几种多糖的分离纯化、结构解析和生物活性研究
|
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、~(13)C 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，~(13)C 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。
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, ~(13)C 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, ~(13)C 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 ~(13)C 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.
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、~(13)C 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，~(13)C 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。
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, ~(13)C 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, ~(13)C 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 ~(13)C 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.
引文
1．郭忠武，王来曦．糖化学研究进展．化学进展，1995，7(1)：10-29
2. Zhang J P, Qian D H. Antitumor activity and tumor necrosis factor production of phytolacca acinosa polysaccharides Ⅰ in mice. Acta pharmacologica Sinica, 1993, 14 (6): 542-545
3. Duan J Y, Wang X S, Dong Q, et al. Structural features of a pectic arabinogalactan with immunological activity from the leaves of Diospyros kaki. Carbohydr Res, 2003, 338: 1291-1297
4．丁保金，金丽琴，吕建新．多糖的生物活性研究进展．中国药学杂志，2004，39(8)：561-564
5．王玲，李俊，李欣等．枸杞多糖2对辐射损伤小鼠免疫功能恢复的影响．上海疫学杂志，1995，15(4)：209-211
6. Kiho T, Ookubo K, Usui S, et al. Structural features and hypoglycemic activity of a polysaccharide (CS-F10) from the cultured mycelium of Cordyceps sinensis. Biol Pharm Bull, 1999, 22 (9): 966-970
7．赵国华，陈宗道，李志孝等．活性多糖的研究进展．食品与发酵工业，2002，27(7)：45-48
8. Kenned Y J F, White C A. Bioactive carbohydrates[M]. Chichester: Ellis Horwood, Ltd, 1993
9．吴东儒．糖类的生物化学[M]．北京：高等教育出版社，1987
10．张惟杰．糖复合物生化研究技术[M]．杭州：浙江大学出版社，1999
11．来鲁华，杨昱婷．寡糖的构象分析．生物化学与生物物理进展，1995，22(4)：290-294
12. Peters T, Meyer B, Stuike-Prill R, et al. A Monte Carlo method for conformational analysis of saccharides. Carbohydr Res, 1993, 238: 49-73
13. Imberty A, Pérez S, Milos H, et al. Flexibility in a tetrasaccharide fragment from the high mannose type of N-linked oligosaccharides. 1993, 15: 17-23
14．方积年．多糖体的结构分析．国外医学·药学分册，1981，7(4)：222-228
15. Rolf S. A chemically bonded stationary phase for carbohydrate analysis in liquid chromatography. J Chromatogr A, 1976: 206-210
16. Bradbury J H, Jenkins G A. Determination of the structures of trisaccharides by ~(13)C-NMR spectroscopy. Carbohydr Res, 1984, 126: 125-156
17．王展，方积年．高场核磁共振波谱在多糖结构研究中的应用．分析化学，2000，28(2)：240-247
18．方一苇．糖结构与质谱分析．质谱学报，1995，16(1)：1-8
19．蒋可，Burl．A L．糖复合物中糖链部分质谱测定．化学通报，1990，(6)：30-38
20. Yamamoto K. Microbial endoglycosidases for analyses of oligosaccharide chains in glycoproteins. J Biochem (Tokyo), 1994, 116: 229-235
21. Edge C J, Rademacher T W, Wormald M R. Fast sequencing of oligosaccharides: the reagent-array analysis method. Proc Natl Acad Sci U S A, 1992, 89(14): 6338-6342
22．王顺春，方积年．X-射线纤维衍射在多糖构型分析中应用的研究进展．天然产物研究与开发，2000，12(2)：75-80
23. McIntire T M, Penner R M, Brant D A. Observations of a circular, triple-helical polysaccharide using noncontact atomic force microscopy. Macromolecules, 1995, 28: 6375-6377
24. Rice K G, Wu P, Brand L, et al. Interterminal distance and flexibility of a triantennary glycopeptide as measured by resonance energy transfer. Biochemistry, 1991, 30: 6646-6655
25. Janaswamy S, Chandrasekaran R. Polysaccharide structures from powder diffraction data: molecular models of arabinan. Carbohydr Res, 2005, 340: 835-839
26. Tao Y Z, Zhang L A, Cheung P C K. Physicochemical properties and antitumor activities of water-soluble native and sulfated hyperbranched mushroom polysaccharides. Carbohydr Res, 2006, 341: 2261-2269
27. Kim H M, Han S B, Oh G T, et al. Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom Phellinus linteus. Int J Immunopharamacol, 1996, 18 (5): 295-303
28. Sakamoto Y, Watanabe H, Nagai M, et al. Lentinula edodes tlgl encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence. Plant Physiol. 2006, 141: 793-801
29．林志彬．灵芝多糖的免疫药理学研究及其意义．北京医科大学学报，1992，24(4)：271-274
30. Chinnah A D, Baig M A, Tizard I R, et al. Antigen dependent adjuvant activity of a polysaceharide β-(1, 4)-linked acetylated mannan (acemannan). Vaccine, 1992, 10 (8): 551-557
31. Duan J Y, Zheng Y, Dong Q, et al. Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki. Phytochemistry, 2004, 65: 609-615
32. Luettig B, Steinmuller C, Gifford G E, et al. Macrophage activation by the polysaccharide arabinogalactan-isolated from" plant cell cultures of Echinacea purpurea. J Natl Cancer Inst, 1989, 81 (9): 669-675
33. Esua M F, Rauwald J W. Novel bioactive maloyl glucans from Aloe vera gel: isolation, structure elucidation and in vitro bioassays. Carbohydr Res, 2006, 341: 355-364
34. Kimura Y, Sumiyoshi M, Suzuki T, et al. Antitumor and antimetastatic activity of a novel water-soluble low molecular weight beta-1, 3-D-glucan (branch beta-1, 6) isolated from Aureobasidium pullulans 1A1 strain black yeast. Anticancer Res, 2006, 26: 4131-4141
35. Gao H, Wang F, Lien E J, et al. Immunostimulating polysaccharides from Panax notoginseng. Pharm Res, 1996, 13 (8): 1196-1200
36．关玉峰．人参、黄芪多糖对免疫功能影响的临床实验研究．航空航天医药，1997，8(2)：4-6
37. Liu F, Fung M C, Ooi V E C, et al. Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complexes. Life Sciences, 1996, 58 (21): 1795-1803
38. Tokunaka K, Ohno N, Adachi Y, et al. Immunopharmacological and immunoto-xicological activities of a water-soluble (1→3)-β-D-glucan, CSBG from Candida spp. Int J Immunopharmacol, 2000, 22 (5): 383-394
39．李宗锴，李电东．牛膝多糖的免疫调节作用．药学学报，1997，32(12)：881-887
40. Czop J K, Austen K F. Properties of glycans that activate the human alternative complement pathway and interact with the human monocyte beta-glucan receptor. J Immunol, 1985, 135: 3388-3393
41. Krizková L, Duracková Z, Sandula J, et al. Antioxidative and antimutagenic activity of yeast cell wall mannans in vitro. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2001, 497: 213-222
42. Olafsdottir E S, Ingólfsdottir K. Polysaccharides from lichens: structural characteristics and biological activity. Planta Med, 2001, 67 (3): 199-208
43. Luettig B, Steinmuller C, Gifford G E, et al. Macrophage activation by the polysaccharide arabinogalactan isolated from plant cell cultures of Echinacea purpurea. J Natl Cancer Inst, 1989, 81 (9): 669-675
44. Kiyohara H, Takemoto N, Zhao J F, et al. Pectic polysaccharides from roots of Glycyrrhiza uralensis: possible contribution of neutral oligosaccharides in the galacturonase-resistant region to anti-complementary and mitogenic activities. Planta Med, 1996, 62 (1): 14-19
45. Kiyohara H, Matsumoto T, Nagai T, et al. The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines. Phytomedicine, 2006, 13: 494-500
