【作者】 李雪莲；

【导师】 张俐娜；

【作者基本信息】 武汉大学 ， 高分子化学与物理， 2004， 硕士

【摘要】 香菇是我国产量丰富的特产资源，也是传统疗养型食用菌，并且具有抗肿瘤和增强免疫等多种功能。近年来已发现多糖的抗肿瘤活性与它在溶液中的链构象（刚性或柔顺、单一、双—及三螺旋和聚集体等形态）有关，而它们主要依靠高分子溶液理论和方法确定。本论文主要通过生物技术从香菇子实体中提取和纯化得到香菇β-葡聚糖，然后研究它的化学结构、溶液中链构象及其构象转变和抗肿瘤活性，并探讨多糖的结构与生物活性之间的构效关系。因此本研究工作属交叉学科领域。 本工作的主要创新点如下：（1）首次利用先进仪器和高分子溶液理论建立了香菇β-葡聚糖在0.9％NaCl水溶液中和在二甲亚砜（DMSO）溶液中的Mark-Houwink方程；（2）证明香菇β-葡聚糖L-FV-IB在水／DMSO混合溶液中会发生螺旋～无规线团的构象转变，转变范围为DMSO的体积分数为0.82～0.85，而且是不可逆行为；（3）评价了香菇β-葡聚糖的抗肿瘤活性，揭示分子量为5.72×10⁵且具有三螺旋链构象的多糖级分显示较高的抗肿瘤活性，且对正常细胞无毒副作用。由实验结果推断，三螺旋链构象有利于多糖抗肿瘤活性的提高。 本论文主要研究内容和结论概括如下。从香菇子实体中提取水溶性香菇多糖L-FV-IB，采用红外光谱（IR）和核磁共振（NMR）检测了它的化学结构。用超声波降解和重沉淀分级法结合得到L-FV-IB试样的不同分子量的级份。用激光光散射（LLS）、尺寸排除色谱与光散射联用装置（SEC-LLS）和粘度法测定了它们的重均分子量（M_w），均方旋转半径(＜S²＞^1/2)和特性粘数（[η]）。结构分析指出，试样L-FV-IB为带有少量（1→6）键接的D-葡聚糖支链的β-（1→3）-D-葡聚糖，并且平均每五个主链糖环上有两个1→6键接的葡萄糖侧基。由一系列L-FV-IB级分的M_w和[η]实验数据得出它在0.9％NaCl水溶液中25℃条件下的Mark-Houwink方程和＜S²＞^1/2与M_w的关系为：[η]=2.94×10^-7M_w^1.58(cm³ g^-1)（M_w＜6.0×10⁵）和[η]=2.30×10^-5M_w^1.25(cm³g^-1)（M_w＞6.0×10⁵）；＜S²＞_z^1/2=（4.35±0.3）×10^-3M_w^0.72±0.02（nm）；而它在DMSO中25℃的Mark-Houwink方程为：[η]=0.112 M_w^0.54(cm³ g^-1)；同时，按YFY高分子溶液理论模型计算出其在水溶液中的构象参数，如单位轮廓长摩尔质量（M_L）、持续长度（q）以及螺旋距（h）依次为2240±100nm^-1，100±10nm，0.30±0.1nm。它在水溶液中的分子参数符合三螺旋链，而在DMSO中的分子参数符合单股柔顺链构象。由此证明L-FV-IB葡聚糖在0.9％NaCl水溶液中为三螺旋链构象，而它在DMSO中呈现无规线团构象。 用SEC-LLS、LLS、粘度法、旋光法和¹³C NMR表征了香菇多糖L-FV-IB在0.2MNaCl水溶液、DMSO和不同W_DMSO的水／DMSO的混合溶液中的链构象。结果表明，L一FV-IB在0.2M NaCI水溶液中为三股螺旋链构象，而在DMSO中则解聚为单股柔顺链。[，]、Mw、扩>z，口、[a]n20和”eN侧叹数据证明L一Fv-IB在水/nMso混合溶液中发生了构象转变，由三股螺旋链解开成为单股柔顺链。这种三螺旋一单股无规线团链的构象转变点是wDMso=0.80一0.85。并且该三螺旋链葡聚糖构象转变不可逆。’3CNM[R谱的明显变化证明L一FV-IB发生了三螺旋链一无规线团链的构象转变，例如三螺旋链构象时，尽（l，3）昔键连接的主链的C3峰信号的消失、侧基取代的C6:峰相对强度的增强;构象发生变化的过程中，C6峰呈现不对称的宽峰。香菇多糖为三螺旋链构象时，其声（l*3）昔键连接的主链被分子内和分子间氢键束缚（创门分别维持链的螺旋状和三股链），因而引起主链上C6和C3信号减弱或消失，而侧基上的C3’及取代的C6:则变得相对自由，而使信号明显增强。并且提出一个圆筒状模型，即三螺旋链主链为芯而侧基为相对自由的圆筒套，来描述香菇多糖的三螺旋链构象及其转变。 用植入BALB/c小鼠体内的s一180实体瘤进行香菇尽葡聚糖的体内生物活性试验。采用s一180细胞株、宫颈癌Hela细胞株和三种不同分化程度的胃腺癌细胞株，低分化的MKN一45、中分化的SGC79OI、高分化的MKN一28进行体外活性试验。同时，对小鼠的肿瘤及脏器进行了病理组织学研究，从而评价香菇中不同分子量和不同链构象对多糖的抗肿瘤活性的影响。香菇多糖L一FV-IB在体外对恶性程度高的低分化胃腺癌细胞株MxN一45和中分化SGC7901有较好的抑制作用，而且分子量为5.71 xl护多糖级分L一FV-IB52具有最高的体外抗胃癌活性。L一FV-IB在体外对贴壁细胞宫颈癌细胞Hela的生长和增殖无明显的抑制作用，而对5180肿瘤细胞的生长和增殖却有较好的抑制作用，表明多糖抗肿瘤活性对癌细胞有选择性。香菇多糖L一FV-IB的分子量范围从3.57x1护到28.4 xl护的级分对s一180肿瘤细胞生长具有一定的抑制作用，而分子量为14.9、l0s和5.71/105的级分具有最高的体内、体外抗肿瘤活性（省=50.5%）。香菇乒（1*3）一D一葡聚糖的三螺旋链构象对其抗肿瘤活性起重要作用。当三螺旋链被破坏后，其抗5180肿瘤活性明显下降（省=123%）甚至消失。香菇尽葡聚糖抗肿瘤活性机理主要还是通过调节宿主机体的免疫反应在体内抑制肿瘤细胞的生长，而不是在体外特异性的产生细胞毒?更多还原

