【作者】 王艺峰；

【导师】 张俐娜；

【作者基本信息】 武汉大学 ， 高分子化学与物理， 2004， 博士

【摘要】 近年发现，不少真菌多糖具有抗肿瘤、抗病毒、抗炎症、降血糖、降胆固醇、降血脂等多种生物活性，它通过免疫调节作用抑制肿瘤或癌细胞的生长，并且对正常细胞无毒副作用。然而，很多多糖不溶于水，从而影响它们在药物方面的应用。通过化学修饰可以增加它们的水溶性，赋予或显著提高多糖的生物活性。据报道，多糖分子在溶液中的分子尺寸和链构象对其生物活性影响很大，但是有关真菌多糖构象研究方面的报道却十分少。为了弄清多糖的生物活性机理和构效关系，首先必须确定多糖的结构和链构象。尤其，我国传统中药茯苓中最重要的活性成分-茯苓多糖在溶液中的分子尺寸和链构象方面的研究报道很少，而且分子量的文献报道值相差很大。本论文主要通过生物技术从茯苓菌核中提取和纯化得到不同组分多糖并制备它们的衍生物，然后研究它们的化学结构、分子量、溶液中链构象及抗肿瘤活性，并探讨茯苓多糖及其衍生物的生物活性机理以及它们的结构、分子量、链构象与生物活性之间的构效关系。因此本工作涉及高分子化学、高分子物理与生物化学和医学交叉学科的新领域，也是国际前沿研究领域之一。 本工作的主要创新包括以下几点。（1）首次全面、系统地弄清了茯苓菌核中六种多糖的化学结构和分子量。（2）成功制备了茯苓水不溶性（1→3）-β-D-葡聚糖的五种衍生物，并用二维核磁共振等方法可靠地确定了这些衍生物的化学结构。（3）首次揭示茯苓葡聚糖硫酸酯对人体胃腺癌（Gastric carcinoma tumor）具有体外和体内抗肿瘤活性。（4）证明在水不溶性茯苓β-D-葡聚糖中通过衍生化反应引入合适的取代基，能显著提高它的水溶性和抗肿瘤活性。（5）应用激光光散射和粘度法并按照现代高分子溶液理论确定了茯苓β-D-葡聚糖的硫酸酯和羧甲基衍生物在0.2 M NaC1水溶液中呈现较伸展的柔顺链构象，而且首次揭示前者的柔顺性大于后者。（6）首次证明茯苓葡聚糖硫酸酯对Sarcoma 180（S-180）肿瘤细胞以及人体胃腺癌细胞株MKN-45和SGC-7901具有较好的体内和体外抗肿瘤活性，并且对正常细胞无毒副作用。（7）首次证明茯苓多糖硫酸酯的抗胃癌活性作用机制是通过诱导细胞凋亡从而抑制胃腺癌细胞的增殖。 本论文主要研究内容和结论概括如下几方面。首先，依次用0.9％氯化钠水溶液（25℃）、热水（120℃）、0.5M氢氧化钠和88％甲酸（4℃）分别从新鲜茯苓菌核中提取出六种多糖（编号为PCS1、PCS2、PCS3-Ⅰ、PCS3-Ⅱ、PCS4-Ⅰ和PCS4-Ⅱ）。采用气相色谱（GC）、红外光谱（IR）、紫外光谱（UV）、气质联用甲基化分析（GC-MS）、¹³C核磁共振谱（NMR）、元素分析（EA）和蛋白质分析检测了它们的化学组成和结构。而且，用粘度法、激光光散射（LLS）和尺寸排除色谱-光散射联用（SEC-LLS）表征了这些多糖的特性粘数（[η]）、重均分子量（M_w）及均方根旋转半径(<S₂>z^1/2)等分子参数。结果表明，茯苓菌核多糖PCS1、PCS2和PCS3-Ⅰ为杂多糖，含有单糖组分D-葡萄糖、D-半乳糖、D-甘露糖、D-岩藻糖、D-木糖和痕量糖醛酸。其中，PCS1的主要单糖组分为D-半乳糖，而其余多糖的主要单糖组分均为D-葡萄糖，而且D-荀萄糖含t大体随着提取分离过程进行而增加。获等菌核中含t最高的多箱成分为一种线型（l，3卜p心荀聚精PCs3一，它具有高纯度.多糖PCs4-I是连有少t队1一6）支链的（l，3）一p一葡聚糖;而PCs4一n主要由（1一3卜卜压葡聚糖构成，并含有少tp一（l，6）和p义z，2）连接的支链.多糖PCsl、PCSZ、pCS3一和pcs4一I在0.2 M NaCI水溶液中的Mw值分别为11.6 x 104、20.9 x 104、17.1 x 104和9.1、104.Pes3一和Pcs4.n为水不溶性多糖，它们在无水二甲亚矾（M勿50）中的几‘值分别为12.3 x10’和2一1 x 104.由分子参数分析得出，PCSI、PCSZ和PCS3一I在0.2 MNaCI水溶液中表现为无规线团链构象，而PCs3一和PCS4.n在二甲亚矾中表现为相对伸展的柔顺链构象。 水不溶性(l‘3卜p公葡聚糖PCs3一分别在无水二甲亚矾中进行硫酸配衍生化以及在20%氢氧化钠和异丙醉的混合溶液中进行狡甲基衍生化，成功制备出硫酸酷和狡甲基衍生物。用一种新溶剂体系（l .SM氢氧化钠/0.5M尿素水溶液）作为均相反应介质，对PCs3一进行经乙基、经丙基及甲基衍生化反应制备出相应的衍生物。上述反应得到五种水溶性p一D-葡聚糖衍生物（编号依次为S一CS3一n、C矛CS3一n、HE一PCS3一11、HI，一PCS3一H和M一PCS3一11）.用红外光谱、元素分析、IH NMR、13C NM[R及二维核磁共振（Zn一eosy、Zn.TOCsY和ZD一，H-de1”改ed’H’3e壬n述QC）表征T它们的化学结构和取代度。结果表明，(1*3卜卜D-葡聚糖糖环上的经基相对反应活性顺序大体为C一6位>C4位>C一2位。试样s-PCs3一n、C一PCs3一n和M一Pcs3一n的取代主要发生在C一6位经墓，其次在C4位和c-2位;而HE一Cs3一取代发生在C-6位和C4位;HP·Pcs3一n的取代几乎全部在C一位，它们均属于非选择性取代。由13c NMR得到它们的取代度（Ds）在0.23到1 .27之间.这些衍生化多糖的水溶性大小的顺序依次为S.PCs3一>C.PCS3一>M一Cs3一>HE一CS3一>1护.PCs3一。本工作对获等p一葡聚糖的衍生化方法进行了较系统的研究，并提供了新的无污染溶剂体系对卜D-葡聚糖进行经乙基、经丙墓?更多还原