46. Mizumoto K, Sugawara I, Ito W, et al. Sulfated homopolysaccharides with immunomodulating activities are more potent anti-HTLV-III agents than sulfated heteropolysaccharides. Jpn J Exp Med, 1988, 58 (3): 145-151
47. Vactor D V, Wall D P, Johnson K G. Heparan sulfate proteoglycans and the emergence of neuronal connectivity. Current Opinion in Neurobiology, 2006, 16: 40-51
48. Pramanik M, Mondal S, Chakraborty I, et al. Structural investigation of a polysaccharide (Fr. II) isolated from the aqueous extract of an edible mushroom, Pleurotus sajor-caju. Carbohydr Res, 2005,340: 629-636
49. Cleary J A, Kelly G E, Husband A J. The effect of molecular weight and β-I, 6-linkages on priming of macrophage function in mice by (1, 3)-β-D-glucan. Immunol Cell Biol, 1999, 77 (5): 395-403
50. Demleitner S, Kraus J, Franz G. Synthesis and antitumour activity of derivatives of curdlan and lichenan branched at C-6. 1992,226: 239-246
51. 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 leukemic U937 cells. Anti-Cancer Drugs, 2001,12: 841-846
52. Misaki A, Kawaguchi K, Miyaji H, et al. Structure of pestalotan, a highly branched (1→3)-β-D-glucan elaborated by Pestalotia sp. 815, and the enhancement of its antitumor activity by polyol modification of the side chains. Carbohydr Res, 1984, 129: 209-227
53. S.one Y, Misaki A, Shibata S. Preparation and characterization of antibodies against 6-O-α-D-xylopyranosyl-β-D-glucopyranose (β-isoprimeverose), the disaccharide unit of xyloglucan in plant cell-walls. Carbohydr Res, 1989, 191: 79-89
54. Groth T, Wagenknecht W. Anticoagulant potential of regioselective derivatized cellulose. Biomaterials, 2001, 22: 2719-2729
55. Bohn J A, BeMiller J N. (1→3)-β-D-Glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohydr Polymer, 1995, 28: 3-14
56. Yang J, Du Y, Huang R, et al. Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides. Int J Biol Macro, 2002, 31: 55-62
57. Sarnuelsen A B, Paulsen B S, Wold J K, et al. Characterization of a biologically active pectin from Plantago major L. Carbohydr Polymer, 1996, 30: 37-44
58. Mulloy B, Mourao P A S, Gray E. Structure/function studies of anticoagulant sulphated polysaccharides using NMR. J Biotech, 2000, 77: 123-135
59. Matsuzaki K, Yamamoto I, Sato T, et al. ~(13)C-NMR investigations of synthetic branched polysaccharides. Carbohydr Polymer, 1986, 6: 155-163
60. Perret J, Bruneteau M, Michel G, et al. Effect of growth conditions on the structure of β-D-glucans from Phytophthora parasitica dastur, a phytopathogenic fungus. Carbohydr Polymer, 1992, 17: 231-236
61．王长云，管华诗．多糖抗病毒作用研究进展Ⅱ．硫酸多糖抗病毒作用．生物工程进展，2000，20(2)：3-8
62. Caducei M J, Pujol C A, Ciancia M, et al. Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: correlation between structure and biological activity. Int J Biol Macro, 1997, 20: 97-105
63．诸葛健，赵振锋，方慧英．功能性多糖的构效关系．无锡轻工大学学报，2002，21(2)：209-212
64. Uryu T, Ikushima N, Katsuraya K, et al. Sulfated alkyl oligosaccharides with potent inhibitory effects on human immunodeficiency virus infection. Biochem Pharmacol, 1992, 43: 2385-2392
65．张健，阳庚元．羟乙基化牛膝多糖的合成及其活性研究，化学学报，2003，61(10)：1692-1696
66．陈惠黎，王克夷．糖复合物的结构与功能[M]，上海：上海医科大学出版社，1997
67. Ohno N, Miura T, Miura N N, et al. Structure and biological activities of hypochlorite oxidized zymosan. Carbohydr Polymer, 2001, 44: 339-349
68. Zhang P, Zhang L, Cheng S. Effects of urea and sodium hydroxide on the molecular weight and conformation of α-(1→3)-D-glucan from Lentinus edodes in aqueous solution. Carbohydr Res, 2000, 327: 431-438
69. Maeda Y Y, Hamuro J, Chihara G. The mechanisms of action of anti-tumour polysaccharides. I. The effects of antilymphocyte serum on the anti-tumour activity oflentinan. Int J Cancer, 1971, 8 (1): 41-46
70. Saito H, Yoshioka Y, Uehara N, et al. Relationship between conformation and biological response for (1→3)-β-D-glucans in the activation of coagulation Factor G from limulus amebocyte lysate and host-mediated antitumor activity. Demonstration of single-helix conformation as a stimulant. Carbohydr Res, 1991, 217: 181-190
71. Alban S, Franz G. Characterization of the anticoagulant actions of a semisynthetic curdlan sulfate. Thromb Res, 2000, 99: 377-388
72. Gao Y, Fukuda A, Katsuraya K, et al. Synthesis of regioselective substituted curdlan sulfates with medium molecular weights and their specific anti-HIV-1 activities. Macromolecules, 1997, 30: 3224-3228
73. Calazans G M T, Lima R C, Franca F P, et al. Molecular weight and antitumour activity of Zymomonas mobilis levans. Int J Biol Macro, 2000, 27: 245-247
74. Dong Q, Jia L M, Fang J N. A β-D-glucan isolated from the fruiting bodies of Hericium erinaceus and its aqueous conformation. Carbohydr Res, 2006, 341: 791-795
75．田庚元，冯宇澄．多糖类免疫调节剂的研究和应用．化学进展，1994，6(2)：114-124
76. Sandula J, Kogan G, Kacuráková M, et al. Microbial (1→3)-β-D-glucans, their preparation, physicochemical characterization and immunomodulatory activity. Carbohydr Polymer, 2006, 38: 247-253
77．郑建仙．功能性食品[M]，北京：中国轻工业出版社，2002
78. Tabata K, Ito W, Kojima T, et al. Ultrasonic degradation of schizophyllan, an antitumor polysaccharide produced by Schizophyllum commune fries. Carbohydr Res, 1981, 89: 121-135
79. Zhao J F, Kiyohara H, Yamada H, et al. Heterogeneity and characterisation of mitogenic and anti-complementary pectic polysaccharides from the roots of Glycyrrhiza uralensis Fisch et D. C. Carbohydr Res, 1991, 219: 149-172
80. Kiyohara H, Takemoto N, Zhao J F, et al. Pectic polysaccharides from roots of Glycyrrhiza uralensis: possible contribution of neutral oligosaccharides in the galacturonase-resistant region to anti-complementary and mitogenic activities. Planta Med, 2006, 72: 14-19
81. Yu K W, Kiyohara H, Matsumoto T, et al. Intestinal immune system modulating polysaccharides from rhizomes of Atractylodes lancea. Planta Med, 1998, 64: 714-719
82．宁君，孔繁祚．具有重要生理活性寡糖的发现及应用．世界科技研究与发展，2001，23(6)：22-25
1．熊文愈，汪计珠，石同岱等．中国木本药用植物[M]，上海：上海科学教育出版社，1993：85-88
2．江苏新医学院．中药大辞典(下册)[M]，上海：上海科学技术出版社，1995：1502-1503
3．程剑华，李以镔．抗癌植物药及其验方[M]，南昌：江西科学技术出版社，1998：62-64
4．薛芳，许占民．中国药物大全(中药卷)，第2版[M]，北京：人民卫生出版社，1998：50-54
5. Fujimoto T, Hano Y, Nomura T. Components of root bark of Cudrania tricuspidata 1. Structures of four new isoPrenylated xanthones, Cudraxanthones A, B, CandD. Planta Medica, 1984, 50 (3): 218-221
6. Hano Y, Matsumoto Y, Sun J Y, et al. Structures of three new isoprenylated xanthones, Cudraxanthones E, F and G. Planta Medica, 1990, 56 (4): 399-402
7. Hano Y, Matsumoto Y, Sun J Y, et al. Structures of four new isoprenylated xanthones, Cudraxanthones H, I, J and K. Planta Medica, 1990, 56 (5): 478-481
8. Hano Y, Matsumoto Y, Shimnohara K, et al. Structures of four new isoprenylated xanthones, Cudraxanthones L, M, N and O from Cudrania tricuspidata. Planta Medica, 1991, 57 (2): 173-175
