【作者】 郑辛甜；

【导师】 瞿海斌；

【作者基本信息】 浙江大学 ， 药物分析学， 2011， 博士

【摘要】 目前中药产业的发展面临着重大的机遇和挑战。为加速中药现代化进程,需要充分运用现代科学的新理论和新技术,进一步加强中药物质基础研究和提升制药工艺水平。中药酚酸类和黄酮类化合物具有抗氧化、抗炎、抗肿瘤、防治心脑血管疾病等多种药理活性,越来越受到人们的关注。中药酚酸类和黄酮类化合物在中药生产加工过程中常会发生降解和氧化等化学反应,影响相关中药质量。因此,深入研究中药酚酸类和黄酮类化合物的降解规律对阐明中药物质基础和提高中药质量控制水平都具有现实意义。本文主要研究内容及成果如下：1建立了基于超高效液相串联飞行时间质谱(UPLC-QqTOF-MS/MS)的中药复方温脾汤化学成分的快速分析方法,在17 min内对温脾汤化学成分完成高效分离分析,并借助QqTOF-MS/MS提供的化合物准确质量数和裂解碎片信息对温脾汤中的64种化学成分进行了推断鉴别,分别归属为大黄中的酚类化合物、附子中的生物碱、人参中的三萜皂苷类化合物和甘草中的黄酮苷类化合物等。该研究为中药复杂体系的物质基础研究提供了可行方法。2建立了一种在线紫外光谱采集系统用于实时记录反应过程光谱,并采用多元曲线分辨方法辨析化学变化信息,获取反应体系中组分的定性和定量信息。将上述方法应用于丹酚酸B、丹酚酸A和黄芩苷的降解和转化过程,发现：1)丹酚酸B在pH 8.5、90℃水浴加热条件下,降解过程为两步连续的一级反应A(?)B(?)C,反应动力学常数k1和k2分别为0.0253 min-1和0.0092 min-1,根据光谱信息和四级杆质谱信息推断丹酚酸B可能的降解产物主要包括丹参素、紫草酸和丹酚酸A。2)丹酚酸A在不同温度下的转化过程推断为一级反应A(?)B,在pH8.5,60℃、70℃、80℃和90℃水浴加热条件下丹酚酸A的转化反应动力学常数k分别为0.00348 min-1、0.00404 min-1、0.00586 min-1和0.00687 min-1。根据Mass WorkTM软件处理直接质谱分析得到的原始数据推断丹酚酸A可能的降解产物主要包括丹酚酸C、异丹酚酸C等4种酚酸类化合物。3)黄芩苷溶液在不同温度水浴加热条件下调节pH过程推断为两步连续一级反应A(?)B(?)C,60℃、70℃、80℃和90℃水浴加热下的反应动力学常数k1,分别为0.183 min-1、0.240 min-1、1.257 min-1和0.280 min-’,k2分别为0.091min-1、0.103 min-1、0.114 min-1和0.137 min-1。根据飞行时间质谱和多级质谱分析结果推断黄芩苷可能的反应产物主要包括黄芩苷的3种同分异构体、黄芩素和黄芩素二聚体。3利用恒温加速法研究了丹参中的丹参素和原儿茶醛对丹酚酸B稳定性的影响,发现丹参素和原儿茶醛的加入会减缓丹酚酸B的降解速度,且随着加入量的增大,丹酚酸B降解速度逐渐减小；丹参素与原儿茶醛加入浓度相同时,原儿茶醛更能抑制丹酚酸B的降解。研究表明丹参素和原儿茶醛均为丹酚酸B的天然稳定剂。4建立了一种基于扩散排序核磁共振技术结合并行因子分析算法监控中药化学成分降解过程的方法。以黄芩苷为例,采集在温度为333K,含25%浓硫酸条件下黄芩苷降解过程中的扩散排序核磁共振数据,实现降解反应混合物在核磁管中的“虚分离”分析,推断黄芩苷的降解产物为黄芩素和葡萄糖醛酸。利用并行因子分析算法分辨得到反应体系各组分的浓度变化趋势和核磁谱图等,推测降解过程为一级反应A(?)B,得到黄芩苷降解反应动力学参数k为0.25 h-1更多还原

