一种灰树花菌丝体水不溶多糖硫酸酯的制备及其理化性质研究
|
摘要
灰树花(Grifola frondosa )是一种名贵药食兼用真菌,灰树花多糖作为其主要活性成分具有提高免疫力、抗肿瘤、抗病毒等功能。然而,用碱溶液从灰树花菌丝体中提取到的大量多糖由于其水不溶性,影响了它在药物方面的开发应用。因此,需要通过化学改性来增加它的水溶性,从而显著提高多糖的生物活性并拓展其应用范围。多糖的硫酸酯化反应是其化学修饰中最吸引人的领域,硫酸酯化多糖由于其抗凝血和抗病毒的作用,而受到广泛关注。多糖硫酸酯的生物活性与结构之间具有密切关系,硫酸酯基的取代位置、硫酸酯基的取代度、分子量、理化性质等都对其生物活性有重大影响。
本文主要对灰树花多糖的提取,灰树花水不溶性多糖GF4A硫酸酯化条件的优化,酯化衍生物SGF4A的理化性质,结构分析和活性测试等进行了研究。系统研究了三氧化硫三甲胺盐-二甲基甲酰胺法对GF4A酯化反应的影响,获得了水溶性良好、取代度高的工艺参数。初步实验结果表明,SGF4A具有较好的免疫调节和抗凝活性,并以SGF4A为原料,进一步研究了该多糖的理化性质。主要结果如下:
1.以灰树花发酵菌丝体为原料,利用碱提酸沉淀法制得一种水不溶性多糖GF4A,经凝胶柱层析获得纯品,糖含量为99.4%。GF4A室温下不溶于水,但溶于0.5 mol/L NaOH中。综合运用化学分析法(酸水解,高碘酸氧化,Smith降解等)和波谱学方法(红外光谱IR,核磁共振波谱NMR等)对其结构进行了表征,结果表明:GF4A是由葡萄糖(Glc)、甘露糖(Man)和木糖(Xyl)三种单糖组成,其摩尔比约为9:2:1;每12个糖残基构成一个结构重复单元,主链由6个α-1,3- D-Glc和2个α-1,3-D-Man构成;每个糖单元中有2个1,4连接的侧链,一分支为三个β-1,4连接的Glc,另一分支为α-1,4连接的Xyl。
2.以GF4A为原料,从事了GF4A的硫酸酯化工艺研究。以产率,硫酸根取代度(DS)和硫酸酯化试剂的性能为指标,比较了氯磺酸-甲酰胺法、三氧化硫三甲胺盐-二甲基甲酰胺法和三氧化硫吡啶盐-吡啶法的酯化特性,确定了三氧化硫三甲胺盐-二甲基甲酰胺法为最佳酯化体系,并通过优化实验,考察了反应温度、反应时间和原料配比对GF4A硫酸酯化产物的影响。最佳制备工艺条件为:硫酸酯化剂与GF4A质量比为5:1、反应时间6 h、反应温度为80℃,该条件下所得酯化产物(SGF4A)水溶解性好,产率153%,取代度为1.6,相对分子量为88 kD。
3.根据《中国药典》,对三批不同批号的SGF4A进行了理化性质研究,结果表明:SGF4A在室温下溶解度高达50%以上,分子量在85~88 kD之间,硫酸基取代度1.5~1.6。紫外光谱分析表明,样品在265 nm处有弱吸收;红外光谱分析表明,样品在1240 cm-1和810 cm-1处有硫酸酯基的特征吸收峰;核磁共振碳谱分析表明,样品C6位羟基完全被酯化, C2和C4位羟基部分被酯化。家兔实验结果表明,SGF4A具有较好的抗凝血活性,在50μg/mL时,其抗凝效果相当于肝素的50%左右;小鼠实验结果显示,SGF4A具有一定的免疫调节作用,可抑制conA诱导T淋巴细胞的增殖,可抑制LPS诱导B淋巴细胞的增殖,在10μmol/L、1μmol/L高浓度可抑制巨噬细胞的活力,而低浓度0.1μmol/L、0.01μmol/L可促进巨噬细胞的吞噬作用。SGF4A的抗S180肿瘤活性测试正在进行中。
本文的研究结果为灰树花多糖及其硫酸酯化产物在医药领域的应用奠定基础,为更好的开发利用灰树花多糖资源,促进我国药用真菌制品的发展具有十分重要的意义。
Grifola frondosa is well known as a highly nutritious and officinal edible fungus. Polysaccharide, one of the main active components extraced from Grifola frondosa, has various functions such as stimulating immune system, anticancer, antivirus and so on. However, a part of these active polysaccharides were extracted alkalinely from the mycelia of submerged fermentation of Grifola frondosa are water-insoluble, which limits their applications in the medicine field. The chemical modification can improve the solubility of natural polysaccharides to enhance their activities, and enlarge their application range. The sulfation of polysaccharides is the most interesting field of its chemical modification. Lot attention has been paid to sulfated polysaccharides because of their anticoagulant and antivirus activity. And the sites of sulfation, sulfate content, molecular weight, physical and chemical characterizations are the key factors for these activities.
This thesis mainly focuses on the isolation of water-insoluble polysaccharide GF4A from Grifola frondosa, the optimized conditions for the sulfation of GF4A, the characterization, structure analysis and bioactivities of the derivative SGF4A. Systemically study is carried out to find out the optimum preparative conditions using Me3N·SO3-DMF. The activity results showes that SGF4A has anticoagulant and immunomodulating activities. The characterizations of SGF4A are studied on the basis of the research above. The main results are shown as follows:
1. Water-insoluble polysaccharide GF4A is extracted in cool alkaline solution from the mycelia of submerged fermentation of Grifola frondosa, and purified with gel-permeation chromatography. A pure water-insoluble polysaccharide GF4A with sugar content of 99.4% is obtained. While it could be dissolved in 0.5mol/L NaOH solution. The primary structure of GF4A is elucidated by chemical method, such as acid hydrolysis, periodate oxidation, Smith degradation, and spectroscopic techniques IR, 13C-NMR etc.. The results show that GF4A contains glucose, mannose and xylose, with the molar ratio of 9:2:1. Every 12 sugar residues consist a repeating unit, in which the backbone is composed of sixα-1,3-D-Glc and twoα-1,3-D-Man. Each unit also has two branches, one is consisted of threeβ-1,4-Glc, and the other is anα-1,4-Xyl.
2. The reaction conditions are studied to prepare sulfated GF4A .The degree of sulfation, yield, Mw and capability of sulfate reagents are used as the guideline to compare three different sulfation system. According to the results, sulfation using Me3N·SO3-DMF is the optimal method. In this reaction, a high yield of sulfation (153%) was achieved with the following optimized conditions: mass ratio of Me3N·SO3 to GF4A, 5:1; temperature, 80oC; reaction time, 6 hours. The degree of sulfation is 1.6 and the relative molecular mass 88 KD. The activity results in vivo showed that sulfated polysaccharide had a good antitumor activity. 3. The physical and chemical characters of three bathes of SGF4A are studied according to the appendix of The Pharmacopoeia of China. The results indicate that SGF4A is water-soluble in room temperature with the highest concentration of 50%, Mw is between 85 kD ~ 88 kD, degree of sulfation is 1.5~1.6. The UV and IR spectra of SGF4A showed that characteristic absorptions of sulfated eater bond at 265nm and 1240cm-1, 810cm-1 respectively, which demonstrate that the modification of sulfation is successful. Relative signal in 13C-NMR spectrum indicate that the C6-OH has been sulfated completely, and the sulfation also occurs at some C2-OH and C4-OH position. The activity results in vivo showed that sulfated polysaccharide had a good anticoagulant activity and immunomodulating activity.
The series of optimal conditions studied in this thesis is suitable for preparing the water-soluble sulfated polysaccharide SGF4A with high content of sulfation and high yield. This study will lay a foundation for more application of Grifola frondosa in medicine fields and accelerate the development of officinal fungus in our country.