9. Fujimoto T, Hano Y, Nomura T, et al. Components of root bark of Cudrania trieuspidata 2. Structures of two new isoprenylated flavones, Cudraflavones A andB. Planta Medica, 1984, 50 (2): 161-163
10. Hano Y, Matsumoto Y, Shimnohara K, et al. Cudraflavones C and D, two new prenylflavones from the root bark of Cudrania tricuspidata (Carr.) Bur. Heterocycles, 1990, 31 (7): 1339-1344
11. Fujimoto T, Nomura T. Components of root bark of Cudrania tricuspidata. Isolation and structure studies on the flavonoids. Planta Medica, 1985, 51 (3): 190-193
12. Lee I K, Kim C J, Song K S, et al. Two benzylated dihydroflavonols from Cudrania tricuspidata. Journal of Natural Products, 1995, 58 (10): 1614-1617
13. Lee I K, Kim C J, Song K S, et al. Cytotoxic benzyl dihydroflavonols from Cudrania tricuspidata. Phytochemistry, 1996, 41 (1): 213-216
14. Chang C H, Lin C C, Kadota S, et al. Flavonoids and a prenylated xanthone from Cudrania cochinchinensis var. gerontogea. Phytoehemistry, 1995, 40 (3): 945-947
15．戴明，野村太郎．柘木成分研究．国外医学中医中药分册，1983，4：53-54
16．张月华，任婉薇，万树文等．柘木的化学成分研究．医药工业，1980，3：15-20
17．缪春辉，顾正兵，杨根金．柘木化学成分的研究．中成药，2002，24(3)：211-212
18．史琪荣，柳润辉，徐希科等．柘木黄酮类成分研究．中国中药杂志，2006，31(1)：77-78
19. Zheng Z P, Liang J Y, Hu L H. Water-soluble constituents of Cudrania tricuspidata (Carr.) Bur. J Integr Plant Biol, 48 (8), 996-1000
20．宫丽华，汪海霞，王先磊等．柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用．山东中医药大学学报，2002，26(2)：145-146
21．江苏新医学院．中药大辞典(下册)[M]，上海：上海科学技术出版社，1995：1731-1732
22．刘建华，高玉琼，霍昕．穿破石挥发性成分的研究．中国中药杂志，2003，28(11)：1047-1049
23．宋耐宝，杨学东，王义明等．柘木化学成分及药理活性研究现状．中成药，2005，27(3)：335-337
24．李贺然，邹忠梅，徐丽珍等．柘属药用植物化学和药理活性研究进展．国外医学中医中药分册，2003，25(4)：203-207
25. Mufti V V S, Seshadri T R, Srvakumaran S. Phytochemistry, 1972, 11 (1): 2089-2092
26. Chang C H, Lin C C, Hattori M, et al. Phytochemistry, 1989, 28 (2): 595-598
27. Chang C H, Lin C C, Kawata Y, et al. Phytochemistry, 1989, 28(10): 2823-2826
28. Hou A J, Fukai, T, Shimazaki M, et al. Benzophenones and xanthones with isoprenoid groups from Cudrania cochinchinensis. Journal of Natural Products, 2001, 64 (1): 65-70
29. Knapp JE, SchiffP L. J Pharm Sci, 1971, 60 (11): 1729-1730
30. Nomura T, Hano Y, Fujimoto T. Three new isoprenylated xanthones, cudraxanthone A, B, C from the root of Cudrania tricuspidata (Carr.) Bur. Heterocycles, 1983, 20 (2): 213-218
31. Chang C H, Lin C C, Hattori M, et al. Journal of Ethnopharmacology, 1994, 44 (1): 79-85
32. Sun NJ, Chang CJ, John MC. Phytochemistry, 1988, 27(3): 951-952
33．柘木糖浆-上海中药制药二厂．中草药通讯，1977，(12)：18
34．曹根生．抗肿瘤新药．柘木注射液．中成药研究，1980，(1)：34-35
35．丁红华，陈栋晖，朱莉菲等．柘木糖浆治疗胃癌疗效观察．中成药，2001，23(2)：151-152
36．杜卫东，沈达明，宋晓华等．柘木糖浆对胃肠道肿瘤手术后化疗病人肿瘤标记物和细胞免疫的影响．临床医药杂志，2004，17(3)：11-14
37．王佩芳，沈辉，唐红敏等．柘木糖浆对胃癌前病变阻断逆转作用的疗效观察．中华医学实践杂志，2003，2(1)：57-58
38．孙菁，袁耀宗，郑舜华等．柘木糖浆治疗活动期慢性胃炎及胃黏膜肠腺化生的疗效观察．中成药，2005，27(5)：12-14
39．毕盛．抗癌中成药-生命之星[P]．中国专利：A61K35／78，1996-03-13
40．王贞丽，姜自彬，钱月中．植物抗肿瘤活性成分研究进展．前卫医药杂志，1999，1：62-63
41．徐誉泰，张可炜，李艳等．柘树黄酮对胃癌细胞株NKM大分子合成的影响．中医药学报，1998，26(5)：47-48
42．张英慧，徐誉泰．柘木根黄酮注射液对小鼠S180肿瘤的抑制作用．佛山科学技术学院学报(自然科学版)，2001，19(3)：75-77
43. Seo W G, Pae H O, Oh G S, et al. Ethyl acetate extract of the stem bark of Cudrania tricuspidata induces apoptosis in human leukemia HL-60 cells. Am J Chin Med, 2001, 29 (2): 313-320
44．张聪敏，萧丽．对柘木黄酮引起细胞凋亡及其机制的初步研究．漳州师范学院学报(自然科学版)，2004，17(4)：98-101
45. Zou Y S, Hou A J, Zhu G F, et al. Cytotoxic isoprenylated xanthones from Cudrania tricuspidata. Bioorganic & Medicinal Chemistry, 2004, 12: 1947-1953
46．陈良儿，谢振家．柘树茎乙醇提取液的抗炎镇痛作用．南京军医学院学报，2002，24(1)：11-13
47．张可炜，徐誉泰，张举仁．银杏叶和柘树提取物的抗氧化作用．山东大学学 报(自然科学版)，2000，35(4)：469-472
48. Cha J Y, Kim H J, Chung C H, et al. Antioxidative activities and contents of polyphenolic compound of Cudrania tricuspidata. Korean Soci F Sci Nutr, 1999, 28 (6): 1310-1315
49. Lee B W, Lee J H, Lee S T, et al. Antioxidant and cytotoxic activities of xanthones from Cudrania tricuspidata. Bioorganic & Medicinal Chemistry Letters, 2005, 15: 5548-5552
50．何泉华，许实波．穿心草口山酮抗氧化作用初探．中国药理学通报，1998，14(2)：130-132
51．何泉华，许实波．口山酮对老年小鼠抗氧化酶活性的影响．广州医学院学报，1999，27(4)：6-9
52. Cho E J, Yokozawa T, Rhyu D Y, et al. The inhibitory effects of 12 medicinal plants and their component compounds on lipid peroxidation. Am J Chin Med, 2003, 31 (6): 907-917
53．王永中，郑良朴，王瑞国．穿破石对小鼠CCl_4急性肝损伤保护作用的实验观察．福建中医药，2004，35(5)：44-46
54．吕强，许国祥．柘木抗结核作用的初步研究．药学通报，1980，15(12)：36
55. Kang D G, Hur T Y, Lee G M, et al. Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension. Life Sciences, 2002, 70 (22): 2599-2609
56. Seo W G, Pae H O, Oh G S, et al. Inhibitory effect of ethyl acetate fraction from Cudrania tricuspidata on the expression of nitric oxide synthase gene in RAW 264.7 macrophages stimulated with interferon-γ and lipopolysaccharide. General Pharmacology, 2001, 35: 21-28
57．孙琳，刘爱芬，李义召等．脑缺血再灌注后热休克蛋白70、c-fos的表达及柘树制剂的神经保护作用．临床神经病学杂志，2005，18(5)：354-356
58．刘福海，常云水，李志成．柘树根皮膏治疗骨折767例临床报告．山东医药，1979，(7)：41
1．宫丽华，汪海霞，王先磊等．柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用．山东中医药大学学报，2002，26(2)：145-146
2. Alsop R M, Vlachogiarmis GJ. Determination of the molecular weight of clinical dextran by gel chromatography on TSK PW type columns. J Chromatogr, 1982, 246: 227-240
3．魏远安，方积年．高效凝胶渗透色谱法测定多糖纯度及分子量．药学学报，1989，24(7)：532-536
4. Dubois M, Gilles K A, Hamilton J K, et al. Colorimetric method for determination of sugars and related substances. Anal Chem, 1956, 28: 350
5. Blumencrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal Biochem, 1973, 54: 484-490
6. Bensadon A, Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem, 1976, 70: 241-250
7. Blakeney A B, Harris P J, Henry R J, et al. A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr Res, 1983, 113: 291-299
8．张惟杰．糖复合物生化研究技术(第2版)[M]．杭州：浙江大学出版社，2003：23
9. Taylor R L, Conrad H E. Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of the carbodiimide-activated carboxyl groups. Biochemistry, 1972, 11: 1383-1388
10. Needs P W, Selvendran R R. Avoiding oxidative degradation during sodium hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydr Res, 1993, 245: 1-12
11. Ciucanu I, Kerek F. A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res, 1984, 131, 209-217
12．董群，张志毅，林颖等．汉防己多糖的研究．生物化学与生物物理学报，1995，27(3)：261-265
13. Redgwell R J, Curti D, Fischer M, et al. Coffee bean arabinogalactans: acidic polymers covalently linked to protein. Carbohydr Res, 2002, 337: 239-253
14．陈伟，林新华，黄丽英等．芦荟多糖中游离蛋白去除的分光光度法．光谱实验室，2005，22(1)：113-115
15．刘成梅，万茵，涂宗财等．百合多糖脱蛋白方法的研究．食品科学，2002，23(1)：89-90
16．酶法脱蛋白技术用于螺旋藻多糖提取工艺的研究．食品与发酵工业，1998，24(3)：24-28
17．张惟杰．糖复合物生化研究技术(第2版)[M]．杭州：浙江大学出版社，2003：194
18．张惟杰．糖复合物生化研究技术(第2版)[M]．杭州：浙江大学出版社，2003：196
19. Gorin R A. J. Carbon-13 nuclear magnetic resonance spectroscopy of polysaccharides. Adv. Carbodydr. Chem. Biochem, 1981, 38: 13-39
