【作者】 高昊东；

【导师】 于广利；

【作者基本信息】 中国海洋大学 ， 药物化学， 2007， 硕士

【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。更多还原

【Abstract】 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.更多还原