【Abstract】 Recently, fungal polysaccharides have been discovered to exhibit many bioactivities, such as antiviral, anti-flammatory, hypoglycemic, hypocholesterolemic and hypotensive. They are good immunomodulators to inhibit the tumor/cancer cells growth without any damage to the normal cells. However, many polysaccharides are water-insoluble, which limits their applications in the medicine field. The chemical modification can improve water solubility of natural polysaccharides to enhance bioactivities, and even give new bioactivities. It has been reported mat the molecular size and chain conformation of polysaccharides in solution affect strongly on their bioactivities. The determination of the structure and chain conformation of the polysaccharides in solution is essential for the understanding of the bioactivity mechanism and the correlation of the structure to bioactivities. Poria cocos is a traditional medicine in China. However, there are few reports on the chain conformation and the molecular size of Poria cocos polysaccharides (the main component of Poria cocos), and some discrepancies among the literature results of molecular weight. In this thesis, several polysaccharides were extracted and purified from Poria cocos sclerotium, and their derivatives were prepared. The chemical structure, weight-average molecular weight (Mw), chain conformation, and antitumor activities of the polysaccharides and their derivatives were studied. The cellular mechanism, as well as the correlation of structure to antitumor bioactivities was investigated. Therefore, this work is in a cross field of polymer chemistry, polymer physics, biochemistry and medicine, and one of the frontiers of polymer science.The innovative points in this work are as the follows. (1) For the first time, the chemical structure and Mw of six polysaccharides extracted from fresh Poria cocos sclerotium were clarified. (2) Five derivatives from water-insoluble (1-3)-B-D-glucan were successfully synthesized, and their structures were reliably determined by two-dimensional NMR spectroscopy (2D NMR). (3) The antitumor bioactivities of sulfated polysaccharides of Poria cocos against gastric carcinoma cells in vitro and in vivo were firstly revealed. (4) It was proved that derivatives from water-insoluble (1-3)-B-D-glucan of Poria cocos possessed good water-solubility and antitumor activities. (S) Based on the modern theory of polymer solution, the chain conformation of the sulfated and carboxymethylated derivatives of (1-3)-B-D-glucan in 0.2 M NaOH solution was determined by light scattering (LS) and viscometry to be expanded flexible chains. Moreover, the chain of the sulfated derivative was more flexible than mat of the carboxymethylated one in the solution. (6) It has been firstly revealed that the sulfated derivatives of (1-3)-B-D-glucan exhibited good antitumor activities against Sarcoma 180(S-180), gastric carcinoma cell strains (MKN-45 and SGC-7901) both in vivo and in vitro, but not damaged the normal cells. (7) It was conformed for the first time that the cellular mechanism of sulfated (1-3)-B-D-glucan against gastric carcinoma tumor cells was to inhibit the proliferation of tumor cells through inducing apoptosis of the cell.The main contents and conclusions in this thesis are divided into the following. Six polysaccharides were extracted sequentially from the fresh sclerotium of Poria cocos cultivated in China using 0.9% NaCl (PCS1), hot water (PCS2), 0.5 M NaOH (PCS3-I and PCS3-II), and 88% formic acid (PCS4-I and PCS4-II). Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), GC-MS methylation analysis, 13C NMR spectroscopy, elementary analysis (EA), protein analysis, size exclusion chromatography combined with laser light scattering (SEC-LLS), light scattering and viscometry. The results indicated that the polysaccharides PCS1, PCS2 and PCS3-I were heteropolysaccharides containing D-glucose, D-galactose, D-mannose, D-fucose and D-xylose; the predominant monosaccharide was D-glucose exc更多还原