【作者】 石磊；

【导师】 陈靠山；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 我国有丰富的中草药资源和微生物资源，以它们为研究材料，分离出免疫活性高、疗效明确的多糖，继而进行化学结构和生物活性研究，一方面可以丰富已有的糖数据库，推动糖化学和糖生物学的发展，另一方面也可为创制具有我国特色的多糖药物奠定基础，因而具有较大意义。柘树（Cudrania tricuspidata（Carr．）Bur．）为桑科柘属植物，广泛分布于我国华东、中南、西南至河北南部。柘树根可治疗风湿关节疼痛、黄疸、淋浊、闭经、跌打损伤以及疔疮痈肿等症，是一种传统的中药材。柘树根具有提高机体免疫功能、抗氧化、抗炎等多方面的药理作用，其已知的化学成分主要有氧杂蒽酮、黄酮、异黄酮、二苯酮、多糖类等。国内外对柘树根多糖的报道很少，并且所用多糖样品为未经纯化的粗多糖，所以无法全面了解柘树根中多糖的类型、含量及它们与柘树根药理作用的关系。本文对柘树根中的多糖组分进行了深入的分离、纯化，并对获得的均一多糖进行化学结构研究和生物活性测定。从柘树根中用热水提取得到一种粗多糖CPS，使用DEAE-纤维素柱层析和多种凝胶过滤柱层析进行分离、纯化，获得了CPS-0A、CPS-1A、CPS-2B、CPS-3A四种均一的多糖组分。应用HPLC、糖基组成分析、甲基化分析、元素分析、GC、GC-MS、部分酸水解、酶水解等化学及生化方法和IR、UV、¹³C NMR、¹H NMR、HMQC、HMBC等光谱学方法对它们的化学结构及理化性质进行了研究，确定了其化学结构或结构特征：①CPS-0A为α构型的葡聚糖。其主链由1，4-连接的葡聚糖组成，分支点位于6位，侧链内部由1，4-连接的葡萄糖残基组成，通过1→6糖苷键取代于分支点的O-6上，其结构式为：（?）②CPS-1A主要以α-1，4-D-Glc为主链，末端糖基为Gal、Glc和Ara，葡萄糖和半乳糖上含有少量的分支，且含有少量的木糖。③CPS-2B的主要部分是一个RG-Ⅰ型的果胶多糖，在主链鼠李糖的4位有复杂的Ⅱ型阿拉伯半乳糖支链，AG支链本身又呈分支结构，以1，3-连接的β-D-半乳聚糖为主链，在部分主链残基的6位有非还原末端、1，5-、1，3，5-连接的阿拉伯糖及1，6-连接的半乳糖构成的进一步分支。④CPS-3A由1，6-连接的β-D-Gal，1，4-连接的β-D-Gal，1，4-连接的β-D-GalA，1，3，6-连接的β-D-Gal，1，2，4-连接的β-L-Rha构成，在1，2，4-连接的β-L-Rha的O-2或O-4位和1，3，6-连接的β-D-Gal的O-3位形成分支点，并以1-连接的Gal为末端。侧链由1-连接的Ara和1，5-连接的Ara和1，3，5-连接的Ara组成，并连接在主链的1，6-连接的β-D-Gal的O-3位。以上4种多糖组分均为首次从柘树中发现。牛蒡（Arctium lappa L．）是菊科牛蒡属直根系二年生大型草本植物，具有良好的医疗保健作用和独特的综合营养价值。牛蒡的肉质根富含蛋白质、氨基酸、多种维生素、矿物质以及菊科（Compositae）植物特有的菊糖。从牛蒡根中用热水提取得到一种粗多糖，使用凝胶过滤柱层析得到一种均一的低聚糖组分ALO。采用HPLC、糖基组成分析、甲基化分析、GC、GC-MS、NMR（1D，2D）、UV、IR等对该牛蒡低聚糖的化学结构进行深入研究，确定它是一种菊糖型的低聚果糖，由13个呋喃型的果糖以β（2→1）糖苷键相连，1个吡喃型的葡萄糖以α（1→2）糖苷键连接在果糖末端的线性直链结构，结构式为：β-D-Fruf-（2[→1）-β-D-Fruf-(2]_n→1-α-D-Glcp n=12拟康氏木霉（Trichoderma pseudokoningii）是本实验室分离到的一株对多种病原菌有较强拮抗作用的生防菌株，已经证明该菌发酵时分泌到胞外的多糖类物质，可使番茄幼苗灰霉病的发病率明显降低，与植物系统抗性相关酶的活性显著升高。用离子交换层析和凝胶过滤层析对拟康氏木霉的胞外多糖组分进行了分离、纯化，得到一个均一多糖组分TPP-0，对其理化性质和化学结构进行了初步分析。TPP-0主要由鼠李糖和葡萄糖醛酸组成，二者的摩尔比为1.6：1.O，¹³C NMR谱中显示有9个异头碳信号，提示这2种组分有多种连接方式，详细结构情况正在研究。用MTS-PMS方法研究了4种柘树根多糖（CPS-0A、CPS-1A、CPS-2B、CPS-3A）在体外直接及协同有丝分裂原ConA或LPS对小鼠脾淋巴细胞增殖的影响，用中性红比色法测定了4种柘树根多糖对腹腔巨噬细胞（MΦ）吞噬功能的影响，并与应用于临床的香菇多糖（LNT）进行了比较。结果表明：在6.25～100μg/mL浓度范围内，CPS-1A、CPS-2B、CPS-3A可直接刺激小鼠脾淋巴细胞增殖，与空白对照相比均达到极显著性差异。CPS-1A在浓度为50μg/mL、100μg/mL时刺激效果高于最佳浓度的LNT（25μg/mL），且达到极显著性差异；CPS-2B在浓度为50μg/mL和100μg/mL时刺激效果高于LNT，且达到极显著性差异；CPS-3A在浓度为25μg/mL、50μg/mL、100μg/mL时刺激效果高于LNT，且达到极显著性差异。在6.25～100μg/mL浓度范围内，CPS-1A、CPS-2B、CPS-3A协同有丝分裂原ConA可显著促进小鼠脾淋巴细胞的增殖，与空白对照相比均达到极显著性差异。CPS-1A在浓度为50μg/mL时刺激效果高于LNT，且达到极显著性差异；CPS-2B在浓度为25μg/mL、50μg/mL时刺激效果高于LNT，且25μg/mL达到显著性差异，50μg/mL达到极显著性差异；CPS-3A在浓度为12.5μg/mL时刺激效果高于LNT，达到显著性差异，且作用浓度小于LNT。在协同有丝分裂原LPS对小鼠脾淋巴细胞增殖的影响中，CPS-1A在浓度为50μg/mL、100μg/mL时与空白对照相比达到极显著性差异，且刺激效果高于LNT，50μg/mL达到显著性差异，100μg/mL达到极显著性差异；CPS-2B在6.25～100μg/mL浓度范围内与空白对照相比均达到极显著性差异，6.25μg/mL、50μg/mL、100μg/mL刺激效果高于LNT，达到显著性差异，且6.25μg/mL作用浓度小于LNT；CPS-3A在浓度为12.5μg/mL时与空白对照相比达到显著性差异，在浓度为6.25μg/mL、25μg/mL、50μg/mL和100μg/mL时与空白对照相比均达到极显著性差异，25ug/mL刺激效果高于LNT，达到显著性差异。CPS-1A、CPS-2B在一定浓度范围内可显著增强小鼠腹腔巨噬细胞吞噬中性红的能力。CPS-1A在100μg/mL与空白对照相比达到显著性差异，50μg/mL与空白对照相比达到极显著性差异，在50μg/mL与最佳浓度（50μg/mL）的LNT作用效果相同；CPS-2B在100μg/mL与空白对照相比达到显著性差异，与LNT作用效果无显著性差异；CPS-3A在此范围内无明显作用。CPS-0A在上述实验中均未表现出活性。CPS-1A、CPS-2B、CPS-3A可能是一种活性高于LNT的免疫增强剂。柘树根中活性较强的多糖组分都具有较多的分支或含有糖醛酸。而不含糖醛酸、分支相对较少的α-1，4-D-葡聚糖，则没有表现出活性，这说明分支的复杂性和糖醛酸基团是多糖免疫活性的重要影响因素。本文还对3种常用的植物多糖脱蛋白的方法，即Sevag法、三氯乙酸法、木瓜蛋白酶-Sevag法的效果进行了比较，结果表明，木瓜蛋白酶-Sevag法除蛋白效果最好。更多还原