本文主要对灰树花多糖的提取,灰树花水不溶性多糖GF4A硫酸酯化条件的优化,酯化衍生物SGF4A的理化性质,结构分析和活性测试等进行了研究。系统研究了三氧化硫三甲胺盐-二甲基甲酰胺法对GF4A酯化反应的影响,获得了水溶性良好、取代度高的工艺参数。初步实验结果表明,SGF4A具有较好的免疫调节和抗凝活性,并以SGF4A为原料,进一步研究了该多糖的理化性质。主要结果如下:
1.以灰树花发酵菌丝体为原料,利用碱提酸沉淀法制得一种水不溶性多糖GF4A,经凝胶柱层析获得纯品,糖含量为99.4%。GF4A室温下不溶于水,但溶于0.5 mol/L NaOH中。综合运用化学分析法(酸水解,高碘酸氧化,Smith降解等)和波谱学方法(红外光谱IR,核磁共振波谱NMR等)对其结构进行了表征,结果表明:GF4A是由葡萄糖(Glc)、甘露糖(Man)和木糖(Xyl)三种单糖组成,其摩尔比约为9:2:1;每12个糖残基构成一个结构重复单元,主链由6个α-1,3- D-Glc和2个α-1,3-D-Man构成;每个糖单元中有2个1,4连接的侧链,一分支为三个β-1,4连接的Glc,另一分支为α-1,4连接的Xyl。
2.以GF4A为原料,从事了GF4A的硫酸酯化工艺研究。以产率,硫酸根取代度(DS)和硫酸酯化试剂的性能为指标,比较了氯磺酸-甲酰胺法、三氧化硫三甲胺盐-二甲基甲酰胺法和三氧化硫吡啶盐-吡啶法的酯化特性,确定了三氧化硫三甲胺盐-二甲基甲酰胺法为最佳酯化体系,并通过优化实验,考察了反应温度、反应时间和原料配比对GF4A硫酸酯化产物的影响。最佳制备工艺条件为:硫酸酯化剂与GF4A质量比为5:1、反应时间6 h、反应温度为80℃,该条件下所得酯化产物(SGF4A)水溶解性好,产率153%,取代度为1.6,相对分子量为88 kD。
3.根据《中国药典》,对三批不同批号的SGF4A进行了理化性质研究,结果表明:SGF4A在室温下溶解度高达50%以上,分子量在85~88 kD之间,硫酸基取代度1.5~1.6。紫外光谱分析表明,样品在265 nm处有弱吸收;红外光谱分析表明,样品在1240 cm-1和810 cm-1处有硫酸酯基的特征吸收峰;核磁共振碳谱分析表明,样品C6位羟基完全被酯化, C2和C4位羟基部分被酯化。家兔实验结果表明,SGF4A具有较好的抗凝血活性,在50μg/mL时,其抗凝效果相当于肝素的50%左右;小鼠实验结果显示,SGF4A具有一定的免疫调节作用,可抑制conA诱导T淋巴细胞的增殖,可抑制LPS诱导B淋巴细胞的增殖,在10μmol/L、1μmol/L高浓度可抑制巨噬细胞的活力,而低浓度0.1μmol/L、0.01μmol/L可促进巨噬细胞的吞噬作用。SGF4A的抗S180肿瘤活性测试正在进行中。
本文的研究结果为灰树花多糖及其硫酸酯化产物在医药领域的应用奠定基础,为更好的开发利用灰树花多糖资源,促进我国药用真菌制品的发展具有十分重要的意义。
Grifola frondosa is well known as a highly nutritious and officinal edible fungus. Polysaccharide, one of the main active components extraced from Grifola frondosa, has various functions such as stimulating immune system, anticancer, antivirus and so on. However, a part of these active polysaccharides were extracted alkalinely from the mycelia of submerged fermentation of Grifola frondosa are water-insoluble, which limits their applications in the medicine field. The chemical modification can improve the solubility of natural polysaccharides to enhance their activities, and enlarge their application range. The sulfation of polysaccharides is the most interesting field of its chemical modification. Lot attention has been paid to sulfated polysaccharides because of their anticoagulant and antivirus activity. And the sites of sulfation, sulfate content, molecular weight, physical and chemical characterizations are the key factors for these activities.
This thesis mainly focuses on the isolation of water-insoluble polysaccharide GF4A from Grifola frondosa, the optimized conditions for the sulfation of GF4A, the characterization, structure analysis and bioactivities of the derivative SGF4A. Systemically study is carried out to find out the optimum preparative conditions using Me3N·SO3-DMF. The activity results showes that SGF4A has anticoagulant and immunomodulating activities. The characterizations of SGF4A are studied on the basis of the research above. The main results are shown as follows:
1. Water-insoluble polysaccharide GF4A is extracted in cool alkaline solution from the mycelia of submerged fermentation of Grifola frondosa, and purified with gel-permeation chromatography. A pure water-insoluble polysaccharide GF4A with sugar content of 99.4% is obtained. While it could be dissolved in 0.5mol/L NaOH solution. The primary structure of GF4A is elucidated by chemical method, such as acid hydrolysis, periodate oxidation, Smith degradation, and spectroscopic techniques IR, 13C-NMR etc.. The results show that GF4A contains glucose, mannose and xylose, with the molar ratio of 9:2:1. Every 12 sugar residues consist a repeating unit, in which the backbone is composed of sixα-1,3-D-Glc and twoα-1,3-D-Man. Each unit also has two branches, one is consisted of threeβ-1,4-Glc, and the other is anα-1,4-Xyl.
2. The reaction conditions are studied to prepare sulfated GF4A .The degree of sulfation, yield, Mw and capability of sulfate reagents are used as the guideline to compare three different sulfation system. According to the results, sulfation using Me3N·SO3-DMF is the optimal method. In this reaction, a high yield of sulfation (153%) was achieved with the following optimized conditions: mass ratio of Me3N·SO3 to GF4A, 5:1; temperature, 80oC; reaction time, 6 hours. The degree of sulfation is 1.6 and the relative molecular mass 88 KD. The activity results in vivo showed that sulfated polysaccharide had a good antitumor activity. 3. The physical and chemical characters of three bathes of SGF4A are studied according to the appendix of The Pharmacopoeia of China. The results indicate that SGF4A is water-soluble in room temperature with the highest concentration of 50%, Mw is between 85 kD ~ 88 kD, degree of sulfation is 1.5~1.6. The UV and IR spectra of SGF4A showed that characteristic absorptions of sulfated eater bond at 265nm and 1240cm-1, 810cm-1 respectively, which demonstrate that the modification of sulfation is successful. Relative signal in 13C-NMR spectrum indicate that the C6-OH has been sulfated completely, and the sulfation also occurs at some C2-OH and C4-OH position. The activity results in vivo showed that sulfated polysaccharide had a good anticoagulant activity and immunomodulating activity.
The series of optimal conditions studied in this thesis is suitable for preparing the water-soluble sulfated polysaccharide SGF4A with high content of sulfation and high yield. This study will lay a foundation for more application of Grifola frondosa in medicine fields and accelerate the development of officinal fungus in our country.
引文
[1] Veksler IG. The effect of zymosan on the toxicity and activity of various antitumor preparations,1966, 1 (2): 80-86.
[2] Kamasuka T, Momoki Y, Sakai S. Antitumor activity of polysaccharide fractions prepared from some strains of Basidiomycetes. Gann, 1968, 59(10): 443-45.
[3] Komatsu N, Okubo S, Kikumoto S, Kimura K, Saito G. Host-mediated antitumor action of schizophyllan, a glucan produced by Schizophyllum commune.Gann,1969, 60(13): 137-144.
[4] Chihara G, Maeda Y, Hamuro J, Sasaki T, Fukuoka F. Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) sing . Nature,1969, 222(3): 687-688.
[5] Yoshida O, Nakashima H, Yoshida T, Kaneko Y, Yamamoto I, Matsuzaki K, Uryu T, Yamamoto N. Sulfation of the immunomodulating polysaccharide lentinan: a novel strategy for antivirals to human immunodeficiency virus (HIV). Biochem Pharmacol,1988, 37(1): 2887-2891.
[6] Ohno N, Suzuki I, Oikawa S, Sato K, Miyazaki T, Yadomae T. Antitumor activity and structural characterization of glucans extracted from cultured fruit bodies of Grifola frondosa. Chem Pharm Bull,1984,32 (6): 1142-1151.
[7] Ohno N, Iino K, Takeyama T, Suzuki I, Sato K, Oikawa S, Miyazaki T, Yadomae T. Structural characterization and antitumor activity of the extracts from matted mycelium of cultured Grifola frondosa. Chem Pharm Bull,1985,33(1): 3395-3401.
[8] Ohno N, Adachi Y, Suzuki I, Sato K, Oikawa S, Yadomae T. Characterization of the antitumor glucan obtained from liquid-cultured Grifola frondosa. Chem Pharm Bull,1986, 34(2): 1709-1715.
[9] Nanba H, Hanmaguchi A, Kuroda H. The chemical structure of antitumour polysaccharide in fruitbodies of Grifola frondasa (maitake). Chem Pharm Bull, 1987, 35(3): 1162-1168.
[10] Hishida I, Nanba H, Kuruda H. Antitumor activity exhibited by orally administered extract fruit body of grifola frondosa. Chem. Pharm. Bull,1988,36(5):1819-1827.
[11] Mark Mayell. Maitake Extracts and Their therapeutic Potential-A Review. Alternative Medicine Review,2001,6(1): 48-60.
[12] Kollar, R., Petrakova, E., Ashwell, G., Robbins, P.W., Cabib, E.. Architecture of the yeastcell wall. The linkage between chitin andβ-(1-3)-glucan. J. Biol. Chem,1995,(270):1170- 178.
[13] Magnelli, Paula; Cipollo, John F.; Abeijon, Claudia. A Refined Method for the Determination of Saccharomyces cerevisiae Cell Wall Composition andβ-1,6-Glucan Fine Structure. Analytical Biochemistry,2002,301(1): 136-150.
[14] Kidd PM., The use of mushroom glucans and proteoglycans in cancer treatment. Altern Med Rev,2000,5(1): 4-7.
[15] Hayakawa K, Mitsuhashi N, Saito Y, et al. E fect of Krestin (PSK) as adjuvant treatment on the prognosis after radical radiotherapy in patients with non-small cell lung cancer. Anticancer Res,1993,3(2): 1815-1820.
