琼,黄滨,邵嘉亮,彭权,马林,古练权.运用圆二色谱研究酶与化合物相互作用的机理.中山大学学报(自然科学版),2006,45(4):62-64.
[111]姚占全,敖敦格日勒,许强,杨体强.应用圆二色光谱研究电场对脂肪酶二级结构的影响.光谱学与光谱分析,2006,26(12):2311-2314.
[112] Paradossi G, Chiessi E, Barbiroli A, & Fessas D. Xanthan and glucomannan mixtures: synergistic interactions and gelation. Biomacromolecules, 2002, 3, 498- 504.
[113] Abu-lail N I, & Camesano T A. Polysaccharide properties probed with atomic force microscopy. Journal of microscopy, 2003, 212(Pt3), 217- 238.
[114] Rief M, Oesterhelt F, Heymann B, & Gaub H E. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science (Washington, D. C.), 1997, 275, 1295-1297.
[115] Rief M, Gautel M, Oesterhelt F, Fernandez J M, & Gaub H E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science (Washington, D. C.), 1997, 276, 1109-1112
[116]张会娟.宽礁膜(Monostroma latissimum)多糖的分离、结构和抗凝血活性研究:[博士学位论文].2007.
[117]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].2008.
[118] Dubois M, Gilles K A, Hamilton J K, Rebers P A, Smith F. Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 1956, 28(3), 350-366.
[119] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 7, 248-254.
[120] Therho T T, Hartiala K. Method for determination of the sulfate content of glycosamino glycans. Analytical Biochemistry, 1971, 41, 471-476.
[121] Bitter T, Muir H M. A modified uronic acid carbazole reaction. Analytical Biochemistry, 1962, 4, 330-334.
[122]张惟杰.糖复合物生化研究技术.第二版.浙江大学出版社.1994:398,417.
[123]邓时锋,刘志礼,李兆兰等.极大螺旋藻多糖的分离纯化及化学结构分析.南京大学学报(自然科学).2000,36(5):579-584.
[124]王皓,于广利,赵峡,郝翠,李欣.几种动物肝脏糖胺聚糖的醋酸纤维素薄膜电泳分析.中国生物化学与分子生物学报. 2009,25(2):193-198.
[125] Lv H Z, Yu G L, Sun L L, Zhang Z, Zhao X, Chai W G. Elevate level of glycosaminoglycans and altered sulfation pattern of chondroitin sulfate are associated with differentiation status and histological type of human primary hepatic carcinoma. Oncology, 2007, 72, 347-356.
[126] Nelson S R, Lyon M, Gallagher J T, Johnson E A, & Pepys M B. Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils. Biochemical Journal, 1991, 275, 67-73.
[127] Sun H H, Mao W J, Chen Y, Guo S D, Li H Y, Qi X H, Chen Y L, Xu J. Isolation, chemical characteristics and antioxidant properties of the polysaccharides from marine fungus Penicillium sp. F23-2. Carbohydrate Polymers, 2009, 78, 117-124.
[128] Falshaw R, Furneaux R H. Structural analysis of carrageenans from the tetrasporic stages of the red algae, Gigartina lanceata and Gigartina chapmanii (Gigartinaceae, Rhodophyta).Carbohydrate Research, 1998, 307, 325-331.
[129] Needs P W, Selvendran R R. Avoiding oxidative degradationduring sodium hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydrate Research, 1993, 245: 1-10.
[130] Mukerjea R, Kim D, Robyt J F. Simplified and improved methylation analysis of saccharides, using a modified procedure and thin-layer chromatography. Carbohydrate Research, 1996, 292, 81-89.
[131] Majdoub H, Mansour M B, Chaubet F, Roudesli M S, Maaroufi R M. Anticoagulant activity of a sulfated polysaccharide from the green alga Arthrospira platensis. Biochimica et Biophysica Acta, 2009, 1790, 1377-1381.
[132] Chen S G, Xue C H, Yin L A, Tang Q J, Yu G L, Chai W G. Comparison of structures and anticoagulant activities of fucosylated chomdroitin sulfates from different sea cucumbers. Carbohydrate Polymers, 2011, 83, 688-696.
[133] Vieira R P, Mulloy B, & Mourao P A S. Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues. Journal of Biological Chemistry, 1991, 266, 13530–13536.