【Abstract】 China has rich resources of TCM and microbe, from which the polysaccharides possessing high activity and curative effect can be isolated and purified. We may use them as materials to carry out the research on chemical structure and biological activity. This work is significant in two aspects. Firstly, it contributes to improve the glycochemistry and glycobiology and the glyco-bank can be enriched. Secondly, it helps to develop polysaccharide drugs in China.Cudrania tricuspidata （Carr.） Bur. is a deciduous shrub or tree distributed over China, Korea, and Japan. Its roots are applied in clinic for the treatment of digestive apparatus tumor, especially gastric carcinoma, and are also used to heal gonorrhea, rheumatism, jaundice, boils, scabies, bruising, and dysmenorrhea. As a widely used traditional Chinese medicine, it can stimulate the immune function, and show antioxidative, anti-inflammatory, and other effects in pharmacological studies. The chemical constituents of C. tricuspidata include xanthones, flavones, isoflavones, benzophenones, polysaccharides, etc. Previous reports on polysaccharides of C. tricuspidata are weak in the preparation of homogeneous fractions, so it is impossible to learn their types, contents, and their relationship with pharmacological effects. This dissertation is focused on the isolation, purification, chemical characterization, and immunological activity of the polysaccharide fractions of C. tricuspidata.The roots of C. tricuspidata were extracted with hot water, giving one crude preparation, from which 4 homogeneous polysaccharide fractions （CPS-OA, CPS-1A, CPS-2B, CPS-3A） were isolated using anion-exchange chromatography and gel permeation chromatography. Their chemical structures and physicochemical properties were investigated using HPLC, glycosyl analysis, methylation analysis, elemental analysis, GC, GC-MS, partial hydrolysis, enzymatic hydrolysis, and spectroscopic methods, such as IR, UV, ¹³C NMR, ¹H NMR, HMQC and HMBC. The structure formula or characteristics were inferred as follow:1) CPS-OA contains glucose residue solely, with an average repeating unit of decasaccharide, having a backbone consisting of 1, 4-linked α-D-glucopyranosyl residues, to which the side chain consisting of terminal and 1, 4-linked α-D-glucopyranosyl residues is attached at position 0-6 of the branching residues.2) CPS-1A has a backbone mainly consisting of α-1, 4-D-glucopyranosyl residues, and the terminals are galactose, glucose, and arabinose residues. Minor branches are attached to galactosyl and glucosyl residues, and CPS-1A also contains trace xylose residue.3) The major composition part of CPS-2B is a kind of pectic polysaccharide called type RG-I. The side chain, composed of complex type II AG, is attached to the backbone consisting of rhamnose at position 0-4. AG itself also has branch structure, which has a backbone consisting of 1, 3-linked β-D-galactosyl residues, and at position 0-6 of partial backbone there were further branches composed of terminal, 1, 5-linked, 1, 3, 5-linked arabinosyl residue and 1,6-linked