[16] Yoshio Ohmura, Kenichi Natsunaga, Isamu Motokawa, et al.. Protective effects of a protein-bound polysaccharide, PSK, on Candida albicans infection in mice via tumor necrosis factor-a induction . International Immunopharmacology,2001,(9-10): 1897- 1811.
[17] Uchiyama Michiharu, Ohno Naohito, Miura Noriko N. et al.. Anti-grifolan antibody reacts with the cell wallβ-glucan and the extracellular mannoprotein-β-glucan complex of C. albican. Carbohydrate Polymers,2002,8(4): 333-340.
[18] Schmid, Frank, tone, Bruce A., cDougall, Barbara M., et al. Sructure of epiglucan, a highly side-chain/branched (1→3;1→6)-β-glucan from the micro fungus Epicoccum nigrum Ehrenb Ex Schlecht. Carbohydrate Research,2001,31(2): 163-71.
[19] Due?as-Chasco, M. Teresa, Rodríguez-Carvajal, Miguel A.,Tejero-Mateo, Pilar. Structural analysis of the exopolysaccharides produced by Lactobacillus spp. G-77. Carbohydrate Research,1998,307(1): 125-33.
[20] Kitamura, Shinichi; Hori, Tsutomu; Kurita, Kaori, akeo, Kenichi, ara, Chihiro; Itoh, Wataru. An antitumor, branched (1→3)-β-d-glucan from a water extract of fruiting bodies of Cryptoporus volvatus. Carbohydrate Research,1994,263 (1): 111-121.
[21] Naohito Ohno, Kazuyoshi Iino,Takahiro Takeyama. Structural Characterization and Antitumor Activity of the Extracts from Matted Mycelium of Cultured Grifola frondosa. Chem. Pharm. Bull,1985,33:3395-3401.
[22] Kazuyoshi Iino, Naohito Ohno, Iwao Suzuki, et al. Structural Characterization of Neutral Antitumorβ-D-Glucan Exracted with Hot Sodium Hydroxide From Cultured Fruit Bodies ofGrifola Frodonsa. Carbohydrate Reseach,1985,141:111-119.
[23] Naohito Ohno, Kazuyoshi Iino, Iwao Suzuki. Neural and Acidic Antitumor Polysaccharides Extracted from Cultured Fruit Bodies of Grifola Frodonsa. Chem. Pharm. Bull,1985,33(3): 1181-1186.
[24] Iwao Suzuki, Koichi Hashimato,Shozo Oikawa. Antitumor and Immunomodulating Activities of aβ-Glucan Obtained from Liquid-Cultured Grifola frondosa. Chem. Pharm. Bull,1989,37: 410-413.
[25] Hishida I, Nanba H, Kuruda H.Antitumor activity exhibited by orally administered extract fruit body of grifola frondosa. Chem Pharm Bull,1988,36(5):1819-1827.
[26] Ken Matsui, Noriko Kodama, Hiroaki Nanba, Effects of Maitake(Grifola Frodonsa)D-Fraction on carcinoma angiogenesis. Cancer Letters,2001,172(2): 193-198.
[27]黄年来.中国大型真菌原色图鉴[M].第一版,中国农业出版社,1998.
[28] C. J.阿历索保罗等著,姚一建等译.菌物学概论[M]第四版,北京:中国农业出版社, 2002.
[29] Manzi, Pamela, Pizzoferrato, Laura. Beta-glucans in edible mushrooms. Food Chemistry, 2000,68(3): 315-318.
[30]姚新生.天然药物化学[M].北京:人民卫生出版社,2001: 268-269.
[31] K.P. R. Kartha, Robert A. Field. Iodine: A versatile reagent in carbohydrate chemistryⅣ. per-O-acetylation, regioselective acylation and acetolysis. Tetrahedron,1997,53(34): 11753 -11766.
[32]蒋可,陈宇东.乙酰解方法测定痕量糖链中α-1,6联接的分枝点.化学学报,1993,51, 502-505.
[33]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): 45-48.
[34] R. Mukerjea, D. Kim, J. F. Robyt. Simplified and improved methylation analysis of saccharides, using a modified procedure and thin-layer chromatography. Carbohydrate Research,1996,292(1) 11-20.
[35] P. Magnelli, J. F. Cipollo, C. Abeijon. A refined method for the determination of saccharomyces cerevisiae cell wall composition andβ-1,6-glucan fine structure. Analytical Biochemistry,2002,301(2): 136-150.
[36] N. G. Karlsson, N. H. Packer. Analysis of O-linked reducing oligosaccharides released by an in-line flow system. Analytical Biochemistry,2002,305(2): 173-185.
[37] Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI Mass and capillary electrophoresis. Analytical Chemistry,2001,73(24).
[38] Chauveau, Christophe, Alaga Philippe, Wieruszeski Jean-Michel, et al.. A water-solubleβ-d-glucan from Boletus erythropus. Phytochemistry,1996,43(2): 413-415.
[39] Kim, Young-Teck, im Eun-Hee, heong, Chaejoon, et al. Structural characterization ofβ-d-(1→3, 1→6)-linked glucans using NMR spectroscopy. Carbohydrate Research,2000, 28(3): 331-41.
[40] Monteiro, Mario A., Slavic Durda, St. Michael Frank, Brisson Jean-Robert; MacInnes, Janet I. et al.. The first description of a (1→6)-β-d-glucan in prokaryotes: (1→6)-β-d-glucan is a common component of Actinobacillus suis and is the basis for a serotyping system. Carohydrate Research,2000,329(1): 121-30.
[41]曾凯宏,等.真菌多糖的结构与功能.食品科技, 2001, 4:68.
[42] Liao H F,Chou C J,Wu S H,et al .. Isolation and characterization of an active compound from black soybean [Glycine max (L.) Merr.] and its effect on proliferation and differentiation of human Ieyke mic U937 cells. Anticancer Drugs,2001,12(10): 841- 846.
[43] Misaki A, Kakuta M, Sakaki T., et al. Studies on interrelation of structure and antitumor effects of polysaccharides: antitumor action of periodate-modified, branched (1→3)β--D-glucan of Auricularia auricular—judae,and other polysaccharides containing (1→3)-glycosidic linkages. Carbohudrate Research,1981,92:115-129.
[44]张丽萍,汉丽萍,王月秋,等.硫酸化高山红景天多糖(RSASL)的制备及鉴定.分子科学学报,1999,15 (4): 205-210.
[45] Kraus J, Blaschek W, Schutz M, et al. Antitumor activity of cell wallβ1,3/1,6-glucans from Phytophthora spp. Planta Med,1992,58(1): 39-42.
[46] Zhang P, Zhang L, Cheng S. Effect of urea and sodium hydroxide on the molecular weight and conformation ofβ-(1-3)-D-glucan from Letinus edodes in aqueous solution . Carbohydr Res,2000,327(2): 431-438.
[47] Young S H, Jacobs R R.Sodium hydroxide-induced conformational change in schizophyllan detected by the fluorescence dye, aniline blue. Carbohydrate Research,1998,310(1): 91-99.
[48] Saito H, Yoshioka Y, Uehara N, et al. Relationship between conformation and biological response for (1→3)-β-D-glucans in the activation of coagylation Factor G from limulus amebocyte lysate and host mediated antitumor activitv. Carbohvdrate Research,1991,217(1):181-190.
[49] Yoshida O, Nakashima H , Yoshida T,et al.Sulfation of the immymomodulation polysaccharide lentinan, a novel strategy for antivirals to human immunodeficiency. Biochem Pharm,1988,37(7): 2887-2891.
[50] Zhang L, Zhang M,Chen J, et al. Solution properties of antitumorcarboxy-methylated derivatives ofβ(1→3)-D-gluean from Ganoderma Lucidum. Chinese J of Polym Sci, 2001,19(3): 283-289.
[51] Carlucci M J, Pujol C A, Ciancia M, et al.. Antiherpetic activity and mode of action of natural carrageenans of diverse stryctural types. Antiviral Res,1999,43(2): 93-102.
[52] Adachi,Y., Ohno,N., Ohsawa, W. et al.. Change of biological activities of (1→3)-β- glucan from Grifola frondosa upon molecular weight reduction by heat treatment.. Chem.Pharrn.Bu11,1990,38: 477-481.
[53] Calazans, G.M.T.,Lima, R.C., de Franca,F.P.,Lopes,C.E.. Molecular weight and antitumour activity of Zymomonas mobilis levans. International Journal of Bio-logical Macro- molecules,2000,27: 245-247.
[54] Gao Y, Fukuda A, Katsuraya K, et al.. Synthesis of regioselective substituted curdlan sulfates with medium molecular weights and their specific anti-HTV-1 activities. Macrom, 1997,30(11): 3224-3228.
[55] Alban S, Franz G. Characterization of the semi-synthetic curdlan sulfate.Thromb Res,2000, 99(4): 377-388.
[56] Oostervckl A et al.. Carbalryrlrate Palynrer.S,2002,48: 73-81.
[57] Ueno Y et al. An antitumor activity of the Sclertia of Grifora umbe12late(Fr) PALIT. Carbohydrate Research,1982,101: 106-107.
[58] Adachi Y et al. Physicochemical properties and antitumor activities of chemical modified derivatives antitumor glucan"Grifora L E" from grifola frondasa. Chem Pham Bull, 1989, 37: 1838-1843.
