论文总字数：21320字

摘 要

寡糖及多糖是中药中的重要活性成分,其活性与结构密切相关。甲基化分析是糖类构造研究的重要手段，它被普遍用于判定寡糖和多糖中单糖残基之间相连位点及糖苷键类型。主要过程为：首先将多糖中的所有自由羟基全部甲基化，进而酸水解成各甲基化的单糖，单糖再经还原、乙酰制备成可挥发的衍生物，最终用GC/MS检测甲基化衍生物的类别和数目。根据色谱出峰时间以及质谱信息可以推断出单糖残基的相连位点以及连接方式。中药苍术中的多糖及寡糖类成分具有良好的药理活性。本论文旨在利用甲基化分析对苍术中的寡糖及多糖类成分进行结构解析，通过分析多批苍术样品建立苍术中寡糖及多糖的甲基化特征图谱。实验结果表明，苍术多糖主要由阿拉伯糖，甘露糖，葡萄糖及半乳糖，且以1,2葡萄糖苷键为主链。几个产地苍术样品多糖结构共性较高，且炮制过程对于苍术中糖类成分结构影响较大。

关键词：甲基化；多糖；GC-MS;寡糖；糖谱

Saccharides mapping based on methylation analysis

Abstract：Oligosaccharide and polysaccharide are important bioactive components in Traditional Chinese Medicine, and its activity is closely related to the structure. Methylation analysis is an important method in the study of carbohydrate structure. It is commonly used to determine the type of glycosides of oligosaccharides and polysaccharides. Methylation analysis traditionally including permethylation- hydrolysis-reduction-acetylation, firstly all the free hydroxyl groups are methylated and then acidic hydrolysis into the methylated monosaccharides. The released monosaccharide was reducted and acetylation to prepare volatile derivatives and analyzed by GC/MS. According to the retention time and mass spectral, the type of glycosides can be identified. Polysaccharide and oligosaccharide in Rhizome Atractylodis have multiple pharmacological activities. In this study, methylation analysis was applied to characterize the structure of oligosaccharides and polysaccharides. According to apply the methylation analysis to different samples from different origins, the characteristic saccharides mapping of Rhizome Atractylodis was established. The results showed that oligosaccharides and polysaccharides in Rhizome Atractylodis were mainly composed of arabinose, mannose, glucose and galactose. 1,2 glucose was the backbone. The consistency of saccharides mapping of different origin samples was high. And processing procedure have significant influence to oligosaccharides and polysaccharides.

Key words: Methylation; polysaccharide; GC-MS; oligosaccharide; saccharides mapping

目 录

第一章 文献综述 1

1.1单糖分析 1

1.1.1 HPTLC 1

1.1.2 HPLC 1

1.1.3 GC 2

1.2 糖苷键类型 2

1.2.1甲基化 3

1.2.2 HPLC-MS 4

1.2.3 NMR 4

1.3 分子量分布 5

1.3.1 GPC 5

1.3.2 HPSEC-MALLSD 6

1.4 多糖质量控制概述 6

1.5 多糖质量控制方法 6

1.6 苍术 7

第二章 实验材料与方法 9

2.1 实验材料 9

2.1.1 实验仪器 9

2.1.2 实验试剂 10

2.2 实验方法 10

2.2.1 材料 10

2.2.2 甲基化反应 11

2.2.3 水解 12

2.2.4 还原 12

2.2.5 乙酰化 12

2.3 分析方法 12

第三章 结果与讨论 13

3.1 甲基化反应注意点 13

3.2甲基化方法的关键问题 13

3.3 结果与讨论 14

第四章 结论与展望 34

4.1 结论 34

4.2 展望 34

参考文献 35

致谢 37

第一章 文献综述

1.1单糖分析

单糖是组成寡糖及多糖的结构单元，组成糖对寡糖及多糖的活性有重要的影响。寡糖及多糖的单糖组成通常采用酸水解后结合色谱法分析的方法。每种色谱方法都有其优缺点，因此采用综合的方法对碳水化合物进行准确的分析是较常用的手段。

1.1.1 HPTLC

薄层色谱（TLC）是一种操作简便、分离迅速的色谱技术，它还能够同时分析多个样品、成本低等优点，已成为鉴别单糖、寡糖成分的常用方法^[1]。高效薄层色谱（high performance thin layer chromatography，简称HPTLC）是薄层色谱法的新进展^[2]。

高效薄层色谱由于吸附剂薄层是微粒且粒度分布狭窄，选择最佳的条件既能获得一定的分离度，又能得到最短的分析时间。

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