[134] Mulloy B, Ribeiro A C, Alves A P, Vieira R P, & Mourao P A S. Sulfated fucans from echinoderms have a regular tetrasaccharide repeating unit defined by specific patterns of sulfation at the O-2 and O-4 positions. Journal of Biological Chemistry, 1994, 269, 22113–22123.
[135] Patankar M S, Oehninger S, Barnett T, Williams R L, & Clark G F. A revised structure for fucoidan may explain some of its biological activities. Journal of Biological Chemistry, 1993, 268, 21770–21776.
[136] Senchenkova S, Shashkov A, Laux P, Knirel Y, & Rudolph K. The O-chain polysaccharide of lipopolysaccharide of Xanthomonas Campestris pv. Begoniae GSPB 525 is a particlly L-xylosylated L-rhamnan. Carbohydrate Research, 1999, 319, 148-153.
[137] Gaur D, Galbraith L, & Wilkinson S G. Structural characterization of a rhamnan and a fucorhamnan, both present in the lipopolysaccharide of Burkholderia vietnamiensis strain LMG 10926. European Journal of Biochemistry, 1998, 258, 696-701.
[138] Gargiulo V, Castro C D, Lanzetta R, Jiang Y, Xu L H, Jiang C L, Molinaro A, & Parrilli M. Structural elucidation of the capsular polysaccharide isolated from Kaistella flava. Carbohydrate Research, 2008, 343, 2401-2405.
[139] Senchenkova S N, Shashkov A S, Laux P, Knirel Y A, & Rudolph K. The O-chain polysaccharide of the lipopolysaccharide of Xanthomonas campestris pv. begoniae GSPB 525 is a partially L-xylosylated L-rhamnan. Carbohydrate Research, 1999, 319, 148-153.
[140] Bedini E, Castro C D, Erbs G, Mangoni L, Dow J M, Newman M A, Parrilli M, & Unverzagt C. Structure-Dependent modulation of a Pathogen response in plants by synthetic O-antigen polysaccharides. Journal of American chemical society, 2005, 127, 2414-2416.
[141] Choma A, Komaniecka I, & Sowinski P. Revised structure of the repeating unit of theO-specific polysaccharide from Azospirillum lipoferum strain SPBr 17. Carbohydrate Research, 2009, 344:936-939.
[142] Ovod V V, Zdorovenko, E. L., Shashkov, A. S., Kocharova, N. A. & Knirel, Yu. A. Structural diversity of O-polysaccharides and serological classification of Pseudomonas syringae pv. garcae and other strains of genomospecies 4. Microbiology, 2004, 73, 666–677.
[143] Knirel Y A, Ovod V V, Zdorovenko G M, Gvozdyak R I, & Krohn K J. Structure of the O-polysaccharide and immunochemical relationships between the lipopolysaccharides of Pseudomonas syringae pathovar tomato and pathovar maculicola. European Journal of Biochemistry, 1998, 258, 657–661.
[144] Senchenkova S N, Shashkov A S, Laux P, Knirel Y A, & Rudolph K. The O-chain polysaccharide of Xanthomonas campestris pv. begoniae GSPB 525 is a partially L-xylosylated L-rhamnan. Carbohydrate Research, 1999, 319, 148–153.
[145] Gargiulo V, Castro C D, Lanzetta R, Jiang Y, Xu L H, Jiang C L, Molinaro A, & Parrilli M. Structural elucidation of the capsular polysaccharide isolated from Kaistella flava. Carbohydrate Research, 2008, 343, 2401–2405.
[146] Gaur D, Galbraith L, & Wilkinson S G. Structural characterization of a rhamnan and a fucorhamnan, both present in the lipopolysaccharide of Burkholderia vietnamiensis strain LMG 10926. European Journal of Biochemistry, 1998, 258, 696–701.
[147]缪月秋,顾袭平,吴国荣.植物多糖水解及其产物研究进展.中国野生植物资源.2005,2(24):4-7.
[148]李红燕,齐晓辉,郭守东,陈荫,陈艳丽,徐健,赵春琦,叶欣,毛文君.宽礁膜多糖酸的水解条件.中国海洋大学学报,2010,40(5): 11-14.