[59]王亚军,郑裕国,吴天星,等.酵母(1→3)-β-D-葡聚糖制备及其化学衍生研究进展.天然产物研究与开发,2005,17(4): 527-31.
[60]孙青,刘长海,李国栋,等.β-环糊精硫酸酯的合成及其在舒必利毛细管电泳拆分中的应用.第二军医大学学报,2004,25(11): 1259-1260.
[61]李东霞,张双全,刘平. SCAMP硫酸酯化多糖的制备及其光谱鉴定.光谱学与光谱分析,2002,22(1): 59-62.
[62]高贵珍,焦庆才,李绪亮.茯苓多糖结构修饰及其与AA相互作用机理的研究.激光生物学报,2005,14(3): 228-232.
[63]邓成华,杨祥良,王雁,等.硫酸酯化虎奶多糖的制备及其抗氧化作用研究.中国生化药物杂志,2001,22(1): 1-4.
[64]陈景耀,吴国荣,王建安,等.正交试验优选白芨多糖硫酸酯化工艺的研究.中草药,2005,36(1): 43-46.
[65]田庚元,李寿桐,宋麦丽,等.牛膝多糖硫酸酯的合成及其抗病毒活性.药学学报,1995,30(2):107-111.
[66]杨铁虹,贾敏,商澎,等.当归多糖硫酸酯的合成及其对脾细胞增殖的作用.第四军医大学学报, 2001, 22(5): 432-434.
[67]赵吉福,么雅娟,陈英杰.磺酰化新茯苓多糖的制备及抗肿瘤作用.沈阳药科大学学报, 1996, 13(2): 125-128.
[68]李文姬,崔浩,李绍顺.唾液酸8,9-二硫酸酯衍生物的合成及唾液酸酶抑制活性.第二军医大学学报, 2000, 21(1): 56-58.
[69]王周玉,蒋珍菊,李富生,等.水溶性N-(2-羧基苯甲酰基)化壳聚糖的合成.化学研究与应用, 2004, 16(1): 8-10.
[70] Petitou M, Coudert C, Level M, et al.. Selectively o-acylated glycosaminoglycan derivatives. Carbohydrate Research, 1992, 236: 107-119.
[71]张灿,丁娅,平其能.水溶性6-O-琥珀酰-N-半乳糖化壳聚糖衍生物的制备与表征.中国药科大学学报, 2005, 36(4): 291-295.
[72]黄理耀,刘超.疏水化长脂肪链酰化壳聚糖的制备.华侨大学学报,2005,26(4): 439-441.
[73]李明春,冯震,辛梅华.十二酰化壳寡糖的制备及其性.化工进展,2005,24(9): 1024-1028.
[74]陈煜,多英全,罗运军. 3,4,5-三甲氧基苯甲酰壳聚糖的制备与表征.高分子材料科学与工程, 2004, 20(3): 210-212.
[75] De Nooy A.E.J., Rori V.,Masci G., Dentini M., Crescenzi V.. Synthesis and preliminarycharacterisation of charged derivatives and hydrogels from scleroglucan. Carbohydrate Research, 2000, 324(2): 116-126.
[76] Ohno N, Durachi K, Radomai T. Physicochemical properties and antitumor activities of carboxymethylated derivatives of glucan from Sclerotinia scleotiorumv. Chem Pharm Bull, 1988, 36(3) : 1016-1021.
[77] Yang, J., Du Y. Chemical modification, Chemical modification, characterization and bioactivity of Chinese lacquer polysaccharides from lac tree Rhus vernicifera against leukopenia induced by cyclophosphamide. Carbohydrate Polymers, 2003, 52: 405-410.
[78] Van Seeventer, M.A. Corsten, M.P. Sanders, J.P. Kamerling and J.F.G. Vliegenthart. Synthesis of two analogues of the Sd. a. determinant. Replacement of the sialic acid residue. by a sulfate or a carboxymethyl group. Carbohydrate. Research, 1997, 229: l71-179.
[79] Dorine L. Verraest, Joop A. Peters, Jan G. Batelaan and Herman van Bekkum, Carboxy- methylation of inulin. Carbohydrate. Research, 1995, 271: 101-112.
[80] Tadashi Kiho, Isao Yoshida, Katsuyuki Nagai, Shigeo Ukai and Chihiro Hara. (1→3)-α-D- glucan from an alkaline extract of Agrocybe cylindracea, and antitumor activity of its O- (carboxymethyl) ated derivatives. Carbohydrate Research, 1989, 189: 273-279.
[81]邓成华,杨祥良,王雁,等.硫酸酯化虎奶多糖的制备及其抗氧化作用研究.中国生化药物杂志, 2001 ,22(1): 1-4.
[82] Mei Zhang, Lina Zhang, et al, Molecular mass and chain conformation of carboxymethylated derivatives ofβ-glucan from sclerotia of Pleurotus tuber-regium. Biopolymers, 2003, 68: 150-159.
[83] Zhang M, Cheung PCK, Zhang LN, et al. Evaluation of mushroom dietary fiber. ( Nonstarch Polysaccharides ) from sclerotia of Pleurotus tuber-regium(Fries) singer as a potential antitumor agent. J Agric Food Chem, 2001, 49(10): 5059-5062.
[84] Jian-Hwa Guo, G. W. Skinner, W. W. Harcum and P. E. Barnum. Pharmaceutical applications of naturally occurring water-soluble polymers. Pharmaceutical Science & Technology Today, 1998, 1: 254-261.
[85]伍焜贤,李敏谊.羧甲基多糖的免疫调节作用.化学通报, 1999, (9): 54-57.
[86] Matsumoto T, Numata M, Anada T, et al.. Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptakeefficiency. Biochim Biophys Acta, 2004, 1670 (2): 91-104.
[87] Okamoto K, Wang H, Ijyuin T, et al. Pervaporation of Aromatic/Non - Aromatic Hydrocarbon Mixtures Through Crosslinked Membranes of Polyimide with Pendant. Phosphonate Ester Groups. J Membr Sci, 1999, 157(1): 97-105.
[88]田庚元,冯宇澄,林颖.植物多糖的研究进展.中国中药杂志, 1995, 20(7): 441.
[89] Lewis A. SLOTIN. Current Methods of Phosphorylation of Biological Molecules. Synthesis, 1977, 737-752.
[90] David L. Williams, Rose B. McNamee, Ernest L. Jones, et al.. A method for the solubilization of a (1→3)-β-D-glucan isolated from Saccharomyces cerevisiae. Carbohydrate Reserch, 1991, 219: 203-213.
[91] J. C. Fricain, P. L. Granja, M. A. Barbosa et al.. Cellulose phosphates as biomaterials. In vivo biocompatibility studies Biomaterials, 2002, 23: 971-980.
[92] Chen, X M, Zhang J., Tian, G. Y. Studies on synthesis and antitumor activity of pho- sphorylated Achyranthes bidentata polysaccharide (P-AbPS). Chin J Chem, 2002, 20(11): 1406-1410.
[93]蒋玉湘,李鹏程.壳聚糖硫酸酯制备方法.海洋科学, 2004, 28(6): 75-77.
[94]魏经建,邵树军,王天元.硫酸酯化多糖化学及临床应用研究进展.中国生化药物杂志, 1999, 20(5): 260-262.
[95] Katsuraya K. Synthesis of sulfonated oligosaccharide glycosides having anti-HIV activity and the relationship between activity and chemical structure. Carbohydater Research, 1999, 315: 234-242.
[96]方积年.硫酸酯化多糖的研究进展.中国药学杂志, 1993, 28(7): 393-395.
[97]董群,方积年.寡糖及多糖甲基化方法的发展及现状.天然产物开发与研究, 1995, 7(2): 60-65.
[98]张翼伸.多糖的结构测定.生物化学与生物物理进展, 1983, 4: 18-23.
[99]黄芳,蒙义文.活性多糖的研究进展.天然产物研究与开发, 1999, 11(5): 90-98.
[100] Caro A D, Perola E, Bartdini B, et al. Fraction of chemically oversulphated galactosaminoglycan sulphates inhibit three enveloped virus: human immunodeficiency virus type 1, herpes simplex virus type 1 and human cytomegalovirus. Antiviral chemistry & Chemotherapy, 1999, 10: 33-38.
[101]陈春英,黄雪华,周井炎,等.硫酸酯化箬叶多糖的结构修饰及其抗艾滋病病毒活性.药学学报, 1998, 33(4): 264-268.
[102] Koyoanagi S, Tanigawa N, Nakagawa H et a1. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochemical Pharmacology,2003, 65(2): 173-179.
[103] Witvrouw M, Schols D, Andrei G et a1. Antiviral activity of low MW dextran sulphate (derived from dextran MW1000)compated to dextran sulfate samples of higher MW. An tiviral Chem Chemother, 1991,2: 71-179.
[104] Mizumoto K, Sugawara I, Ito W et a1. Sulfated homopolysaccharide with immunomodulating activities are more potent anti-HTLV2 agents than sulfated beteropolysaccharides. Japan J BxpMed, 1998, 58(3): 145-151.
