淫羊藿类及蛇类药材的鉴定和质量评价研究

【作者】 曹树萍；

【导师】 林瑞超；

【作者基本信息】 北京中医药大学 ， 中药化学， 2014， 博士

【摘要】 目的：2010版药典收载的植物药材淫羊藿基源复杂,动物药材蛇类混伪品多,现行药典方法难以对其进行全面、有效、客观的质量评价。因此本文选取这两种药材分别作为药典植物类药材和动物类药材的代表进行多维化研究,为药典植物药和动物药的鉴定和质量评价模式提供参考,同时也为提高药典标准提供实验依据。方法：本研究首先选取了含药典淫羊藿药材5种植物基源在内的18个品种120份淫羊藿类植物与药材样本和20个品种79份蛇类药材样本作为研究对象,从外观形状鉴别、分子生物学鉴定、化学鉴定及质量控制等方面进行质量评价研究。分别采用BLAST.遗传距离分析、系统发育树(NJ、MP和ML)树和‘’barcoding gap”检验4种方法对DNA条形码技术在药典淫羊藿类药材鉴定领域应用的可行性进行了详细的分析和探讨。采用RRLC-DAD-ESI-MSn釉UPLC-Q-TOF-HDMS法对淫羊藿属18个种的样本(含药典5种基源)进行化学成分分析。建立了18种113份淫羊藿类药材样本(含药典5种基源)的指纹图谱,结合化学计量学(相似度分析、聚类分析、PCA主成分分析)对其进行分析。同时结合5种主要黄酮类成分(朝藿定A、朝藿定B、朝藿定C、淫羊藿苷和宝藿苷I)的含量测定,对药典品种和非药典品种进行了质量评价的比较研究。并采用QAMS法对淫羊藿中的朝藿定A、朝藿定B、朝藿定C和宝藿苷I四种成分进行了含量测定。在国家药典品种乌梢蛇检品的PCR检验过程中发现存在鉴定方法的缺陷,为规避其出现的假阳性结果,采用BLAST、系统发育树(NJ)、遗传距离分析和“barcoding gap’检验4种方法对线粒体DNA条形码技术在药典中蛇类药材鉴定领域应用的可行性进行了详细的探讨。结果：从ITS、psbA-trnH、rbcL、matK和rpoCl五个基因片段中筛选出鉴定成功率最高的条形码psbA-trnH基因片段。其中基于ITS、psbA-trnH釉matK序列的条形码能够实现朝鲜淫羊藿的完全鉴定。通过高分辨质谱得到化合物精确分子量、多级质谱串联提供的碎片离子信息,与标准品或文献比对后,共鉴定出23个化合物,包括22个黄酮苷类成分和1个生物碱类成分。化学实验表明,柔毛淫羊藿和心叶淫羊藿药材质量最好,其5种黄酮类成分含量黄酮类有效成分总和要高于朝鲜淫羊藿。朝鲜淫羊藿品质稳定,但是有效成分含量相对较低,药典达标率仅为5.88%,远低于其他药典淫羊藿。说明朝鲜淫羊藿品种稳定但质量欠佳。箭叶淫羊藿分布范围虽广,但质量参差不齐,特别是资源量较大的安徽省,其部分地区淫羊藿苷类成分含量低微。按照相似度值的高低对5个药典淫羊藿的野生产地进行排序比较,为选择优质种质作为人工栽培的种源提供依据。基于淫羊藿化学成分为基础建立的聚类分析和基于ITS、psbA-trnH和matK DNA序列的条形码聚类分析,均显示朝鲜淫羊藿可以实现完全聚类,易于其它药典品种的淫羊藿区分开。黄酮类含量测定结果和指纹图相似度评价显示朝鲜淫羊藿化学成分和含量稳定,相似度高,而其他淫羊藿品种内部差异分化较大,种间种内区分不明显。对一测多评法应用于药典淫羊藿样品的测定结果进行考察,药典淫羊藿样品不同种种间差异较大,其中巫山和箭叶淫羊藿部分样本由于内参物(淫羊藿苷)含量普遍过低,或不含有其他黄酮类成分,不适宜用QAMS法进行含量测定。建立了基于16S、Cytb和COI的药典蛇类药材(乌梢蛇、金钱白花蛇和蕲蛇)的线粒体DNA条形码检验平台。首次提出以16S基因片段作为药典蛇类药材检验的最佳DNA条形码候选序列,建议以此作为蛇类药材快速检验的首选序列。结论：本文对淫羊藿和蛇类药材进行了多维化研究,为药典植物药和动物药的鉴定和质量评价模式提供参考,同时也为提高药典标准提供实验依据。更多还原