20. Wang Xue-Song, Dong Qun, Zuo Jian-Ping, et al. Structure and potential immunological activity of a pectin from Centella asiatica (L.) Urban. Carbohydr. Res, 2003, 338: 2393-2402
21．王镜岩，朱圣庚，徐长法．生物化学(第三版)[M]．北京：高等教育出版社，2002：42-43
22. Strasser G R, Amado R. Pectic substances from red beet (Beta vulgaris conditiva). Part Ⅰ. Structural analysis of rhamnogalacturonan I using enzymic degradation and methylation analysis. Carbohydr Polym, 200.1, 44: 63-70
23. Pinto G L D, Martinez M, Mendoza J A, et al. Structural studies of the polysaccharide isolated from Spondias purpurea gum exudates. Carbohydr Res, 1996, 290: 97-103
24. Pinto G L D, Martinez M, Corredor A L D, et al. Chemical and ~(13)C NMR studies of Enterolobium cyclocarpum gum and in degradation products. Phytochemistry, 1994, 37: 1311-1315
25. Shimizu N, Tomoda M, Kanari M, et al. An acidic polysaccharide having activity on the reticuloendothelial system from the root ofAstragalus mongholicus. Chem Pharm Bull, 1991, 39: 2969-2972
26. An J, O'Neill M A, Albersherm P, et al. Isolation and structural characterization of β-D-glucosyluronic acid and 4-O-methyl β-D-glucosyluronic acid-containing oligosaccharides from the cell-wall pectic polysaccharide, rhamnogalacturonan Ⅰ. Carbohydr Res, 1994, 252: 235-245
27. Cui W W, Eskin M N A, Biliaderis C G, et al. NMR characterization of a 4-O-methyl-β-D-glucuronic acid containing rhamnogalacturonan from yellow mustard (Sinapis alba L.) mucilage. Carbohydr Res, 1996, 292: 173-183
28. Rauvala H, Finne J, Krusius T, et al. Methylation techniques in the structural analysis of glycoproteins and glycolipids. Advances in carbohydrate chemistry and biochemistry, 1981: 389-416
29．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)：60-65
30. Kim J S, Reuhs B L, Michon F, et al. Addition of glycerol for improved methylation linkage analysis of polysaccharides. Carbohydr Res, 2006, 341: 1061-1064
1. Liu F, Fung M C, Ooi V E C, et al. Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complex. Life Sci, 1996, 58: 1795-1803
2. Yamaoka Y, Kawakita T, Kaneko M, et al. A polysaccharide fraction of shosaiko-to active in augmentation of natural killer activity by oral administration. Biol Pharm Bull, 1995, 18(6): 846-849
3. Oka S, Ohno N, Iwanaga S, et al. Characterization of mitogenic substances in the hot water extracts of bupleuri radix. Biol Pharm Bull, 1995, 18: 757-765
4. Kim K H, Lee Y S, Jung I S, et al. Acid polysaccharide from Pangxginseng, ginsan, induces TH1 cell and macrophage cytokines and generates LAK cells in synergy with rIL-2. Planta Med, 1998, 64: 110-115
5. Ohtani K, Mizutani S, Hatono S, et al. Sanchinan-A, a reticuloendothelial system activating arabinogalactan from sanchi-ginseng (roots of Panax notoginseng). Planta Med, 1987, 53: 166-169
6. Shimizu N, Tomoda M, Kanari M, et al. An acidic polysaccharide having activity on the reticuloendothelial system from the root of Astragalus mongholicus. Chem Pharm Bull, 1991, 39: 2969-2972
7．王玲，杜守英，钱玉昆．枸杞多糖的免疫调节研究进展．上海免疫学杂志，1995，15(2)：118-120
8．宫丽华，汪海霞，王先磊等．柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用．山东中医药大学学报，2002，26(2)：145-146
9．徐叔云，卞如濂，陈修．药理实验方法学(第3版)[M]．北京：人民卫生出版社，2002：1420-1455
10．宋锦平，钟萍，汪涛等．测定淋巴细胞增殖的MTS和XTT比色法的建立．细胞与分子免疫学杂志，2001，17(3)：292-295
11. de Barros C M, Andrade L R, Allodi S, et al. The hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma. J Biol Chem, 2007, 282 (3): 1615-1626
1．程肖蕊，李彦舫．食药俱佳植物——牛蒡．植物杂志，2001，(1)：10
2．魏瑞兰．牛蒡活性成分研究进展．中国中医药信息杂志，1997，4(5)：22
3. Oktay A., Mahmut E., Selma S., et al. Purification of mulberry (Morus alba L.) polyphenol oxidase by affinity chromatography and investigation of its kinetic and electrophoretic properties. Food Chemistry, 2004, 88 (3): 479-484
4. Lin S. C., Chung T. C., Lin C. C., et al. Hepatoprotective effects ofArctium Lappa on carbon tetrachloride- and acetaminophen-induced liver damage. American Journal of Chinese Medicine, 2000, 28 (2): 163-173
5. Lin S. C., Lin C. H., Lin C. C. et al. Hepatoprotective effects ofArctium lappa linne on liver injuries induced by chronic ethanol consumption and potentiated by carbon tetrachloride. Journal &Biomedical Science, 2002, 9 (5): 401-409
6. Duh P. D. Journal of the American Oil Chemists' Society, 1998, 75 (4): 455-461
7. ChowL. W., WangS. J., Duh P. D. Food Science, 1997, 24 (2): 195-202
8. Yoshihiko M., Jun K., Ryoya N. Journal of Agriculture and Food Chemistry, 1995, 43: 2592-2595
9. Ishimaru M., Kagoroku K., Chachin K. et al. Scientia Horticulturae,. 2004, 101: 1-10
10．郝林华，陈磊，仲娜等．牛蒡寡糖的分离纯化及结构研究．高等学校化学学报，2005，26(7)：1242-1247
11．董群，张志毅，林颖等．汉防己多糖的研究．生物化学与生物物理学报，1995，27(3)：261-265
12．张惟杰．糖复合物生化研究技术(第二版)[M]，杭州：浙江大学出版社，1999：12-13
13. Gorin P. A. J. Carbon-13 nuclear magnetic resonance spectroscopy of polysaccharides. Adv. Carbodydr. Chem. Biochem, 1981, 38: 13-39
14. Kardosova A, Ebringerova A, Alfoldi J, et al. A biologically active fructan from the roots ofArctium lappa L., var. Herkules. Int. J. Biol. Macromol., 2003, 33: 135-140
15. Chen X. M, Tian G. Y. Structural elucidation and antitumor activity of a fructan from Cyathula officinalis Kuan. Carbohydrate Research, 2003, 338: 1235-1241
1. Kuc J. Induced systemic resistance in plants to diseases caused by fungi and bacteria [M]. London: Bailey J A and Deverall B J(ed) The Dynamics of Host Defense. Academic Press Inc, 1983, 191-221
2. Ishiba C, Tani T, Murata M. Protection of cucumber against anthracnose by hypovilurent strain of Fusarium oxysporum f. sp. Cucumerium. Annual Phytopathological Society of Japan. 1981, 47: 352-359
3. Yan Zhi-nong, Reddy M S, Choong-Min Ryu, et al. Induced systemic protection against tomato late blight elicited by plant-promoting rhizobacteria. Phytopathology, 2002, 92: 1329-1333
4. Dong He-zhong, Li Wei-jiang, Zhang Dong-mei, et al. Differential expression of induced resistance by an aqueous extract of killed Penicillium chrysogenum against Verticilliun wilt of cotton. Crop Protection, 2003, 22: 129-134
5. Koike N, Hyakumachi M, Kageyama K, et al. Induction of systemic resistance in cucumber against several diseases by plant growth-promoting fungi: lignification and superoxide generation. European Journal of Plant Pathology, 2001, 107: 523-533
6．岳东霞，张要武，庄勇等．酵母胞壁多糖对黄瓜植株防御酶系活性的影响．华北农学报，2004，19(1)：37-39
7．李默怡，余龙江，陈超等．黑曲霉及其多糖组分诱导红豆杉叶子产生抗病反应之比较．植物研究，2003，23(1)：72-76
8．郭敏，柳春燕，陈靠山等．拟康氏木霉对几种病原菌的拮抗作用及对番茄灰霉病的防效．中国生物防治，2005(增刊)
9．郭敏，石磊，陈靠山．拟康氏木霉胞外多糖对番茄灰霉病的诱抗作用及对几种防御酶活性的影响．曲阜师范大学学报，2005，31(2)：99-102
10．董群，张志毅，林颖等．汉防己多糖的研究．生物化学与生物物理学报，1995，27(3)：261-265
11. Delgobo C L, Gorin P A J, Tischer C A, et al. The free reducing oligosaccharides of angico branco (Anadenanthera colubrina) gum exudate: an aid for structural assignments in the heteropolysaccharide. Carbohydr Res, 1999, 320: 167-175