[105] Chaidedgumjom A, Toyoda H, Woo ER et a1. Effect of (1→3)- and(1→4)- linkages of fully sulfated polysaccharides on their anti-coagulant activity. Carbohydrate Research, 2002, 337(10): 925-933.
[106] Hirohashi N, VilelaS, AC. Structural requirements for species specific induction of the sperm acrosome reaction by sea urchin egg sulfated fucan. Biochemical and Biophysical Research Communications, 2002, 298(3):403-407.
[107] Okazaki M, Adachi Y, Ohno N et al. Structure-activity relationship of(1→3)-beta-D-glucans in the induction of cytokine production from macrophages, in vitro. Biol Pharm Bull, 1995, 18: 1320.
[108] Adachi Y, Ohno N, Ohsawa M, et al.. Physiochemical properties and antitumor activities of chemically modified derivatives of antitumor glucan‘grifolan LE’from Grifola frondosa. Chem Pharm Bul1, 1989, 37: 1838.
[109] Aggarwal B, Kohr J, Hass E, et al. Human tumornecrosis factor. J Biol Chem, 1998, 260(4): 2345-2352.
[110] Pross HF. Continuous IL-2 and LAK cells for advanced cancer:a national biotherapy study group trial. Nat Immunol Cell Growth Regul, 1997, 7: 51.
[111] Nanha H. M ailakc mushroom immune lhcrapc In prcecnl from canccr grnwlh and mclaslasis. Ecplnre, 1995, 6(1): 74-78.
[112] Adachi Y, Ohno N, Yadomae T. Preparation and antigen specificity of an anti-(1,3)-beta-D-glucan antibody. Biol Pharm Bull, 1994, Nov.17(11): 1508-1512.
[113]孙震,陈石良,谷文英,等.灰树花多糖体内抗肿瘤作用的实验研究.药物生物技术, 2001, 8(5): 279-283.
[114]邢增涛,周昌艳,潘迎捷,等.灰树花多糖研究进展.食用菌学报, 1999, 6(3): 54.
[115]王顺春,方积年.香菇多糖硫酸化衍生物的制备及其结构分析.生物化学与生物物理学报, 1999, 31(5): 594-597.
[116]方积年.硫酸酯化多糖的研究进展.中国药学杂志,1993, 28(7): 393-95.
[117]黄玉萍.多糖的研究.华南师范大学学报(自然科学版), 1991, (2): 107-114.
[118]肖建辉,蒋侬辉,梁宗琦,等.食药用真菌多糖研究进展.生命的化学,2002,22(2): 148-151.
[119]李师鹏,安利国.真菌多糖免疫活性的研究进展.菌物系统, 2001, 20(4): 581-587.
[120] Wang YF, Zhang LN, Li YQ, Hou XH, Zeng FB. Correlation of structure to antitumor activities of five derivatives ofβ-glucan from Poria cocos sclerotium.Carbohydrate Research, 2004, 339: 2567-2574.
[121]李春林,于广利,赵峡,等.灰树花菌丝体中几种多糖的分离及其结构表征.中国生化药物杂志, 2004, 25(1): 20-22.
[122]于广利,王莹,赵峡,等.一种碱溶性灰树花菌丝体多糖GFM2A的制备和结构表征.高等学校化学学报,2007, 28(1):87-91.
[123] Ohno N, Suzuki I, Oikawa S, etal.. Antitumor activity and structural characterization of Glucans extracted from cultured fruit bodies of Grifola frondosa . Chem. Pharm. Bull, 1984, 32(3): 1142-1151.
[124] Ohno N, Iino K, Takeyama T, etal. Structural characterization and antitumor activity if extracts from matted mycelium of cultured Grifola frondosa . Chem. Pharm. Bul, 1985, 33(8): 3395-3401.
[125]栗衍华,谭成玉,王秀武,等.硫酸化多糖的制备及其生物活性的研究进展.精细与专用化学品, 2005, 14(16): 6-9.
[126]李小定,吴谋成,曾晓波.灰树花多糖的分离、纯化和理化性质.华中农业大学学报, 2002, 21(2): 186-188.
[127]陈石良,马青,谷文英.灰树花胞外多糖的性质与结构.无锡轻工业大学学报, 2002, 21(3): 244-248.
[128]林颖,黄琳娟,田庚元.一种改良的糖醛酸含量测定方法.中草药,1999,30(11):817-819.
[129] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances . Anal. Chem., 1956, 28: 350-356.
[130]徐桂云,陈汝贤,常理文.用毛细管气相色谱法测定多糖中单糖的组成.分析测试学报, 2000, 19(3): 71-73.
[131]李铁林,吴昌贤,张燕霞.糖和糖醇的气相色谱分析研究II,糖腈乙酸酯衍生物气相色谱分析的改进[M].分析化学, 1982, 10(5): 272-276.
[132]张芳,王旻,尹鸿萍.螺旋藻多糖PSP的化学结构研究.中国天然药物,2005,3(3): 155-157.
[133]谢强胜,尹鸿萍,陈昕,等.盐藻多糖初级结构及性质测定.药物生物技术, 2005, 12(4): 242-247.
[134] Chichoke A. Maitake - the king of mushrooms . Townsend Letter for Doctors, 1994, 130: 432-433.
[135] Kubo K, Aoki H, Nanba H. Anti-diabetic activity present in the fruit body of Grifola frondosa (maitake) . J. Biol Pharm Bull, 1994, 17:1106-1110.
[136] Bao X, Duan J, Fang X, Fang J. Chemical modifications of the (1→3)-α-D-glucan from spores of Ganoderma lucidum and investigation of their physicochemical properties and immunological activity . Carbohydrate Research, 2001, 336, 127-140.
[137] Gorin P A J. Cabon-13 nuclear magnetic resonance spectroscopy of polysaccharide from Acacia Senegal . Adv Carbohydr Chem Biochem, 1981,38:13-39.
[138]方积年. 13C-NMR在多糖结构分析上的应用[M].国外医药(抗生素分册), 1982, 2: 107
[139] Michae HJK, Peter SR, Jennifer AM et al.. A 13CNMR study of sugar beet pectin. Carbohydrate Research, 1985, 138(1): 168-170.
[140] JH Yang, YM Du, Y Wen, etal. Sulfation of Chinese lacquers polysaccharides in different solvents . Carbohydr Polym, 2003, 52(4): 397-403.
[141]李绍顺,崔浩,小仓治夫.唾液酸衍生物的合成及生物活性研究. 1,2-去氧-2,3-去氧-N-乙酰基神经氨酸的合成.中国药物化学杂志, 1997, 7(3): 167-170.
[142] S. Alban, A. Schauerte, G. Franz. Anticoagulant sulfated polysaccharides: Part I. Synthesis and structure-activity relationships of new pullulan sulfates . Carbohydrate Polymers, 2002, 47(3): 267-276.
[143] L. J. Silvestri, R. E. Hurst, L. Simpson, etal.. Analysis of sulfate in complex carbohydrate . Anal. Biochem, 1982, 123: 303-309.
[144] Dodgson K S, Price R G. A Note on the Determination of Ester Sulfate Content of Sulphated Polysaccharides . Biochem. J., 1962, 84: 106-10.
[145]张惟杰主编.糖复合物生化研究技术[M].杭州:浙江大学出版社,1994: 398.
[146]徐叔云,卞如濂,陈修主编.药理实验方法学[M].第三版,北京:人民卫生出版社, 2002, 1270-1272.
[147] UenoY, Okamoto Y, Yamauchi R. An zantitumor activity of the alkali-soluble polysaccharide and its derivatives obtained from the Sclerotia of Grifora. umbellate . Research, 1982, 101: 160-167.
[2] Kamasuka T, Momoki Y, Sakai S. Antitumor activity of polysaccharide fractions prepared from some strains of Basidiomycetes. Gann, 1968, 59(10): 443-45.
[3] Komatsu N, Okubo S, Kikumoto S, Kimura K, Saito G. Host-mediated antitumor action of schizophyllan, a glucan produced by Schizophyllum commune.Gann,1969, 60(13): 137-144.
[4] Chihara G, Maeda Y, Hamuro J, Sasaki T, Fukuoka F. Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) sing . Nature,1969, 222(3): 687-688.
[5] Yoshida O, Nakashima H, Yoshida T, Kaneko Y, Yamamoto I, Matsuzaki K, Uryu T, Yamamoto N. Sulfation of the immunomodulating polysaccharide lentinan: a novel strategy for antivirals to human immunodeficiency virus (HIV). Biochem Pharmacol,1988, 37(1): 2887-2891.
[6] Ohno N, Suzuki I, Oikawa S, Sato K, Miyazaki T, Yadomae T. Antitumor activity and structural characterization of glucans extracted from cultured fruit bodies of Grifola frondosa. Chem Pharm Bull,1984,32 (6): 1142-1151.
[7] Ohno N, Iino K, Takeyama T, Suzuki I, Sato K, Oikawa S, Miyazaki T, Yadomae T. Structural characterization and antitumor activity of the extracts from matted mycelium of cultured Grifola frondosa. Chem Pharm Bull,1985,33(1): 3395-3401.
[8] Ohno N, Adachi Y, Suzuki I, Sato K, Oikawa S, Yadomae T. Characterization of the antitumor glucan obtained from liquid-cultured Grifola frondosa. Chem Pharm Bull,1986, 34(2): 1709-1715.