[149] Yu G L, Guan H S, Ioanoviciu A S, Sikkander S A, Thanawiroon C, Tobacman J K, Toida T, & Linhardt R J. Structural studies onκ-carrageenan derived oligosaccharides. Carbohydrate Research, 2002, 337, 433-440.
[150]迟连利,Robert J Linhardt.κ-卡拉胶五糖的制备及结构研究.中国海洋药物.2002,21(3):21-27.
[151]付海宁,赵峡,于广利,陈娥功,文松松.盐藻多糖单糖组成分析的四种色谱方法比较.中国海洋药物杂志.2008,27(4):30-34.
[152]戴军,朱松,汤坚,王旻,尹鸿萍,陈尚卫.PMP柱前衍生高效液相色谱法分析杜氏盐藻多糖的单糖组成.分析测试学报.2007,26(2):206-210.
[153] Bahr U, Pfenninger A, Karas M, & Stahl B. High-sensitivity analysis of neutral underivatized oligosacchardies by nanoelectrospray mass spectrometry. Analytical Chemistry, 1997, 69, 4530–4535.
[154] Suzuki H, Muller O, Guttman A, & Karger B L. Analysis of 1-aminopyrene -3,6,8-trisulfonate-derivatized oligosaccharides by capillary electrophoresis with matric-assisted laser desorption/ionization time-of-flight mass spectrometry. Analytical Chemistry, 1997, 69, 4554–4559.
[155] Viseux N, Hoffmann E D, & Domon B. Structural analysis of permethylated oligosaccharides by electrospray tandem mass spectrometry. Analytical Chemistry, 1997, 69, 3193–3198.
[156] Chai W G, Piskarev V, & Lawson A M. Negative-ion electrospray mass spectrometry of neutral underivatized oligosaccharides. Analytical Chemistry, 2001, 73, 651–657.
[157] Chai W G, Luo J L, Lim C K, & Lawson A M. Characterization of heparin oligosaccharide mixtures as ammonium salts using electrospray mass spectrometry. Analytical Chemistry, 1998, 70, 2060–2066.
[158] Thomsson K A, Karlsson H, & Hanssom G C. Sequencing of sulfated oligosaccharides from mucins by liquid chromatography and electrospray ionization tandem mass spectrometry. Analytical Chemistry, 2000, 72, 4543–4549.
[159] Cancilla M T, Gaucher S P, Desaire H, & Leary J A. Combined partial acid hydrolysis and electrospray ionization-mass spectrometry for the structural determination of oligosaccharides. Analytical Chemistry, 2000, 72, 2901–2907.
[160] Zaia J, Mcclellan J E, & Costello C E. Tandem mass spectrometric determination of the4S/6S sulfation sequence in chondroitin sulfate oligosaccharides. Analytical Chemistry, 2001, 73, 6030–6039.
[161] Huang L H, & Riggin R M. Analysis of nonderivatized neutral and sialylated oligosaccharides by electrospray mass spectrometry. Analytical Chemistry, 2000, 72, 3539–3546.
[162]张真庆.质谱分析外源性和内源性寡糖的方法学研究:[博士学位论文].2006.
[163] Daniel R, Chevolot L, Carrascal M, Tissot B, Mourao P A S, & Abian J. Electrospray ionization mass spectrometry of oligosaccharides derived from fucoidan of Ascophyllum nodosum. Carbohydrate Research, 2007, 342, 826-834.
[164] Saad O M, & Leary J A. Delineating mechanisms of dissociation for isomeric heparin disaccharides using isotope labeling and ion trap tandem mass spectrometry. Journal of the American Society for Mass Spectrometry, 2004, 15, 1274-1286.
[165] Tissot B, Salpin J Y, Martinez M, Gaigeot M P, & Daniel R. Differentitation of the fucoidan sulfated L-fucose isomers constituents by CE-ESIMS and molecular modeling. Carbohydrate Research, 2006, 341, 598-609.
[166] Pomin V H, Pereira M S, Valente A P, Tollefsen D M, Pavao M S G, & Mourao P A S. Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removel of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity. Glycobiology, 2005, 15, 369-381.
[167] Pereira M S, Mulloy B, & Mourao P A S. Structure and anticoagulat activity of sulfated fucan. The journal of Biological Chemistry, 1999, 274, 7656-7667.