12. Miguel A R C, Catherine H P, Karim M, et al. The three-dimensional structure of the mega-oligosaccharide rhamnogalacturonan Ⅱ monomer: a combined molecular modeling and NMR investigation. Carbohydr Res, 2003, 338: 651-671
13. Harding L P, Marshall V M, Elvin M, et al. Structural characterization of a perdeuteriomethylated exopolysaccharide by NMR spectroscopy: characterization of the novel exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus EU23. Carbohydr Res, 2003, 338: 61-67
2. Zhang J P, Qian D H. Antitumor activity and tumor necrosis factor production of phytolacca acinosa polysaccharides Ⅰ in mice. Acta pharmacologica Sinica, 1993, 14 (6): 542-545
3. Duan J Y, Wang X S, Dong Q, et al. Structural features of a pectic arabinogalactan with immunological activity from the leaves of Diospyros kaki. Carbohydr Res, 2003, 338: 1291-1297
4．丁保金，金丽琴，吕建新．多糖的生物活性研究进展．中国药学杂志，2004，39(8)：561-564
5．王玲，李俊，李欣等．枸杞多糖2对辐射损伤小鼠免疫功能恢复的影响．上海疫学杂志，1995，15(4)：209-211
6. Kiho T, Ookubo K, Usui S, et al. Structural features and hypoglycemic activity of a polysaccharide (CS-F10) from the cultured mycelium of Cordyceps sinensis. Biol Pharm Bull, 1999, 22 (9): 966-970
7．赵国华，陈宗道，李志孝等．活性多糖的研究进展．食品与发酵工业，2002，27(7)：45-48
8. Kenned Y J F, White C A. Bioactive carbohydrates[M]. Chichester: Ellis Horwood, Ltd, 1993
9．吴东儒．糖类的生物化学[M]．北京：高等教育出版社，1987
10．张惟杰．糖复合物生化研究技术[M]．杭州：浙江大学出版社，1999
11．来鲁华，杨昱婷．寡糖的构象分析．生物化学与生物物理进展，1995，22(4)：290-294
12. Peters T, Meyer B, Stuike-Prill R, et al. A Monte Carlo method for conformational analysis of saccharides. Carbohydr Res, 1993, 238: 49-73
13. Imberty A, Pérez S, Milos H, et al. Flexibility in a tetrasaccharide fragment from the high mannose type of N-linked oligosaccharides. 1993, 15: 17-23
14．方积年．多糖体的结构分析．国外医学·药学分册，1981，7(4)：222-228
15. Rolf S. A chemically bonded stationary phase for carbohydrate analysis in liquid chromatography. J Chromatogr A, 1976: 206-210
16. Bradbury J H, Jenkins G A. Determination of the structures of trisaccharides by ~(13)C-NMR spectroscopy. Carbohydr Res, 1984, 126: 125-156
17．王展，方积年．高场核磁共振波谱在多糖结构研究中的应用．分析化学，2000，28(2)：240-247
18．方一苇．糖结构与质谱分析．质谱学报，1995，16(1)：1-8
19．蒋可，Burl．A L．糖复合物中糖链部分质谱测定．化学通报，1990，(6)：30-38
20. Yamamoto K. Microbial endoglycosidases for analyses of oligosaccharide chains in glycoproteins. J Biochem (Tokyo), 1994, 116: 229-235
21. Edge C J, Rademacher T W, Wormald M R. Fast sequencing of oligosaccharides: the reagent-array analysis method. Proc Natl Acad Sci U S A, 1992, 89(14): 6338-6342
22．王顺春，方积年．X-射线纤维衍射在多糖构型分析中应用的研究进展．天然产物研究与开发，2000，12(2)：75-80
23. McIntire T M, Penner R M, Brant D A. Observations of a circular, triple-helical polysaccharide using noncontact atomic force microscopy. Macromolecules, 1995, 28: 6375-6377
24. Rice K G, Wu P, Brand L, et al. Interterminal distance and flexibility of a triantennary glycopeptide as measured by resonance energy transfer. Biochemistry, 1991, 30: 6646-6655
25. Janaswamy S, Chandrasekaran R. Polysaccharide structures from powder diffraction data: molecular models of arabinan. Carbohydr Res, 2005, 340: 835-839
26. Tao Y Z, Zhang L A, Cheung P C K. Physicochemical properties and antitumor activities of water-soluble native and sulfated hyperbranched mushroom polysaccharides. Carbohydr Res, 2006, 341: 2261-2269
27. Kim H M, Han S B, Oh G T, et al. Stimulation of humoral and cell mediated immunity by polysaccharide from mushroom Phellinus linteus. Int J Immunopharamacol, 1996, 18 (5): 295-303
28. Sakamoto Y, Watanabe H, Nagai M, et al. Lentinula edodes tlgl encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence. Plant Physiol. 2006, 141: 793-801
29．林志彬．灵芝多糖的免疫药理学研究及其意义．北京医科大学学报，1992，24(4)：271-274
30. Chinnah A D, Baig M A, Tizard I R, et al. Antigen dependent adjuvant activity of a polysaceharide β-(1, 4)-linked acetylated mannan (acemannan). Vaccine, 1992, 10 (8): 551-557
31. Duan J Y, Zheng Y, Dong Q, et al. Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki. Phytochemistry, 2004, 65: 609-615
32. Luettig B, Steinmuller C, Gifford G E, et al. Macrophage activation by the polysaccharide arabinogalactan-isolated from" plant cell cultures of Echinacea purpurea. J Natl Cancer Inst, 1989, 81 (9): 669-675
33. Esua M F, Rauwald J W. Novel bioactive maloyl glucans from Aloe vera gel: isolation, structure elucidation and in vitro bioassays. Carbohydr Res, 2006, 341: 355-364
34. Kimura Y, Sumiyoshi M, Suzuki T, et al. Antitumor and antimetastatic activity of a novel water-soluble low molecular weight beta-1, 3-D-glucan (branch beta-1, 6) isolated from Aureobasidium pullulans 1A1 strain black yeast. Anticancer Res, 2006, 26: 4131-4141
35. Gao H, Wang F, Lien E J, et al. Immunostimulating polysaccharides from Panax notoginseng. Pharm Res, 1996, 13 (8): 1196-1200
36．关玉峰．人参、黄芪多糖对免疫功能影响的临床实验研究．航空航天医药，1997，8(2)：4-6
37. Liu F, Fung M C, Ooi V E C, et al. Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complexes. Life Sciences, 1996, 58 (21): 1795-1803
38. Tokunaka K, Ohno N, Adachi Y, et al. Immunopharmacological and immunoto-xicological activities of a water-soluble (1→3)-β-D-glucan, CSBG from Candida spp. Int J Immunopharmacol, 2000, 22 (5): 383-394
39．李宗锴，李电东．牛膝多糖的免疫调节作用．药学学报，1997，32(12)：881-887
40. Czop J K, Austen K F. Properties of glycans that activate the human alternative complement pathway and interact with the human monocyte beta-glucan receptor. J Immunol, 1985, 135: 3388-3393
41. Krizková L, Duracková Z, Sandula J, et al. Antioxidative and antimutagenic activity of yeast cell wall mannans in vitro. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2001, 497: 213-222
42. Olafsdottir E S, Ingólfsdottir K. Polysaccharides from lichens: structural characteristics and biological activity. Planta Med, 2001, 67 (3): 199-208
43. Luettig B, Steinmuller C, Gifford G E, et al. Macrophage activation by the polysaccharide arabinogalactan isolated from plant cell cultures of Echinacea purpurea. J Natl Cancer Inst, 1989, 81 (9): 669-675
44. Kiyohara H, Takemoto N, Zhao J F, et al. Pectic polysaccharides from roots of Glycyrrhiza uralensis: possible contribution of neutral oligosaccharides in the galacturonase-resistant region to anti-complementary and mitogenic activities. Planta Med, 1996, 62 (1): 14-19
45. Kiyohara H, Matsumoto T, Nagai T, et al. The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines. Phytomedicine, 2006, 13: 494-500
46. Mizumoto K, Sugawara I, Ito W, et al. Sulfated homopolysaccharides with immunomodulating activities are more potent anti-HTLV-III agents than sulfated heteropolysaccharides. Jpn J Exp Med, 1988, 58 (3): 145-151
47. Vactor D V, Wall D P, Johnson K G. Heparan sulfate proteoglycans and the emergence of neuronal connectivity. Current Opinion in Neurobiology, 2006, 16: 40-51