[9] Nanba H, Hanmaguchi A, Kuroda H. The chemical structure of antitumour polysaccharide in fruitbodies of Grifola frondasa (maitake). Chem Pharm Bull, 1987, 35(3): 1162-1168.
[10] Hishida I, Nanba H, Kuruda H. Antitumor activity exhibited by orally administered extract fruit body of grifola frondosa. Chem. Pharm. Bull,1988,36(5):1819-1827.
[11] Mark Mayell. Maitake Extracts and Their therapeutic Potential-A Review. Alternative Medicine Review,2001,6(1): 48-60.
[12] Kollar, R., Petrakova, E., Ashwell, G., Robbins, P.W., Cabib, E.. Architecture of the yeastcell wall. The linkage between chitin andβ-(1-3)-glucan. J. Biol. Chem,1995,(270):1170- 178.
[13] Magnelli, Paula; Cipollo, John F.; Abeijon, Claudia. A Refined Method for the Determination of Saccharomyces cerevisiae Cell Wall Composition andβ-1,6-Glucan Fine Structure. Analytical Biochemistry,2002,301(1): 136-150.
[14] Kidd PM., The use of mushroom glucans and proteoglycans in cancer treatment. Altern Med Rev,2000,5(1): 4-7.
[15] Hayakawa K, Mitsuhashi N, Saito Y, et al. E fect of Krestin (PSK) as adjuvant treatment on the prognosis after radical radiotherapy in patients with non-small cell lung cancer. Anticancer Res,1993,3(2): 1815-1820.
[16] Yoshio Ohmura, Kenichi Natsunaga, Isamu Motokawa, et al.. Protective effects of a protein-bound polysaccharide, PSK, on Candida albicans infection in mice via tumor necrosis factor-a induction . International Immunopharmacology,2001,(9-10): 1897- 1811.
[17] Uchiyama Michiharu, Ohno Naohito, Miura Noriko N. et al.. Anti-grifolan antibody reacts with the cell wallβ-glucan and the extracellular mannoprotein-β-glucan complex of C. albican. Carbohydrate Polymers,2002,8(4): 333-340.
[18] Schmid, Frank, tone, Bruce A., cDougall, Barbara M., et al. Sructure of epiglucan, a highly side-chain/branched (1→3;1→6)-β-glucan from the micro fungus Epicoccum nigrum Ehrenb Ex Schlecht. Carbohydrate Research,2001,31(2): 163-71.
[19] Due?as-Chasco, M. Teresa, Rodríguez-Carvajal, Miguel A.,Tejero-Mateo, Pilar. Structural analysis of the exopolysaccharides produced by Lactobacillus spp. G-77. Carbohydrate Research,1998,307(1): 125-33.
[20] Kitamura, Shinichi; Hori, Tsutomu; Kurita, Kaori, akeo, Kenichi, ara, Chihiro; Itoh, Wataru. An antitumor, branched (1→3)-β-d-glucan from a water extract of fruiting bodies of Cryptoporus volvatus. Carbohydrate Research,1994,263 (1): 111-121.
[21] Naohito Ohno, Kazuyoshi Iino,Takahiro Takeyama. Structural Characterization and Antitumor Activity of the Extracts from Matted Mycelium of Cultured Grifola frondosa. Chem. Pharm. Bull,1985,33:3395-3401.
[22] Kazuyoshi Iino, Naohito Ohno, Iwao Suzuki, et al. Structural Characterization of Neutral Antitumorβ-D-Glucan Exracted with Hot Sodium Hydroxide From Cultured Fruit Bodies ofGrifola Frodonsa. Carbohydrate Reseach,1985,141:111-119.
[23] Naohito Ohno, Kazuyoshi Iino, Iwao Suzuki. Neural and Acidic Antitumor Polysaccharides Extracted from Cultured Fruit Bodies of Grifola Frodonsa. Chem. Pharm. Bull,1985,33(3): 1181-1186.
[24] Iwao Suzuki, Koichi Hashimato,Shozo Oikawa. Antitumor and Immunomodulating Activities of aβ-Glucan Obtained from Liquid-Cultured Grifola frondosa. Chem. Pharm. Bull,1989,37: 410-413.
[25] Hishida I, Nanba H, Kuruda H.Antitumor activity exhibited by orally administered extract fruit body of grifola frondosa. Chem Pharm Bull,1988,36(5):1819-1827.
[26] Ken Matsui, Noriko Kodama, Hiroaki Nanba, Effects of Maitake(Grifola Frodonsa)D-Fraction on carcinoma angiogenesis. Cancer Letters,2001,172(2): 193-198.
[27]黄年来.中国大型真菌原色图鉴[M].第一版,中国农业出版社,1998.
[28] C. J.阿历索保罗等著,姚一建等译.菌物学概论[M]第四版,北京:中国农业出版社, 2002.
[29] Manzi, Pamela, Pizzoferrato, Laura. Beta-glucans in edible mushrooms. Food Chemistry, 2000,68(3): 315-318.
[30]姚新生.天然药物化学[M].北京:人民卫生出版社,2001: 268-269.
[31] K.P. R. Kartha, Robert A. Field. Iodine: A versatile reagent in carbohydrate chemistryⅣ. per-O-acetylation, regioselective acylation and acetolysis. Tetrahedron,1997,53(34): 11753 -11766.
[32]蒋可,陈宇东.乙酰解方法测定痕量糖链中α-1,6联接的分枝点.化学学报,1993,51, 502-505.
[33]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): 45-48.
[34] R. Mukerjea, D. Kim, J. F. Robyt. Simplified and improved methylation analysis of saccharides, using a modified procedure and thin-layer chromatography. Carbohydrate Research,1996,292(1) 11-20.
[35] P. Magnelli, J. F. Cipollo, C. Abeijon. A refined method for the determination of saccharomyces cerevisiae cell wall composition andβ-1,6-glucan fine structure. Analytical Biochemistry,2002,301(2): 136-150.
[36] N. G. Karlsson, N. H. Packer. Analysis of O-linked reducing oligosaccharides released by an in-line flow system. Analytical Biochemistry,2002,305(2): 173-185.
[37] Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI Mass and capillary electrophoresis. Analytical Chemistry,2001,73(24).
[38] Chauveau, Christophe, Alaga Philippe, Wieruszeski Jean-Michel, et al.. A water-solubleβ-d-glucan from Boletus erythropus. Phytochemistry,1996,43(2): 413-415.
[39] Kim, Young-Teck, im Eun-Hee, heong, Chaejoon, et al. Structural characterization ofβ-d-(1→3, 1→6)-linked glucans using NMR spectroscopy. Carbohydrate Research,2000, 28(3): 331-41.
[40] Monteiro, Mario A., Slavic Durda, St. Michael Frank, Brisson Jean-Robert; MacInnes, Janet I. et al.. The first description of a (1→6)-β-d-glucan in prokaryotes: (1→6)-β-d-glucan is a common component of Actinobacillus suis and is the basis for a serotyping system. Carohydrate Research,2000,329(1): 121-30.
[41]曾凯宏,等.真菌多糖的结构与功能.食品科技, 2001, 4:68.
[42] Liao H F,Chou C J,Wu S H,et al .. Isolation and characterization of an active compound from black soybean [Glycine max (L.) Merr.] and its effect on proliferation and differentiation of human Ieyke mic U937 cells. Anticancer Drugs,2001,12(10): 841- 846.
[43] Misaki A, Kakuta M, Sakaki T., et al. Studies on interrelation of structure and antitumor effects of polysaccharides: antitumor action of periodate-modified, branched (1→3)β--D-glucan of Auricularia auricular—judae,and other polysaccharides containing (1→3)-glycosidic linkages. Carbohudrate Research,1981,92:115-129.
[44]张丽萍,汉丽萍,王月秋,等.硫酸化高山红景天多糖(RSASL)的制备及鉴定.分子科学学报,1999,15 (4): 205-210.
[45] Kraus J, Blaschek W, Schutz M, et al. Antitumor activity of cell wallβ1,3/1,6-glucans from Phytophthora spp. Planta Med,1992,58(1): 39-42.
[46] Zhang P, Zhang L, Cheng S. Effect of urea and sodium hydroxide on the molecular weight and conformation ofβ-(1-3)-D-glucan from Letinus edodes in aqueous solution . Carbohydr Res,2000,327(2): 431-438.
[47] Young S H, Jacobs R R.Sodium hydroxide-induced conformational change in schizophyllan detected by the fluorescence dye, aniline blue. Carbohydrate Research,1998,310(1): 91-99.
[48] Saito H, Yoshioka Y, Uehara N, et al. Relationship between conformation and biological response for (1→3)-β-D-glucans in the activation of coagylation Factor G from limulus amebocyte lysate and host mediated antitumor activitv. Carbohvdrate Research,1991,217(1):181-190.
[49] Yoshida O, Nakashima H , Yoshida T,et al.Sulfation of the immymomodulation polysaccharide lentinan, a novel strategy for antivirals to human immunodeficiency. Biochem Pharm,1988,37(7): 2887-2891.
