低极性化合物的电喷雾质谱检测方法及几种中藏药材的活性成分研究

本学位论文首先报道了为解决低极性化合物的电喷雾质谱（ESI-MS）分析难题而建立的一种衍生化分析方法；然后从色谱-质谱联用分析、分离纯化和结构鉴定等方面分别报道了几种中藏药材的活性成分研究。论文由下述六章组成： 第一章报道了盐酸羟胺衍生化方法在电喷雾质谱 (ESI-MS) 分析中的应用。该方法利用盐酸羟胺和羰基成肟的快速反应，建立了针对三萜酮等含酮或醛羰基低极性化合物的ESI-MS 信号增强技术。此方法不仅可应用于增强羰基化合物的ESI-MS 质谱信号，还可检测化合物中羰基的个数以及辨别涉及羰基官能团的同分异构体。此外，通过简单的氧化反应，还可将该方法拓展到三萜醇、甾醇等含羟基的低极性化合物，增强它们的ESI-MS 信号。对比已报道的相关ESI-MS 增强质谱信号的衍生化方法，此方法有经济、实用、快速和简便的显著特点。 第二章是关于野生羌活及其栽培品种化学成分的色谱-质谱联用分析。对不同产地野生羌活生长过程中活性成分的动态变化、野生羌活不同形态部位和人工栽培羌活中的活性成分含量进行了HPLC 定量分析。结果表明主要活性成分羌活醇和异欧前胡素都随生长期存在规律性变化，羌活不同形态部位中的活性成分含量也有明显不同。这些实验结果有些较好地印证了传统中医的用药理论，有些也对羌活的传统使用方法提出了新的建议。 第三章介绍了几种传统中藏药材的色谱-质谱联用及串联质谱分析。通过GC-MS 方法，从藏药材长花党参挥发油中共分离鉴定出45 个化合物；利用HPLC方法测定了该藏药材中的主要化学成分——木犀草素的含量（0.7%）；利用串联质谱技术，对西番莲和射干中的主要成分进行了快速鉴定，从西番莲中鉴定了4个黄酮碳苷；从不同产地的射干和川射干中鉴定了8 个主要异黄酮成分，其中包括一个未见报道的化合物。 第四章的内容为藏药材石莲叶点地梅的活性成分研究。从植物石莲叶点地梅(Androsace integra (Maxim.) Hand.-Mazz.) 乙醇提取物的正丁醇萃取部分共分离和鉴定了6 个化合物，利用MS 和NMR 等现代波谱学技术阐明了它们的结构：其中包括4 个三萜类化合物：分别是androsacin (1)、 ardisiacrispin A (2) 、saxifragifolin A (3) 和20(29)-lupen-3-one (4)；一个神经酰胺：4-羟基-Δ8,9(Z)-鞘氨醇-2'-羟基正二十四碳酸酰胺(5)；一个甾体类化合物：胡萝卜苷(6)。化合物1为新的13,28-epoxy-oleanane 型三萜皂苷，在其结构表征的过程中，采用LC-MS 进行糖分析，获得了值得推广的好结果。通过活性筛选发现化合物1~3 对HepG2肝癌细胞表现出不同程度的抑制活性，其中化合物2 活性最好，其IG50 为1.65μg/mL。 第五章是关于一些传统中藏药材的农药活性筛选。利用Syngenta 公司的活性筛选平台对68 种传统中藏药材醇提物进行了抗菌和除草的生物源农药活性筛选。结果表明所筛选的68 种植物提取物中，共有14 种样品表现出明显的除草/杀虫活性，其中水母雪莲花、松萝和茯神木等植物提取物还具有多种生物活性。活性成分还有待进一步追踪分离、纯化和结构鉴定。 第六章为文献综述，概述了羌活药材的研究进展。对羌活属及药用羌活植物从分类学、本草学、品质评价、人工栽培、化学成分及药理作用等方面进行了文献归纳和总结。 In this dissertation, an electrospray ionization mass spectrometry (ESI-MS) signal enhancement method, as well as the work of bioactive components study, HPLC-MS/MS application, bioassay screening, chromatograph separation and structure identification of the metabolites in several medicinal herbs have been reported. First chapter expounded a rapid, simple ESI-MS sensitivity enhancement method for detecting carbonyl groups in natural products has been developed by using hydroxylamine hydrochloride (NH2OH·HCl) as a derivatization reagent. We use the oxime formed during the derivatization reactions and its Beckmann rearrangement intermediates as a means of detecting the carbonyl groups originally present in these triterpenoids. In comparison with other derivatization methods in the literature, this method is simple, specific and can be used to detect carbonyl groups in triterpenoids which have low polarity and are poorly or non-ionizable. Moreover, it can also be used to detect hydroxyl groups by using the Dess-Martin periodinane (DMP) to convert primary and secondary hydroxyls into carbonyl groups. Chapter 2 reported an HPLC-MS method for analyzing the main bioactive compounds in both wild and cultured Notopterygium incisum. The results indicated that the main bioactive compounds varied through different seasons regularly, and in different commercial parts of this herb the content of these compounds also differed from each other. The quantitative analysis results showed that in the traditional commercial parts, the content of main chemical constitutes in Silkworm Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are higher than that in Striped Notopterygium. This result