48. Pramanik M, Mondal S, Chakraborty I, et al. Structural investigation of a polysaccharide (Fr. II) isolated from the aqueous extract of an edible mushroom, Pleurotus sajor-caju. Carbohydr Res, 2005,340: 629-636
49. Cleary J A, Kelly G E, Husband A J. The effect of molecular weight and β-I, 6-linkages on priming of macrophage function in mice by (1, 3)-β-D-glucan. Immunol Cell Biol, 1999, 77 (5): 395-403
50. Demleitner S, Kraus J, Franz G. Synthesis and antitumour activity of derivatives of curdlan and lichenan branched at C-6. 1992,226: 239-246
51. 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 leukemic U937 cells. Anti-Cancer Drugs, 2001,12: 841-846
52. Misaki A, Kawaguchi K, Miyaji H, et al. Structure of pestalotan, a highly branched (1→3)-β-D-glucan elaborated by Pestalotia sp. 815, and the enhancement of its antitumor activity by polyol modification of the side chains. Carbohydr Res, 1984, 129: 209-227
53. S.one Y, Misaki A, Shibata S. Preparation and characterization of antibodies against 6-O-α-D-xylopyranosyl-β-D-glucopyranose (β-isoprimeverose), the disaccharide unit of xyloglucan in plant cell-walls. Carbohydr Res, 1989, 191: 79-89
54. Groth T, Wagenknecht W. Anticoagulant potential of regioselective derivatized cellulose. Biomaterials, 2001, 22: 2719-2729
55. Bohn J A, BeMiller J N. (1→3)-β-D-Glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohydr Polymer, 1995, 28: 3-14
56. Yang J, Du Y, Huang R, et al. Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides. Int J Biol Macro, 2002, 31: 55-62
57. Sarnuelsen A B, Paulsen B S, Wold J K, et al. Characterization of a biologically active pectin from Plantago major L. Carbohydr Polymer, 1996, 30: 37-44
58. Mulloy B, Mourao P A S, Gray E. Structure/function studies of anticoagulant sulphated polysaccharides using NMR. J Biotech, 2000, 77: 123-135
59. Matsuzaki K, Yamamoto I, Sato T, et al. ~(13)C-NMR investigations of synthetic branched polysaccharides. Carbohydr Polymer, 1986, 6: 155-163
60. Perret J, Bruneteau M, Michel G, et al. Effect of growth conditions on the structure of β-D-glucans from Phytophthora parasitica dastur, a phytopathogenic fungus. Carbohydr Polymer, 1992, 17: 231-236
61．王长云，管华诗．多糖抗病毒作用研究进展Ⅱ．硫酸多糖抗病毒作用．生物工程进展，2000，20(2)：3-8
62. Caducei M J, Pujol C A, Ciancia M, et al. Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: correlation between structure and biological activity. Int J Biol Macro, 1997, 20: 97-105
63．诸葛健，赵振锋，方慧英．功能性多糖的构效关系．无锡轻工大学学报，2002，21(2)：209-212
64. Uryu T, Ikushima N, Katsuraya K, et al. Sulfated alkyl oligosaccharides with potent inhibitory effects on human immunodeficiency virus infection. Biochem Pharmacol, 1992, 43: 2385-2392
65．张健，阳庚元．羟乙基化牛膝多糖的合成及其活性研究，化学学报，2003，61(10)：1692-1696
66．陈惠黎，王克夷．糖复合物的结构与功能[M]，上海：上海医科大学出版社，1997
67. Ohno N, Miura T, Miura N N, et al. Structure and biological activities of hypochlorite oxidized zymosan. Carbohydr Polymer, 2001, 44: 339-349
68. Zhang P, Zhang L, Cheng S. Effects of urea and sodium hydroxide on the molecular weight and conformation of α-(1→3)-D-glucan from Lentinus edodes in aqueous solution. Carbohydr Res, 2000, 327: 431-438
69. Maeda Y Y, Hamuro J, Chihara G. The mechanisms of action of anti-tumour polysaccharides. I. The effects of antilymphocyte serum on the anti-tumour activity oflentinan. Int J Cancer, 1971, 8 (1): 41-46
70. Saito H, Yoshioka Y, Uehara N, et al. Relationship between conformation and biological response for (1→3)-β-D-glucans in the activation of coagulation Factor G from limulus amebocyte lysate and host-mediated antitumor activity. Demonstration of single-helix conformation as a stimulant. Carbohydr Res, 1991, 217: 181-190
71. Alban S, Franz G. Characterization of the anticoagulant actions of a semisynthetic curdlan sulfate. Thromb Res, 2000, 99: 377-388
72. Gao Y, Fukuda A, Katsuraya K, et al. Synthesis of regioselective substituted curdlan sulfates with medium molecular weights and their specific anti-HIV-1 activities. Macromolecules, 1997, 30: 3224-3228
73. Calazans G M T, Lima R C, Franca F P, et al. Molecular weight and antitumour activity of Zymomonas mobilis levans. Int J Biol Macro, 2000, 27: 245-247
74. Dong Q, Jia L M, Fang J N. A β-D-glucan isolated from the fruiting bodies of Hericium erinaceus and its aqueous conformation. Carbohydr Res, 2006, 341: 791-795
75．田庚元，冯宇澄．多糖类免疫调节剂的研究和应用．化学进展，1994，6(2)：114-124
76. Sandula J, Kogan G, Kacuráková M, et al. Microbial (1→3)-β-D-glucans, their preparation, physicochemical characterization and immunomodulatory activity. Carbohydr Polymer, 2006, 38: 247-253
77．郑建仙．功能性食品[M]，北京：中国轻工业出版社，2002
78. Tabata K, Ito W, Kojima T, et al. Ultrasonic degradation of schizophyllan, an antitumor polysaccharide produced by Schizophyllum commune fries. Carbohydr Res, 1981, 89: 121-135
79. Zhao J F, Kiyohara H, Yamada H, et al. Heterogeneity and characterisation of mitogenic and anti-complementary pectic polysaccharides from the roots of Glycyrrhiza uralensis Fisch et D. C. Carbohydr Res, 1991, 219: 149-172
80. Kiyohara H, Takemoto N, Zhao J F, et al. Pectic polysaccharides from roots of Glycyrrhiza uralensis: possible contribution of neutral oligosaccharides in the galacturonase-resistant region to anti-complementary and mitogenic activities. Planta Med, 2006, 72: 14-19
81. Yu K W, Kiyohara H, Matsumoto T, et al. Intestinal immune system modulating polysaccharides from rhizomes of Atractylodes lancea. Planta Med, 1998, 64: 714-719
82．宁君，孔繁祚．具有重要生理活性寡糖的发现及应用．世界科技研究与发展，2001，23(6)：22-25
1．熊文愈，汪计珠，石同岱等．中国木本药用植物[M]，上海：上海科学教育出版社，1993：85-88
2．江苏新医学院．中药大辞典(下册)[M]，上海：上海科学技术出版社，1995：1502-1503
3．程剑华，李以镔．抗癌植物药及其验方[M]，南昌：江西科学技术出版社，1998：62-64
4．薛芳，许占民．中国药物大全(中药卷)，第2版[M]，北京：人民卫生出版社，1998：50-54
5. Fujimoto T, Hano Y, Nomura T. Components of root bark of Cudrania tricuspidata 1. Structures of four new isoPrenylated xanthones, Cudraxanthones A, B, CandD. Planta Medica, 1984, 50 (3): 218-221
6. Hano Y, Matsumoto Y, Sun J Y, et al. Structures of three new isoprenylated xanthones, Cudraxanthones E, F and G. Planta Medica, 1990, 56 (4): 399-402
7. Hano Y, Matsumoto Y, Sun J Y, et al. Structures of four new isoprenylated xanthones, Cudraxanthones H, I, J and K. Planta Medica, 1990, 56 (5): 478-481
8. Hano Y, Matsumoto Y, Shimnohara K, et al. Structures of four new isoprenylated xanthones, Cudraxanthones L, M, N and O from Cudrania tricuspidata. Planta Medica, 1991, 57 (2): 173-175
9. Fujimoto T, Hano Y, Nomura T, et al. Components of root bark of Cudrania trieuspidata 2. Structures of two new isoprenylated flavones, Cudraflavones A andB. Planta Medica, 1984, 50 (2): 161-163
10. Hano Y, Matsumoto Y, Shimnohara K, et al. Cudraflavones C and D, two new prenylflavones from the root bark of Cudrania tricuspidata (Carr.) Bur. Heterocycles, 1990, 31 (7): 1339-1344