[50] Zhang L, Zhang M,Chen J, et al. Solution properties of antitumorcarboxy-methylated derivatives ofβ(1→3)-D-gluean from Ganoderma Lucidum. Chinese J of Polym Sci, 2001,19(3): 283-289.
[51] Carlucci M J, Pujol C A, Ciancia M, et al.. Antiherpetic activity and mode of action of natural carrageenans of diverse stryctural types. Antiviral Res,1999,43(2): 93-102.
[52] Adachi,Y., Ohno,N., Ohsawa, W. et al.. Change of biological activities of (1→3)-β- glucan from Grifola frondosa upon molecular weight reduction by heat treatment.. Chem.Pharrn.Bu11,1990,38: 477-481.
[53] Calazans, G.M.T.,Lima, R.C., de Franca,F.P.,Lopes,C.E.. Molecular weight and antitumour activity of Zymomonas mobilis levans. International Journal of Bio-logical Macro- molecules,2000,27: 245-247.
[54] Gao Y, Fukuda A, Katsuraya K, et al.. Synthesis of regioselective substituted curdlan sulfates with medium molecular weights and their specific anti-HTV-1 activities. Macrom, 1997,30(11): 3224-3228.
[55] Alban S, Franz G. Characterization of the semi-synthetic curdlan sulfate.Thromb Res,2000, 99(4): 377-388.
[56] Oostervckl A et al.. Carbalryrlrate Palynrer.S,2002,48: 73-81.
[57] Ueno Y et al. An antitumor activity of the Sclertia of Grifora umbe12late(Fr) PALIT. Carbohydrate Research,1982,101: 106-107.
[58] Adachi Y et al. Physicochemical properties and antitumor activities of chemical modified derivatives antitumor glucan"Grifora L E" from grifola frondasa. Chem Pham Bull, 1989, 37: 1838-1843.
[59]王亚军,郑裕国,吴天星,等.酵母(1→3)-β-D-葡聚糖制备及其化学衍生研究进展.天然产物研究与开发,2005,17(4): 527-31.
[60]孙青,刘长海,李国栋,等.β-环糊精硫酸酯的合成及其在舒必利毛细管电泳拆分中的应用.第二军医大学学报,2004,25(11): 1259-1260.
[61]李东霞,张双全,刘平. SCAMP硫酸酯化多糖的制备及其光谱鉴定.光谱学与光谱分析,2002,22(1): 59-62.
[62]高贵珍,焦庆才,李绪亮.茯苓多糖结构修饰及其与AA相互作用机理的研究.激光生物学报,2005,14(3): 228-232.
[63]邓成华,杨祥良,王雁,等.硫酸酯化虎奶多糖的制备及其抗氧化作用研究.中国生化药物杂志,2001,22(1): 1-4.
[64]陈景耀,吴国荣,王建安,等.正交试验优选白芨多糖硫酸酯化工艺的研究.中草药,2005,36(1): 43-46.
[65]田庚元,李寿桐,宋麦丽,等.牛膝多糖硫酸酯的合成及其抗病毒活性.药学学报,1995,30(2):107-111.
[66]杨铁虹,贾敏,商澎,等.当归多糖硫酸酯的合成及其对脾细胞增殖的作用.第四军医大学学报, 2001, 22(5): 432-434.
[67]赵吉福,么雅娟,陈英杰.磺酰化新茯苓多糖的制备及抗肿瘤作用.沈阳药科大学学报, 1996, 13(2): 125-128.
[68]李文姬,崔浩,李绍顺.唾液酸8,9-二硫酸酯衍生物的合成及唾液酸酶抑制活性.第二军医大学学报, 2000, 21(1): 56-58.
[69]王周玉,蒋珍菊,李富生,等.水溶性N-(2-羧基苯甲酰基)化壳聚糖的合成.化学研究与应用, 2004, 16(1): 8-10.
[70] Petitou M, Coudert C, Level M, et al.. Selectively o-acylated glycosaminoglycan derivatives. Carbohydrate Research, 1992, 236: 107-119.
[71]张灿,丁娅,平其能.水溶性6-O-琥珀酰-N-半乳糖化壳聚糖衍生物的制备与表征.中国药科大学学报, 2005, 36(4): 291-295.
[72]黄理耀,刘超.疏水化长脂肪链酰化壳聚糖的制备.华侨大学学报,2005,26(4): 439-441.
[73]李明春,冯震,辛梅华.十二酰化壳寡糖的制备及其性.化工进展,2005,24(9): 1024-1028.
[74]陈煜,多英全,罗运军. 3,4,5-三甲氧基苯甲酰壳聚糖的制备与表征.高分子材料科学与工程, 2004, 20(3): 210-212.
[75] De Nooy A.E.J., Rori V.,Masci G., Dentini M., Crescenzi V.. Synthesis and preliminarycharacterisation of charged derivatives and hydrogels from scleroglucan. Carbohydrate Research, 2000, 324(2): 116-126.
[76] Ohno N, Durachi K, Radomai T. Physicochemical properties and antitumor activities of carboxymethylated derivatives of glucan from Sclerotinia scleotiorumv. Chem Pharm Bull, 1988, 36(3) : 1016-1021.
[77] Yang, J., Du Y. Chemical modification, Chemical modification, characterization and bioactivity of Chinese lacquer polysaccharides from lac tree Rhus vernicifera against leukopenia induced by cyclophosphamide. Carbohydrate Polymers, 2003, 52: 405-410.
[78] Van Seeventer, M.A. Corsten, M.P. Sanders, J.P. Kamerling and J.F.G. Vliegenthart. Synthesis of two analogues of the Sd. a. determinant. Replacement of the sialic acid residue. by a sulfate or a carboxymethyl group. Carbohydrate. Research, 1997, 229: l71-179.
[79] Dorine L. Verraest, Joop A. Peters, Jan G. Batelaan and Herman van Bekkum, Carboxy- methylation of inulin. Carbohydrate. Research, 1995, 271: 101-112.
[80] Tadashi Kiho, Isao Yoshida, Katsuyuki Nagai, Shigeo Ukai and Chihiro Hara. (1→3)-α-D- glucan from an alkaline extract of Agrocybe cylindracea, and antitumor activity of its O- (carboxymethyl) ated derivatives. Carbohydrate Research, 1989, 189: 273-279.
[81]邓成华,杨祥良,王雁,等.硫酸酯化虎奶多糖的制备及其抗氧化作用研究.中国生化药物杂志, 2001 ,22(1): 1-4.
[82] Mei Zhang, Lina Zhang, et al, Molecular mass and chain conformation of carboxymethylated derivatives ofβ-glucan from sclerotia of Pleurotus tuber-regium. Biopolymers, 2003, 68: 150-159.
[83] Zhang M, Cheung PCK, Zhang LN, et al. Evaluation of mushroom dietary fiber. ( Nonstarch Polysaccharides ) from sclerotia of Pleurotus tuber-regium(Fries) singer as a potential antitumor agent. J Agric Food Chem, 2001, 49(10): 5059-5062.
[84] Jian-Hwa Guo, G. W. Skinner, W. W. Harcum and P. E. Barnum. Pharmaceutical applications of naturally occurring water-soluble polymers. Pharmaceutical Science & Technology Today, 1998, 1: 254-261.
[85]伍焜贤,李敏谊.羧甲基多糖的免疫调节作用.化学通报, 1999, (9): 54-57.
[86] Matsumoto T, Numata M, Anada T, et al.. Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptakeefficiency. Biochim Biophys Acta, 2004, 1670 (2): 91-104.
[87] Okamoto K, Wang H, Ijyuin T, et al. Pervaporation of Aromatic/Non - Aromatic Hydrocarbon Mixtures Through Crosslinked Membranes of Polyimide with Pendant. Phosphonate Ester Groups. J Membr Sci, 1999, 157(1): 97-105.
[88]田庚元,冯宇澄,林颖.植物多糖的研究进展.中国中药杂志, 1995, 20(7): 441.
[89] Lewis A. SLOTIN. Current Methods of Phosphorylation of Biological Molecules. Synthesis, 1977, 737-752.
[90] David L. Williams, Rose B. McNamee, Ernest L. Jones, et al.. A method for the solubilization of a (1→3)-β-D-glucan isolated from Saccharomyces cerevisiae. Carbohydrate Reserch, 1991, 219: 203-213.
[91] J. C. Fricain, P. L. Granja, M. A. Barbosa et al.. Cellulose phosphates as biomaterials. In vivo biocompatibility studies Biomaterials, 2002, 23: 971-980.
[92] Chen, X M, Zhang J., Tian, G. Y. Studies on synthesis and antitumor activity of pho- sphorylated Achyranthes bidentata polysaccharide (P-AbPS). Chin J Chem, 2002, 20(11): 1406-1410.
[93]蒋玉湘,李鹏程.壳聚糖硫酸酯制备方法.海洋科学, 2004, 28(6): 75-77.
[94]魏经建,邵树军,王天元.硫酸酯化多糖化学及临床应用研究进展.中国生化药物杂志, 1999, 20(5): 260-262.
[95] Katsuraya K. Synthesis of sulfonated oligosaccharide glycosides having anti-HIV activity and the relationship between activity and chemical structure. Carbohydater Research, 1999, 315: 234-242.
[96]方积年.硫酸酯化多糖的研究进展.中国药学杂志, 1993, 28(7): 393-395.