is tally with the traditionally concept that the quality of Notopterygium, Bamboo Notopterygium and Irregular-nodal Notopterygium are better than that of Striped Notopterygium, which means that the quality of rhizomes is better than main roots. The chemical constituents of cultured N. incisum is reported for the first time in this dissertation and the analysis results showed some growth curves of chemical constituents in this plant, but still left some questions unanswered. Chapter 3 discussed the GC/LC-MS analysis of the traditional Chinese medicines Codonopsis thalictrifolis, Passiflora incarnate, Belamcanda chinensis and Passiflora incarnate. The main constituent, luteolin was isolated and identified from the traditional Tibet medicine of C. thalictrifolis. The quantitative analysis by HPLC has revealed that the content of luteolin in this herb is 0.7%. GC-MS was employed to analyzed chemical constituents of the essential oil from the flower of C. thalictrifolis. More than 60 peaks were detected and 45 of them were identified by comparing their spectra with that of the standards in the database and literatures. ESI-MS/MS was used to analyze the n-butanol extract of Passiflora incarnate. Based on the information of pseudo molecular ions and fragment ions of the glycosides, four major flavone-C-glycosides have been detected and identified as 7-methoxyluteolin-6-C-β-D-glucopyranoside, vitexin, swertisin and orientin. The isoflavone compounds in theextracts of three samples of B. chinensis collected in Gansu, Sichuan and Hunan, and the extract of Iris tectorum collected in Sichuan were analyzed by using TOF-HRMS and IT-MS. From the extracts of these herbs, a new isoflavone, identified as 5’,5,6,7-tetrahydroxy-3’4’-dimethoxyl isoflavon, and 7 known ones have been identified by analyzing the fragmentation patterns and their molecular formulas given by HRMS and the tandem mass spectrometry acquired by IT-MS. Chapter 4 elucidated the isolation and identification of a new triterpene saponin, androsacin (1), along with five known compounds (2-6) were isolated from the whole plants of Androsace integra (Maxim.) Hand.-Mazz., an herb used in traditional Chinese and Tibetan medicine. The chemical structure of the new compound was established as 3β-O-{β-D-glucopyranosyl-(1→4)-O-β-D-xylopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-[O-β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl}-16α-hydroxy-13β,28-epoxy-olean-30-al by analyzing its MS, 1D- and 2D-NMR spectra. Compound 2 was cytotoxic toward HepG2 cancer cell with the GI50 value of 1.65 μg/mL. Chapter 5 described the biogenic pesticide activity screening of 68 traditional Chinese and Tibetan medicine extractions. The intention of this study is to explore bioactive natural compounds from these traditional medicinal herbs for biogenic insecticides use. Based on Syngenta’s bioassay, 14 extractions of these traditional medicines showed pesticide activities, and some of them had multi-activities on antibacterial and insecticidal. Chapter 6 is a review on the chemical and bioactivity research progress of Notopterygium incisum and N. forbesii.