11. Fujimoto T, Nomura T. Components of root bark of Cudrania tricuspidata. Isolation and structure studies on the flavonoids. Planta Medica, 1985, 51 (3): 190-193
12. Lee I K, Kim C J, Song K S, et al. Two benzylated dihydroflavonols from Cudrania tricuspidata. Journal of Natural Products, 1995, 58 (10): 1614-1617
13. Lee I K, Kim C J, Song K S, et al. Cytotoxic benzyl dihydroflavonols from Cudrania tricuspidata. Phytochemistry, 1996, 41 (1): 213-216
14. Chang C H, Lin C C, Kadota S, et al. Flavonoids and a prenylated xanthone from Cudrania cochinchinensis var. gerontogea. Phytoehemistry, 1995, 40 (3): 945-947
15．戴明，野村太郎．柘木成分研究．国外医学中医中药分册，1983，4：53-54
16．张月华，任婉薇，万树文等．柘木的化学成分研究．医药工业，1980，3：15-20
17．缪春辉，顾正兵，杨根金．柘木化学成分的研究．中成药，2002，24(3)：211-212
18．史琪荣，柳润辉，徐希科等．柘木黄酮类成分研究．中国中药杂志，2006，31(1)：77-78
19. Zheng Z P, Liang J Y, Hu L H. Water-soluble constituents of Cudrania tricuspidata (Carr.) Bur. J Integr Plant Biol, 48 (8), 996-1000
20．宫丽华，汪海霞，王先磊等．柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用．山东中医药大学学报，2002，26(2)：145-146
21．江苏新医学院．中药大辞典(下册)[M]，上海：上海科学技术出版社，1995：1731-1732
22．刘建华，高玉琼，霍昕．穿破石挥发性成分的研究．中国中药杂志，2003，28(11)：1047-1049
23．宋耐宝，杨学东，王义明等．柘木化学成分及药理活性研究现状．中成药，2005，27(3)：335-337
24．李贺然，邹忠梅，徐丽珍等．柘属药用植物化学和药理活性研究进展．国外医学中医中药分册，2003，25(4)：203-207
25. Mufti V V S, Seshadri T R, Srvakumaran S. Phytochemistry, 1972, 11 (1): 2089-2092
26. Chang C H, Lin C C, Hattori M, et al. Phytochemistry, 1989, 28 (2): 595-598
27. Chang C H, Lin C C, Kawata Y, et al. Phytochemistry, 1989, 28(10): 2823-2826
28. Hou A J, Fukai, T, Shimazaki M, et al. Benzophenones and xanthones with isoprenoid groups from Cudrania cochinchinensis. Journal of Natural Products, 2001, 64 (1): 65-70
29. Knapp JE, SchiffP L. J Pharm Sci, 1971, 60 (11): 1729-1730
30. Nomura T, Hano Y, Fujimoto T. Three new isoprenylated xanthones, cudraxanthone A, B, C from the root of Cudrania tricuspidata (Carr.) Bur. Heterocycles, 1983, 20 (2): 213-218
31. Chang C H, Lin C C, Hattori M, et al. Journal of Ethnopharmacology, 1994, 44 (1): 79-85
32. Sun NJ, Chang CJ, John MC. Phytochemistry, 1988, 27(3): 951-952
33．柘木糖浆-上海中药制药二厂．中草药通讯，1977，(12)：18
34．曹根生．抗肿瘤新药．柘木注射液．中成药研究，1980，(1)：34-35
35．丁红华，陈栋晖，朱莉菲等．柘木糖浆治疗胃癌疗效观察．中成药，2001，23(2)：151-152
36．杜卫东，沈达明，宋晓华等．柘木糖浆对胃肠道肿瘤手术后化疗病人肿瘤标记物和细胞免疫的影响．临床医药杂志，2004，17(3)：11-14
37．王佩芳，沈辉，唐红敏等．柘木糖浆对胃癌前病变阻断逆转作用的疗效观察．中华医学实践杂志，2003，2(1)：57-58
38．孙菁，袁耀宗，郑舜华等．柘木糖浆治疗活动期慢性胃炎及胃黏膜肠腺化生的疗效观察．中成药，2005，27(5)：12-14
39．毕盛．抗癌中成药-生命之星[P]．中国专利：A61K35／78，1996-03-13
40．王贞丽，姜自彬，钱月中．植物抗肿瘤活性成分研究进展．前卫医药杂志，1999，1：62-63
41．徐誉泰，张可炜，李艳等．柘树黄酮对胃癌细胞株NKM大分子合成的影响．中医药学报，1998，26(5)：47-48
42．张英慧，徐誉泰．柘木根黄酮注射液对小鼠S180肿瘤的抑制作用．佛山科学技术学院学报(自然科学版)，2001，19(3)：75-77
43. Seo W G, Pae H O, Oh G S, et al. Ethyl acetate extract of the stem bark of Cudrania tricuspidata induces apoptosis in human leukemia HL-60 cells. Am J Chin Med, 2001, 29 (2): 313-320
44．张聪敏，萧丽．对柘木黄酮引起细胞凋亡及其机制的初步研究．漳州师范学院学报(自然科学版)，2004，17(4)：98-101
45. Zou Y S, Hou A J, Zhu G F, et al. Cytotoxic isoprenylated xanthones from Cudrania tricuspidata. Bioorganic & Medicinal Chemistry, 2004, 12: 1947-1953
46．陈良儿，谢振家．柘树茎乙醇提取液的抗炎镇痛作用．南京军医学院学报，2002，24(1)：11-13
47．张可炜，徐誉泰，张举仁．银杏叶和柘树提取物的抗氧化作用．山东大学学 报(自然科学版)，2000，35(4)：469-472
48. Cha J Y, Kim H J, Chung C H, et al. Antioxidative activities and contents of polyphenolic compound of Cudrania tricuspidata. Korean Soci F Sci Nutr, 1999, 28 (6): 1310-1315
49. Lee B W, Lee J H, Lee S T, et al. Antioxidant and cytotoxic activities of xanthones from Cudrania tricuspidata. Bioorganic & Medicinal Chemistry Letters, 2005, 15: 5548-5552
50．何泉华，许实波．穿心草口山酮抗氧化作用初探．中国药理学通报，1998，14(2)：130-132
51．何泉华，许实波．口山酮对老年小鼠抗氧化酶活性的影响．广州医学院学报，1999，27(4)：6-9
52. Cho E J, Yokozawa T, Rhyu D Y, et al. The inhibitory effects of 12 medicinal plants and their component compounds on lipid peroxidation. Am J Chin Med, 2003, 31 (6): 907-917
53．王永中，郑良朴，王瑞国．穿破石对小鼠CCl_4急性肝损伤保护作用的实验观察．福建中医药，2004，35(5)：44-46
54．吕强，许国祥．柘木抗结核作用的初步研究．药学通报，1980，15(12)：36
55. Kang D G, Hur T Y, Lee G M, et al. Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension. Life Sciences, 2002, 70 (22): 2599-2609
56. Seo W G, Pae H O, Oh G S, et al. Inhibitory effect of ethyl acetate fraction from Cudrania tricuspidata on the expression of nitric oxide synthase gene in RAW 264.7 macrophages stimulated with interferon-γ and lipopolysaccharide. General Pharmacology, 2001, 35: 21-28
57．孙琳，刘爱芬，李义召等．脑缺血再灌注后热休克蛋白70、c-fos的表达及柘树制剂的神经保护作用．临床神经病学杂志，2005，18(5)：354-356
58．刘福海，常云水，李志成．柘树根皮膏治疗骨折767例临床报告．山东医药，1979，(7)：41
1．宫丽华，汪海霞，王先磊等．柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用．山东中医药大学学报，2002，26(2)：145-146
2. Alsop R M, Vlachogiarmis GJ. Determination of the molecular weight of clinical dextran by gel chromatography on TSK PW type columns. J Chromatogr, 1982, 246: 227-240
3．魏远安，方积年．高效凝胶渗透色谱法测定多糖纯度及分子量．药学学报，1989，24(7)：532-536
4. Dubois M, Gilles K A, Hamilton J K, et al. Colorimetric method for determination of sugars and related substances. Anal Chem, 1956, 28: 350
5. Blumencrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal Biochem, 1973, 54: 484-490
6. Bensadon A, Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem, 1976, 70: 241-250
7. Blakeney A B, Harris P J, Henry R J, et al. A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydr Res, 1983, 113: 291-299
8．张惟杰．糖复合物生化研究技术(第2版)[M]．杭州：浙江大学出版社，2003：23
9. Taylor R L, Conrad H E. Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of the carbodiimide-activated carboxyl groups. Biochemistry, 1972, 11: 1383-1388
10. Needs P W, Selvendran R R. Avoiding oxidative degradation during sodium hydroxide/methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide. Carbohydr Res, 1993, 245: 1-12
11. Ciucanu I, Kerek F. A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res, 1984, 131, 209-217
12．董群，张志毅，林颖等．汉防己多糖的研究．生物化学与生物物理学报，1995，27(3)：261-265
13. Redgwell R J, Curti D, Fischer M, et al. Coffee bean arabinogalactans: acidic polymers covalently linked to protein. Carbohydr Res, 2002, 337: 239-253
14．陈伟，林新华，黄丽英等．芦荟多糖中游离蛋白去除的分光光度法．光谱实验室，2005，22(1)：113-115
15．刘成梅，万茵，涂宗财等．百合多糖脱蛋白方法的研究．食品科学，2002，23(1)：89-90
16．酶法脱蛋白技术用于螺旋藻多糖提取工艺的研究．食品与发酵工业，1998，24(3)：24-28
17．张惟杰．糖复合物生化研究技术(第2版)[M]．杭州：浙江大学出版社，2003：194
18．张惟杰．糖复合物生化研究技术(第2版)[M]．杭州：浙江大学出版社，2003：196
19. Gorin R A. J. Carbon-13 nuclear magnetic resonance spectroscopy of polysaccharides. Adv. Carbodydr. Chem. Biochem, 1981, 38: 13-39
20. Wang Xue-Song, Dong Qun, Zuo Jian-Ping, et al. Structure and potential immunological activity of a pectin from Centella asiatica (L.) Urban. Carbohydr. Res, 2003, 338: 2393-2402
21．王镜岩，朱圣庚，徐长法．生物化学(第三版)[M]．北京：高等教育出版社，2002：42-43
22. Strasser G R, Amado R. Pectic substances from red beet (Beta vulgaris conditiva). Part Ⅰ. Structural analysis of rhamnogalacturonan I using enzymic degradation and methylation analysis. Carbohydr Polym, 200.1, 44: 63-70