[97]董群,方积年.寡糖及多糖甲基化方法的发展及现状.天然产物开发与研究, 1995, 7(2): 60-65.
[98]张翼伸.多糖的结构测定.生物化学与生物物理进展, 1983, 4: 18-23.
[99]黄芳,蒙义文.活性多糖的研究进展.天然产物研究与开发, 1999, 11(5): 90-98.
[100] Caro A D, Perola E, Bartdini B, et al. Fraction of chemically oversulphated galactosaminoglycan sulphates inhibit three enveloped virus: human immunodeficiency virus type 1, herpes simplex virus type 1 and human cytomegalovirus. Antiviral chemistry & Chemotherapy, 1999, 10: 33-38.
[101]陈春英,黄雪华,周井炎,等.硫酸酯化箬叶多糖的结构修饰及其抗艾滋病病毒活性.药学学报, 1998, 33(4): 264-268.
[102] Koyoanagi S, Tanigawa N, Nakagawa H et a1. Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities. Biochemical Pharmacology,2003, 65(2): 173-179.
[103] Witvrouw M, Schols D, Andrei G et a1. Antiviral activity of low MW dextran sulphate (derived from dextran MW1000)compated to dextran sulfate samples of higher MW. An tiviral Chem Chemother, 1991,2: 71-179.
[104] Mizumoto K, Sugawara I, Ito W et a1. Sulfated homopolysaccharide with immunomodulating activities are more potent anti-HTLV2 agents than sulfated beteropolysaccharides. Japan J BxpMed, 1998, 58(3): 145-151.
[105] Chaidedgumjom A, Toyoda H, Woo ER et a1. Effect of (1→3)- and(1→4)- linkages of fully sulfated polysaccharides on their anti-coagulant activity. Carbohydrate Research, 2002, 337(10): 925-933.
[106] Hirohashi N, VilelaS, AC. Structural requirements for species specific induction of the sperm acrosome reaction by sea urchin egg sulfated fucan. Biochemical and Biophysical Research Communications, 2002, 298(3):403-407.
[107] Okazaki M, Adachi Y, Ohno N et al. Structure-activity relationship of(1→3)-beta-D-glucans in the induction of cytokine production from macrophages, in vitro. Biol Pharm Bull, 1995, 18: 1320.
[108] Adachi Y, Ohno N, Ohsawa M, et al.. Physiochemical properties and antitumor activities of chemically modified derivatives of antitumor glucan‘grifolan LE’from Grifola frondosa. Chem Pharm Bul1, 1989, 37: 1838.
[109] Aggarwal B, Kohr J, Hass E, et al. Human tumornecrosis factor. J Biol Chem, 1998, 260(4): 2345-2352.
[110] Pross HF. Continuous IL-2 and LAK cells for advanced cancer:a national biotherapy study group trial. Nat Immunol Cell Growth Regul, 1997, 7: 51.
[111] Nanha H. M ailakc mushroom immune lhcrapc In prcecnl from canccr grnwlh and mclaslasis. Ecplnre, 1995, 6(1): 74-78.
[112] Adachi Y, Ohno N, Yadomae T. Preparation and antigen specificity of an anti-(1,3)-beta-D-glucan antibody. Biol Pharm Bull, 1994, Nov.17(11): 1508-1512.
[113]孙震,陈石良,谷文英,等.灰树花多糖体内抗肿瘤作用的实验研究.药物生物技术, 2001, 8(5): 279-283.
[114]邢增涛,周昌艳,潘迎捷,等.灰树花多糖研究进展.食用菌学报, 1999, 6(3): 54.
[115]王顺春,方积年.香菇多糖硫酸化衍生物的制备及其结构分析.生物化学与生物物理学报, 1999, 31(5): 594-597.
[116]方积年.硫酸酯化多糖的研究进展.中国药学杂志,1993, 28(7): 393-95.
[117]黄玉萍.多糖的研究.华南师范大学学报(自然科学版), 1991, (2): 107-114.
[118]肖建辉,蒋侬辉,梁宗琦,等.食药用真菌多糖研究进展.生命的化学,2002,22(2): 148-151.
[119]李师鹏,安利国.真菌多糖免疫活性的研究进展.菌物系统, 2001, 20(4): 581-587.
[120] Wang YF, Zhang LN, Li YQ, Hou XH, Zeng FB. Correlation of structure to antitumor activities of five derivatives ofβ-glucan from Poria cocos sclerotium.Carbohydrate Research, 2004, 339: 2567-2574.
[121]李春林,于广利,赵峡,等.灰树花菌丝体中几种多糖的分离及其结构表征.中国生化药物杂志, 2004, 25(1): 20-22.
[122]于广利,王莹,赵峡,等.一种碱溶性灰树花菌丝体多糖GFM2A的制备和结构表征.高等学校化学学报,2007, 28(1):87-91.
[123] Ohno N, Suzuki I, Oikawa S, etal.. Antitumor activity and structural characterization of Glucans extracted from cultured fruit bodies of Grifola frondosa . Chem. Pharm. Bull, 1984, 32(3): 1142-1151.
[124] Ohno N, Iino K, Takeyama T, etal. Structural characterization and antitumor activity if extracts from matted mycelium of cultured Grifola frondosa . Chem. Pharm. Bul, 1985, 33(8): 3395-3401.
[125]栗衍华,谭成玉,王秀武,等.硫酸化多糖的制备及其生物活性的研究进展.精细与专用化学品, 2005, 14(16): 6-9.
[126]李小定,吴谋成,曾晓波.灰树花多糖的分离、纯化和理化性质.华中农业大学学报, 2002, 21(2): 186-188.
[127]陈石良,马青,谷文英.灰树花胞外多糖的性质与结构.无锡轻工业大学学报, 2002, 21(3): 244-248.
[128]林颖,黄琳娟,田庚元.一种改良的糖醛酸含量测定方法.中草药,1999,30(11):817-819.
[129] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances . Anal. Chem., 1956, 28: 350-356.
[130]徐桂云,陈汝贤,常理文.用毛细管气相色谱法测定多糖中单糖的组成.分析测试学报, 2000, 19(3): 71-73.
[131]李铁林,吴昌贤,张燕霞.糖和糖醇的气相色谱分析研究II,糖腈乙酸酯衍生物气相色谱分析的改进[M].分析化学, 1982, 10(5): 272-276.
[132]张芳,王旻,尹鸿萍.螺旋藻多糖PSP的化学结构研究.中国天然药物,2005,3(3): 155-157.
[133]谢强胜,尹鸿萍,陈昕,等.盐藻多糖初级结构及性质测定.药物生物技术, 2005, 12(4): 242-247.
[134] Chichoke A. Maitake - the king of mushrooms . Townsend Letter for Doctors, 1994, 130: 432-433.
[135] Kubo K, Aoki H, Nanba H. Anti-diabetic activity present in the fruit body of Grifola frondosa (maitake) . J. Biol Pharm Bull, 1994, 17:1106-1110.
[136] Bao X, Duan J, Fang X, Fang J. Chemical modifications of the (1→3)-α-D-glucan from spores of Ganoderma lucidum and investigation of their physicochemical properties and immunological activity . Carbohydrate Research, 2001, 336, 127-140.
[137] Gorin P A J. Cabon-13 nuclear magnetic resonance spectroscopy of polysaccharide from Acacia Senegal . Adv Carbohydr Chem Biochem, 1981,38:13-39.
[138]方积年. 13C-NMR在多糖结构分析上的应用[M].国外医药(抗生素分册), 1982, 2: 107
[139] Michae HJK, Peter SR, Jennifer AM et al.. A 13CNMR study of sugar beet pectin. Carbohydrate Research, 1985, 138(1): 168-170.
[140] JH Yang, YM Du, Y Wen, etal. Sulfation of Chinese lacquers polysaccharides in different solvents . Carbohydr Polym, 2003, 52(4): 397-403.
[141]李绍顺,崔浩,小仓治夫.唾液酸衍生物的合成及生物活性研究. 1,2-去氧-2,3-去氧-N-乙酰基神经氨酸的合成.中国药物化学杂志, 1997, 7(3): 167-170.
[142] S. Alban, A. Schauerte, G. Franz. Anticoagulant sulfated polysaccharides: Part I. Synthesis and structure-activity relationships of new pullulan sulfates . Carbohydrate Polymers, 2002, 47(3): 267-276.
[143] L. J. Silvestri, R. E. Hurst, L. Simpson, etal.. Analysis of sulfate in complex carbohydrate . Anal. Biochem, 1982, 123: 303-309.
[144] Dodgson K S, Price R G. A Note on the Determination of Ester Sulfate Content of Sulphated Polysaccharides . Biochem. J., 1962, 84: 106-10.
[145]张惟杰主编.糖复合物生化研究技术[M].杭州:浙江大学出版社,1994: 398.
[146]徐叔云,卞如濂,陈修主编.药理实验方法学[M].第三版,北京:人民卫生出版社, 2002, 1270-1272.
[147] UenoY, Okamoto Y, Yamauchi R. An zantitumor activity of the alkali-soluble polysaccharide and its derivatives obtained from the Sclerotia of Grifora. umbellate . Research, 1982, 101: 160-167.