galactosyl residues.4) CPS-3A is composed of 1, 6-linked β-D-galactosyl residues, 1, 4-linked β-D-galactosyl residues, 1, 4-linked β-D-galacturonic acid residues, 1, 3, 6-linked β-D-galactosyl residues, and 1, 2, 4-linked β-L-rhamnosyl residues. There are branch points at position 0-2 or 0-4 of 1, 2, 4-linked β-L-rhamnosyl residues, and at position 0-3 of 1, 3, 6-linked β-D-galactosyl residues, furthermore, the terminal is 1-linked galactosyl residues. The side chain consists of 1-linked arabinosyl residues, 1, 5-linked arabinosyl residues, and 1, 3, 5-linked arabinosyl residues. The side chain is attached to the backbone at position 0-3 of 1, 6-linked β-D-galactosyl residues.It is the first time that the presence of the four polysaccharides in C. tricuspidata is reported.Arctium lappa L. is a member of the Compositae family, which is a kind of biennial giant plant and possesses excellent value for medical care and special nutrition. The roots of A. lappa L. contain rich proteins, amino acids, vitamins, miner- als, and inulin, which is special for Compositae plants. The roots were extracted with hot water, giving one crude preparation, from which one homogeneous oligosacch-aride fraction （ALO） was isolated using gel permeation chromatography. Its chemical structure and physicochemical property were investigated using HPLC, glycosyl analysis, methylation analysis, GC, GC-MS, and spectroscopic methods, such as IR, UV, ¹³C NMR, ¹H NMR, HMQC and HMBC. ALO is composed of D-fructose and D-glucose in the molar ratio of about 13:1, which is confirmed by the composition of 12 fructose residues linked by β （2→1） glycosidic bond and 1 glucose residue linked by a （1→2） glycosidic bond at the end of linear straight sugar chain. The structure formula was inferred as follow:The extracellular polysaccharide produced by Trichoderma pseudokoningii was isolated and purified using anion-exchange chromatography and gel permeation chromatography. Its chemical structure and physicochemical property were elementarily investigated. The homogeneous polysaccharide named TPP-0 was composed of rhamnose and glucuronic acid in the molar ratio of 1.6:1.0. There were nine anomeric carbon signals in the ¹³C NMR spectrum, which suggested that TPP-0 has many linkage types. Its detailed structure is still in research.The effects of CPS-OA, CPS-1 A, CPS-2B, and CPS-3A on proliferation of lymphocytes and lymphocytes induced by ConA or LPS were studied with MTS-PMS assay. They were also studied on phagocytosis of peritoneal macrophages （MO） of mice in vitro with colorimetric method of neutral red. At the same time, the effects of CPS-OA, CPS-1 A, CPS-2B, and CPS-3A were compared with that of lentinan widely used in clinic.The results showed that CPS-1 A, CPS-2B, and CPS-3A directly stimulated lymphocytes proliferation at concentrations of 6.25 ?100 礸/mL. Compared with the control group, the mean differences of these effects were very significant. Effects of CPS-1A and CPS-2B at 50 μg/mL and 100 μg/mL were better than that of lentinan at the best concentration of 25 μg/mL and the mean differences were very significant. Effects of CPS-3A at 25 μg/mL, 50 