23. Pinto G L D, Martinez M, Mendoza J A, et al. Structural studies of the polysaccharide isolated from Spondias purpurea gum exudates. Carbohydr Res, 1996, 290: 97-103
24. Pinto G L D, Martinez M, Corredor A L D, et al. Chemical and ~(13)C NMR studies of Enterolobium cyclocarpum gum and in degradation products. Phytochemistry, 1994, 37: 1311-1315
25. Shimizu N, Tomoda M, Kanari M, et al. An acidic polysaccharide having activity on the reticuloendothelial system from the root ofAstragalus mongholicus. Chem Pharm Bull, 1991, 39: 2969-2972
26. An J, O'Neill M A, Albersherm P, et al. Isolation and structural characterization of β-D-glucosyluronic acid and 4-O-methyl β-D-glucosyluronic acid-containing oligosaccharides from the cell-wall pectic polysaccharide, rhamnogalacturonan Ⅰ. Carbohydr Res, 1994, 252: 235-245
27. Cui W W, Eskin M N A, Biliaderis C G, et al. NMR characterization of a 4-O-methyl-β-D-glucuronic acid containing rhamnogalacturonan from yellow mustard (Sinapis alba L.) mucilage. Carbohydr Res, 1996, 292: 173-183
28. Rauvala H, Finne J, Krusius T, et al. Methylation techniques in the structural analysis of glycoproteins and glycolipids. Advances in carbohydrate chemistry and biochemistry, 1981: 389-416
29．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)：60-65
30. Kim J S, Reuhs B L, Michon F, et al. Addition of glycerol for improved methylation linkage analysis of polysaccharides. Carbohydr Res, 2006, 341: 1061-1064
1. Liu F, Fung M C, Ooi V E C, et al. Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide-protein complex. Life Sci, 1996, 58: 1795-1803
2. Yamaoka Y, Kawakita T, Kaneko M, et al. A polysaccharide fraction of shosaiko-to active in augmentation of natural killer activity by oral administration. Biol Pharm Bull, 1995, 18(6): 846-849
3. Oka S, Ohno N, Iwanaga S, et al. Characterization of mitogenic substances in the hot water extracts of bupleuri radix. Biol Pharm Bull, 1995, 18: 757-765
4. Kim K H, Lee Y S, Jung I S, et al. Acid polysaccharide from Pangxginseng, ginsan, induces TH1 cell and macrophage cytokines and generates LAK cells in synergy with rIL-2. Planta Med, 1998, 64: 110-115
5. Ohtani K, Mizutani S, Hatono S, et al. Sanchinan-A, a reticuloendothelial system activating arabinogalactan from sanchi-ginseng (roots of Panax notoginseng). Planta Med, 1987, 53: 166-169
6. Shimizu N, Tomoda M, Kanari M, et al. An acidic polysaccharide having activity on the reticuloendothelial system from the root of Astragalus mongholicus. Chem Pharm Bull, 1991, 39: 2969-2972
7．王玲，杜守英，钱玉昆．枸杞多糖的免疫调节研究进展．上海免疫学杂志，1995，15(2)：118-120
8．宫丽华，汪海霞，王先磊等．柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用．山东中医药大学学报，2002，26(2)：145-146
9．徐叔云，卞如濂，陈修．药理实验方法学(第3版)[M]．北京：人民卫生出版社，2002：1420-1455
10．宋锦平，钟萍，汪涛等．测定淋巴细胞增殖的MTS和XTT比色法的建立．细胞与分子免疫学杂志，2001，17(3)：292-295
11. de Barros C M, Andrade L R, Allodi S, et al. The hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma. J Biol Chem, 2007, 282 (3): 1615-1626
1．程肖蕊，李彦舫．食药俱佳植物——牛蒡．植物杂志，2001，(1)：10
2．魏瑞兰．牛蒡活性成分研究进展．中国中医药信息杂志，1997，4(5)：22
3. Oktay A., Mahmut E., Selma S., et al. Purification of mulberry (Morus alba L.) polyphenol oxidase by affinity chromatography and investigation of its kinetic and electrophoretic properties. Food Chemistry, 2004, 88 (3): 479-484
4. Lin S. C., Chung T. C., Lin C. C., et al. Hepatoprotective effects ofArctium Lappa on carbon tetrachloride- and acetaminophen-induced liver damage. American Journal of Chinese Medicine, 2000, 28 (2): 163-173
5. Lin S. C., Lin C. H., Lin C. C. et al. Hepatoprotective effects ofArctium lappa linne on liver injuries induced by chronic ethanol consumption and potentiated by carbon tetrachloride. Journal &Biomedical Science, 2002, 9 (5): 401-409
6. Duh P. D. Journal of the American Oil Chemists' Society, 1998, 75 (4): 455-461
7. ChowL. W., WangS. J., Duh P. D. Food Science, 1997, 24 (2): 195-202
8. Yoshihiko M., Jun K., Ryoya N. Journal of Agriculture and Food Chemistry, 1995, 43: 2592-2595
9. Ishimaru M., Kagoroku K., Chachin K. et al. Scientia Horticulturae,. 2004, 101: 1-10
10．郝林华，陈磊，仲娜等．牛蒡寡糖的分离纯化及结构研究．高等学校化学学报，2005，26(7)：1242-1247
11．董群，张志毅，林颖等．汉防己多糖的研究．生物化学与生物物理学报，1995，27(3)：261-265
12．张惟杰．糖复合物生化研究技术(第二版)[M]，杭州：浙江大学出版社，1999：12-13
13. Gorin P. A. J. Carbon-13 nuclear magnetic resonance spectroscopy of polysaccharides. Adv. Carbodydr. Chem. Biochem, 1981, 38: 13-39
14. Kardosova A, Ebringerova A, Alfoldi J, et al. A biologically active fructan from the roots ofArctium lappa L., var. Herkules. Int. J. Biol. Macromol., 2003, 33: 135-140
15. Chen X. M, Tian G. Y. Structural elucidation and antitumor activity of a fructan from Cyathula officinalis Kuan. Carbohydrate Research, 2003, 338: 1235-1241
1. Kuc J. Induced systemic resistance in plants to diseases caused by fungi and bacteria [M]. London: Bailey J A and Deverall B J(ed) The Dynamics of Host Defense. Academic Press Inc, 1983, 191-221
2. Ishiba C, Tani T, Murata M. Protection of cucumber against anthracnose by hypovilurent strain of Fusarium oxysporum f. sp. Cucumerium. Annual Phytopathological Society of Japan. 1981, 47: 352-359
3. Yan Zhi-nong, Reddy M S, Choong-Min Ryu, et al. Induced systemic protection against tomato late blight elicited by plant-promoting rhizobacteria. Phytopathology, 2002, 92: 1329-1333
4. Dong He-zhong, Li Wei-jiang, Zhang Dong-mei, et al. Differential expression of induced resistance by an aqueous extract of killed Penicillium chrysogenum against Verticilliun wilt of cotton. Crop Protection, 2003, 22: 129-134
5. Koike N, Hyakumachi M, Kageyama K, et al. Induction of systemic resistance in cucumber against several diseases by plant growth-promoting fungi: lignification and superoxide generation. European Journal of Plant Pathology, 2001, 107: 523-533
6．岳东霞，张要武，庄勇等．酵母胞壁多糖对黄瓜植株防御酶系活性的影响．华北农学报，2004，19(1)：37-39
7．李默怡，余龙江，陈超等．黑曲霉及其多糖组分诱导红豆杉叶子产生抗病反应之比较．植物研究，2003，23(1)：72-76
8．郭敏，柳春燕，陈靠山等．拟康氏木霉对几种病原菌的拮抗作用及对番茄灰霉病的防效．中国生物防治，2005(增刊)
9．郭敏，石磊，陈靠山．拟康氏木霉胞外多糖对番茄灰霉病的诱抗作用及对几种防御酶活性的影响．曲阜师范大学学报，2005，31(2)：99-102
10．董群，张志毅，林颖等．汉防己多糖的研究．生物化学与生物物理学报，1995，27(3)：261-265
11. Delgobo C L, Gorin P A J, Tischer C A, et al. The free reducing oligosaccharides of angico branco (Anadenanthera colubrina) gum exudate: an aid for structural assignments in the heteropolysaccharide. Carbohydr Res, 1999, 320: 167-175
12. Miguel A R C, Catherine H P, Karim M, et al. The three-dimensional structure of the mega-oligosaccharide rhamnogalacturonan Ⅱ monomer: a combined molecular modeling and NMR investigation. Carbohydr Res, 2003, 338: 651-671
13. Harding L P, Marshall V M, Elvin M, et al. Structural characterization of a perdeuteriomethylated exopolysaccharide by NMR spectroscopy: characterization of the novel exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus EU23. Carbohydr Res, 2003, 338: 61-67