【Abstract】 Nowadays, the development of Chinese medicine industries faces with great opportunities and challenges. In order to accelerate the modernization of Chinese medicine, novel theory and techniques are required in material basis research of Chinese medicine and improvement of pharmaceutical technological level. Phenolic acids and flavonoids in Traditional Chinese Medicines (TCMs) have obtained more and more attention for their significant pharmacological activities, such as antioxidant, anti-inflammatory, anti-tumor, prevention of cardiovascular and cerebrovascular diseases etc. Phenolic acids and flavonoids undergo degradation and oxidation reactions during the manufacture process of Chinese medicine sometimes, and these reactions will influence the quality of Chinese medicine final products. Thus, the research of degradation of phenolic acids and flavonoids plays an important role in material basis research of Chinese medicine and the improvement of quality control of Chinese medicine. The main contents and achievements of this thesis are summarized as follows:1 A rapid method based on UPLC-QqTOF-MS/MS was developed for the analysis of Chinese herbal prescription Wen-Pi-Tang. The constituents in Wen-Pi-Tang were well separated in only 17 minutes.64 constituents of this prescription were identified and deduced according to accurate mass and mass spectrometric fragmentation mechanisms. The major constituents of this prescription included phenolic compounds from rhubarb, alkaloids from aconite root, triterpenoid saponins from ginseng and flavonoid glycoside from licorice. This method was suitable for material basis research of complex chemical system such as Chinese medicines. 2 An on-line UV spectroscopic collection system was established for recording reaction process spectra. Resolution of complex process spectra was performed with multivariate curve resolution (MCR), and spectra and kinetic profiles of reactant species were reconstructed. On-line UV spectra combined with MCR was applied to the degradation and transformation research of salvianolic acid B, salvianolic acid A and baicalin. The results included:1) the degradation process of salvianolic acid B at pH 8.5,90℃is likely a consecutive two-steps reaction in the form of A(?)B(?)C, the calculated first-order rate constants k1=0.0253 min-1, and k2 =0.0092 min-1. Based on spectra profiles and quadrupole mass spectrometry analy results, the possible degradation products of salvianolic acid B are mainly proposed to be lithospermic acid, danshensu and salvianolic acid A; 2) transformation processes of salvianolic acid A at different temperatures are likely one-order reactions in the form of A(?)B. The calculated rate constants at pH8.5,60℃,70℃,80℃and 90℃were 0.00348 min-1,0.00404 min-1,0.00586 min-1 and 0.00687 min-1, respectively. Based on direct mass spectrometry analysis and Mass workTM software process results, the possible transformation products of salvianolic acid A are mainly proposed to be four polyphenolic acids included salvianolic acid C, isosalvianolic acid C etc; 3) the research of baicalin transformation processes at different conditions indicated that these processes are likely consecutive two-steps reactions in the form of A(?)B(?)C, the calculated first-order rate constants k1 at 60℃,70℃,80℃and 90℃were 0.183 min-1,0.240 min-1,0.257 min-1 and 0.280 min-1, while k2 were 0.091 min-1,0.103 min-1,0.114 min-1 and 0.137 min-1, respectively. The possible transformation products of baicalin are mainly deduced to be 3 isomerides of baicalin, baicalein and dimmer of baicalein. They were deduced based on the analysis results of time-of-flight mass spectrometry and multistage mass spectrometry.3 Isothermal acceleration method was used to investigate the effects of danshensu and protocatechualdehyde on the stability of salvianolic acid B. It was indicated that addition of danshensu and protocatechualdehyde inhibits the degradation of salvianolic acid B. The degradation rate of salvianolic acid B decreased with the increase of amount of danshensu and protocatechualdehyde. Furthermore, with the same concentration of danshensu and protocatechualdehyde, the latter provided stronger protection effect. It was suggested that danshensu and protocatechualdehyde might be natural stabilizing agents of salvianolic acid B.4 Diffusion ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) combined with parallel factor analysis (PARAFAC) was applied for monitoring degradation process of Chinese medicine constituents. As an example, the DOSY-NMR spectra of baicalin at 333 K, with 25% sulphuric acid were collected during its degradation process, pseudo-isolation and analysis of degradation mixture in NMR tube was implemented, and possible degradation products were deduced as baicalein and glucuronic acid. DOSY spctra data of degradation of baicalin was further processed with PARAFAC to resolve spectra and concentration time courses of components, and the results indicated that degradation process of baicalin is likely one-order reaction in the form of A(?)B, the calculated rate constant was 0.25 h-1.更多还原