μg/mL, and 100 μg/mL were better than that of lentinan and the mean differences were very significant.CPS-0A, CPS-1A, CPS-2B, and CPS-3A stimulated lymphocytes proliferation induced by ConA at concentrations of 6.25 ～100 μg/mL. Compared with the control group, the mean differences of these effects were very significant. The effect of CPS-1A at 50 μg/mL was better than that of lentinan and the mean difference was very significant. The effects of CPS-2B at 25 μg/mL and 50 μg/mL were better than that of lentinan. The mean difference was significant at 25 μg/mL and very significant at 50 μg/mL. Effect of CPS-3A at 12.5 μg/mL was better than that of lentinan, the mean difference was significant and the concentration was less than that of lentinan.In lymphocytes proliferation induced by LPS, effects of CPS-1 A at concentrations of 50 μg/mL and 100 μg/mL were better than those of the control group and lentinan. Compared with the control group, the mean differences were very significant. Compared with lentinan, the mean difference was significant at 50 μg/mL and very significant at 100 μg/mL. CPS-2B stimulated lymphocytes proliferation at concentrations of 6.25～100 μg/mL. Compared with the control group, the mean differences were very significant. Effects of CPS-2B at 6.25 μg/mL, 50 μg/mL and 100 μg/mL were better than that of lentinan, the mean differences were significant and the concentration of 6.25 μg/mL was less than that of lentinan. CPS-3 strengthened lymphocytes proliferation at concentrations of 6.25～100 μg/mL. Compared with the control group, the mean difference was significant at 12.5μg/mL and very significant at 6.25μg/mL, 25μg/mL, 50μg/mL and 100μg/mL. Effect of CPS-3 A at 25 μg/mL was better than that of lentinan and the mean difference was significant.In phagocytosis of peritoneal MO experiments, CPS-1 A promoted phagocytosis of MO on neutral red. Compared with the control group, the mean difference was significant at 100 μg/mL and very significant at 50 μg/mL. The effect of CPS-1 A at 50 μg/mL was equal with that of lentinan at the best concentration of 50 μg/mL. CPS-2B promoted phagocytosis of MO on neutral red. Compared with the control group, the mean difference was significant at 100μg/mL. The effect of CPS-2B at 100 μg/mL and that of lentinan were insignificant. Compared with the control group, CPS-3 A had no effect on phagocytosis of MO.In the above immunological assays, CPS-OA showed no activity. CPS-1 A, CPS-2B, and CPS-3A might be better immunomodulators than lentinan.The polysaccharide fractions showing high activity from C. tricuspidata are those highly branched or possessing uronic acid at nonreducing terminals. CPS-OA with few branches and without uronic acid showed no activity. The results suggested that complex branches and uronic acid have significant effect on immune activity for polysaccharides.Three commonly used methods in elimination of proteins, i.e. Sevag method, TCA method, and papain-Sevag method, were compared in this dissertation. The result showed that papain-Sevag method had the best effect on the elimination of proteins in preparing the polysaccharide from C. tricuspidata.更多还原