【Abstract】 Lentinus edodes as the special local product is an abundant natural food resource in China. It has long been used in traditional herb except for its nutritional values. It is well established that the polysaccharide fractions of lentinan has many functions such as antitumor and immunological enhancement. It has been found in recent years that the antitumor activities of polysaccharides are correlated to their chain conformations in solution (stiff or coil, single-, double- or triple helix, aggregate), which are mainly characterized by polymer solution theory and modern method in polymer science. In this work, lentinan, a kind of water-soluble polysaccharide, was extracted from Lentinus edodes by biotechnics. The chemical structure, chain conformation in solution, and antitumor activity of polysaccharide were studied, and the correlation between structure and bioactivities were discussed. Therefore the research is in a cross field of polymer physics and biochemistry.The creative points of this work are as follows. (1) The dependences of intrinsic viscosity [] and radius of gyration <S2>1/2 on Mw for Lentinan in 0.9% NaCl aqueous solution and in DMSO were established based on the theory of polymer solution and advanced instruments. (2) It is confirmed that the helix-coil transition for the B-D-glucan in the mixture of water / DMSO occurred from 0.82 to 0.85 of the volume fraction of the DMSO. (3) The anti-tumor activity of Lentinan with different molecular weights was evaluated, showing that the triple helix Lentinan with Mw of 5.71 x105 exhibited higher antitumor activity in vivo and vitro without any damage to the normal cell.The main contents and conclusions in this thesis are divided into the following parts.B-(1-3)-D-glucan was isolated from Lentinus edodes by extracting with 0.8% NaOH / 0.02% NaBH4 and 5% NaOH / 0.05% NaBH4 respectively to obtain glucans L-FV-IA and L-FV-IB. The chemical structure of the sample L-FV-IB was determined by infrared (IR) and 13C NMR to be a B-(1-3)-D-glucan with (1-6) branching , namely two branched residues for every five D-gluopyranosyl residues. The glucan L-FV-IB was degraded into seven fractions of different molecular weights by ultrasonic irradiation, and further fractionated, each into three parts, by precipitation from water to acetone at the room temperature. Weight-average molecular weight (Mw), radius of gyration (<Sa>1/2), and intrinsic viscosity ([]) of the B-D-glucan and its fractions in 0.9% NaCl aqueous solution and dimethylsulfoxide (DMSO) were measured by multi-angle laser light scattering (LLS), size exclusionchromatography combined with multi-angel laser light scattering (SEC-LLS), and viscometry. Mw dependence of [] and radius of gyration <S2>1/2 for the glucan in 0.9% NaCl aqueous solution at 25 C were established to be [17] = 2.94 x 10-7Mw1.58 (cm3 g-1) (Mw < 6.0 x 105), [] = 2.30x10-5Mw125(cm3 g-1) (Mw>6.0x 105) and <S2>1/2= (4.35+0.3) x 10-3Mw0.75+0.02(nm), respectively. However, the dependences of intrinsic viscosity [] on Mw for the glucan in DMSO was established to be [] = 0.112 Mwa54 (cm3 g-1). Based on YFY theory for wormlike chain model, the conformational parameters of the glucan L-FV-IB were obtained to be 2240 + 100 nm-1, 100 + 10 nm and 0.30 + 0.1nm for molar mass per unit contour length (ML), persistence length (q), and contour length per main-chain glucose residue (h), respectively, which agree closely with theory data of triple-helical chains and reported parameters for triple-helix schizophyllan in 0.01M NaOH aqueous solution. However, the parameters of the B-D-glucan agree with theory data of single-flexible chains. The results indicated that the glucan in 0.9% NaCl aqueous solution exist as triple-helical chains with high rigidity, and in DMSO as single-flexible chains.The values of Mw, <S2>1/2, [] and the special optical rotations at 589nm [a]D of lentinan in 0.2M NaCl aqueous solution, dimethylsulfoxide (DMSO) and water / DMSO mixtures were measured by LLS, SEC-LLS, viscometry, and polarimeter. The results indicated that the更多还原