【Abstract】 Goal:The Chinese Pharmacopoeia (ChP,2010version) includes Herba Epimedii that has various complex species, and snakes that has many counterfeits. It’s quite challenging to conduct comprehensive, effective, and objective quality control of them. Therefore, these two Chinese Medicines are chosen as representatives from plants and animals representatively for multi-dimentional analysis, to provide reference for authentication and quality control of Chinese Medicine made from plants and animals included in the ChP, as well as to provide supplement for the improvement of the standards used in ChP.Methodology:In this research,120samples were chosen from18species of Epimedii with5of them included in ChP; while79samples from20species of snakes were collected. The analysis of these samples included morphological characteristics authentication, molecular authentication, chemical authentication, and quality control, etc. The feasibility of applying DNA barcoding in the authentication of Herba Epimedii was further investigated with validation using BLAST, Kimura-2-parameter (K-2P) distances analysis, phylogenetic tree (NJ, MP, and ML), and "barcoding gap". The RRLC-DAD-ESI-MSn and UPLC-Q-TOF-HDMS methods were adopted to analyze the chemical composition of the samples from18species (5of them are included in ChP) of herba Epimedii. In combination with chemometrics analysis (similarity analysis, cluster analysis, and PCA for component analysis), the fingerprints of113samples of Herba Epimedii from18species were established and quantitative determination of five flavonoids (Epimedin A\B\C, Icariin, Baohuoside I) were used to evaluate the quality of Epimedium. In the meanwhile, QAMS method was adopted to measure the content of these five flavonoids (Epimedin A\B\C, Icariin, Baohuoside I). The quality of Epimedium included and excluded in the ChP was compared and evaluated. To avoid "false positive" that was found in the authentification of Zaocys using PCR which is recommended by ChP, the feasibility of applying mitochondrial DNA barcoding in the authentication of snakes was further investigated and validated using BLAST, Neighbor-Joining tree, K-2P distance, and "barcoding gap"Results:Five DNA sequences, ITS, psbA-trnH, rbcL, matK and rpoCl, were used for analysis and it was found that psbA-trnH had the highest rate of success. The DNA barcodes using ITS, psbA-trnH amd matK sequence could all have fully accurate identification of Epimedium koreanum. High Definition Mass Spectrometry (HDMS) was used to obtain the precise molecular mass of the compounds and the characteristics of fragment ions provided by MSn. These two pieces of information were then compared with standard substances or literature to identify23compounds including22flavonoids and one alkaloid. The chemical experiment tests showed that Epimedium pubescens and Epimedium brevicornu had the best quality; their contens in total five flavonoids were higher compared with Epimedium koreanum. Epimedium koreanum had stable quality, but its content in effective flavonoids was much lower compared with other Epimedium included in ChP, with qualification rate of5.88%only according to the standards of ChP. This meant the Epimedium koreanum had lower quality though it had stable species. Epimedium sagittatum has a broad distribution, but its quality is not stable, especially in Anhui province where it has a big production; the content of Icariin of Epimedium sagittatum in other locations was much lower. The source places of five wild Epimedium included in ChP were ranked based on the high-low order of similarity, and this could provide reference for their cultivation in selecting high-quality species. The cluster analysis based on both chemical componets and DNA barcoding using ITS, psbA-trnH, matK sequences showed that Epimedium koreanum could have complete cluster, and could be distinguished from other Epimedium. According to chemical experiment test and similarity analysis using fingerprints, Epimedium koreanum was stable in chemical components and their contents; Epimedium koreanum had higher similarity, while other Epimedium had large intra-specicies variations, theirfore, the inter-and intraspecies differences were not obvious. The QAMS method in the measurement of Epimedium included in ChP was investigated. It was found that different Epimedium included in ChP had large variations; Epimedium wushanense and Epimedium sagittatum were not suitable for contens measurement using QAMS method, due to their low contents in Icariin or lack of other flavonoids components. The platform using mitochondrial DNA barcoding based on16S, Cytb, and COI was established for snakes (Zaocys dhumnades, Bungarus multicinctus, and Agkistrodon acutus) included in ChP, and it is the first that16S is recommended as the best candidate sequence in quick assessment of snake medicine using DNA barcoding technique.Conclusion:This research had explored multi-dimensional analysis of Epimedium and snake medicine. The research findings could provide reference in the authentication and quality control mode for plant and snake medicine included in ChP, and could also provide experimental test reference in improving the standards used in ChP.更多还原