摘 要

中国自古都以银杏果作为入药的首选。本文通过HPLC-ELSD法检测银杏果仁粗多糖水解后的单糖组成，并用高分辨质谱检测银杏果仁和银杏叶中的生物活性成分差异和提取溶剂对提取产物的影响。在用一级质谱信号强度对比化合物浓度的同时，通过部分化合物的二级质谱图确认它的存在。具体工作内容和结果如下：

对银杏叶和银杏果仁以200 mg/mL的固液比，分别在70%甲醇，纯甲醇，纯乙腈提取后，混合物直接进行质谱检测，部分化合物在质谱软件的自动筛选下进行了二级质谱检测，通过与数据库中标准化合物的一级、二级质谱图比对，确定了单黄酮12种、双黄酮5种和氨基酸12种的存在。同时以误差绝对值小于10 ppm，信噪比大于8为筛选条件，筛选除了确定存在的化合物外可能含有的物质——单黄酮3种，双黄酮1种，萜内酯7种氨基酸5种。通过对一级质谱图信号的分析，可以发现银杏果仁中生物活性物质的种类和含量普遍低于银杏叶，其中金松双黄酮在全部银杏果仁提取物中均没检测到。

另外，通过对比三种提取剂对同一种物质提取后的质谱图分析可得，纯甲醇提取的产物种类和浓度最高，纯乙腈提取的产物少，其中70%甲醇提取银杏果的样品中5种双黄酮均没发现对应的峰，综上可以得出纯甲醇提取的效果最好。

对鼠李糖、木糖、果糖、甘露糖、阿拉伯糖、葡萄糖和半乳糖共7种单糖进行HPLC-ELSD法检测，对HPLC分离的色谱条件进行了选择优化，以乙腈：乙酸乙酯：水=60：30:10等度洗脱，柱温30℃，流速0.8 mL/min，20 min内可将7种单糖较好的分离，其分离度除最后一个峰（半乳糖）为1.194外，其余都在1.8以上。将本方法用于检测银杏果仁粗多糖在2 mol/mL TFA水解3h后的单糖组成成分，得出其组成成分为：葡萄糖、木糖和果糖。

关键词：银杏叶 银杏果仁 单糖 HPLC-ELSD法 高分辨率质谱

Abstract

Since ancient times, Ginkgo semen has been used as the first choice for medicine. In this paper, HPLC-ELSD method was used to detect the monosaccharide composition of crude polysaccharides after hydrolysis of Ginkgo biloba semens. High-resolution mass spectrometry was used to detect the difference of bioactive components in Ginkgo biloba semens and Ginkgo biloba leaves and the effect of extraction solvent on the extracted products. While comparing the signal intensity of the MS with the concentration of a compound, its presence was confirmed by the MS/MS of a portion of the compound. The specific work content and results are as follows:

Ginkgo biloba leaves and ginkgo biloba semens were extracted with 70% methanol, pure methanol, and pure acetonitrile at a solid-liquid ratio of 200 mg/mL, and the mixture was directly detected with MS. Some compounds were subjected to the MS/MS by automated mass spectrometry software. The MS and MS/MS of standard compounds in the database were compared to determine the presence of 12 kinds of flavonoids, 5 kinds of biflavones, and 12 kinds of amino acids. At the same time, the absolute value of the error was less than 10 ppm, and the signal to noise ratio was greater than 8 as screening conditions. The substances that may be contained in addition to the identified compounds were listed: 3 kind of flavonoids, 1 kind of biflavones, 5 kinds of alopecuronide and 7 kinds of amino acids. Through the analysis of the MS signal, it can be found that the type and content of bioactive substances in Ginkgo biloba semens are generally lower than Ginkgo biloba leaves, and gold pine biflavonoids are not detected in all Ginkgo biloba extracts.

In addition, by comparing the MS of the same substance extracted which are respectively extracted by the three solvents, it can be summarized that the pure methanol extract has the highest productivity of type and concentration, and the pure acetonitrile extract has less productivity. What’s more, the corresponding peaks of Sciadopitysin were not found in the samples of 70% methanol extracted ginkgo biloba semens, and it was concluded that the pure methanol extraction had the best effect.

7 kinds of monosaccharides such as rhamnose, xylose, fructose, mannose, arabinose,glucose and galactose were detected by HPLC-ELSD method. The chromatographic conditions for HPLC separation were optimized and selected to acetonitrile: ethyl acetate: water=60. : 30:10 isocratic elution, column temperature is 30 °C, flow rate is 0.8 mL/min. 7 kinds of monosaccharides can be better separated in 20 minutes, and the degree of separation except the last peak (galactose) is 1.194, the rest are above 1.8. This method was used to detect the monosaccharide composition of the crude polysaccharide of Ginkgo biloba L. after 3 h hydrolysis in 2 mol/mL TFA. Finally, we would conclude that the composition of the crude polysaccharide was: glucose, xylose and fructose.

Keywords：Ginkgo biloba leaves Ginkgo Semen monosaccharide HPLC-ELSD

high-resolution mass spectrometer

目录

摘 要 I

Abstract II

第1章 绪论 1

1.1 人体消化吸收机制与内服药物药效关系 1

1.1.1 人体的消化机制 1

1.1.2 人体对银杏主要活性物质的吸收 2

1.2 银杏生物活性物质药理研究及其活性与毒性问题 2

1.2.1 黄酮类化合物 3

1.2.2 萜内酯 6

1.2.3 非淀粉多糖、蛋白质及氨基酸 7

1.2.4 酚酸类化合物 10

1.3 溶剂提取法简介及其原理 12

1.4 四极杆-静电场轨道阱质谱 12

1.4.1 四极杆-静电场轨道阱质谱原理 12

1.4.2 四极杆-静电场轨道阱质谱特点 13

1.5 课题研究（设计）的基本内容 13

1.5.1 课题研究（设计）的目的意义及创新点 13

1.5.2 课题研究（设计）的主要内容 13

第2章 四极杆静电场轨道阱质谱检测银杏叶和银杏果的活性成分 14

2.1 实验方案 14

2.2 结果与讨论 15

2.2.1 银杏叶和银杏果仁中化合物的质谱分析 15

2.2.2 银杏叶和银杏果仁的成分对比及溶剂对提取成分的影响 26

2.3 本章小结 28

第3章 银杏果多糖提取及其结构单元的HPLC-ELSD检测 30

3.1 实验方案 30

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