【作者】 范捷；

【导师】 傅承新；

【作者基本信息】 浙江大学 ， 药用植物资源学， 2007， 硕士

【摘要】 元胡(Rhizoma Coridalis)和浙贝母(Bulbus Fritillariae Thunbergii)是浙江省所产著名药材“磐五味”中的两种。磐安因其独特的地理环境和气候条件，使其所产的这两种药材质量上乘，质地稳定，目前已经形成一定规模的生产基地。但是，药材来源除了部分来自种植基地外，大量原药材仍收购于松散农户，生产条件的不规范导致质量参差不齐，品种间可能存在相当的混杂度，这就需要建立道地药材的标准指纹图谱，从而指导GAP标准化生产。基于此目的，本文通过高效液相(HPLC)色谱技术和傅立叶变换红外(FTIR)光谱技术，对浙江省产的道地元胡和浙贝母药材与全国其它产地所产的进行指纹图谱分析和比较，综合建立了浙产道地药材元胡和浙贝母的化学指纹图谱。1．元胡化学指纹图谱的构建元胡为紫堇科植物延胡索(Corydalis yanhusuo W．T．Wang ex Z．Y．Su et C．Y．Wu)的干燥块茎，本文以延胡索乙素标准品为对照，考察了样品的不同提取方法和HPLC检测过程中不同的流动相，并通过加入小分子有机酸和碱(乙酸、三乙胺)等优化流动相系统，以改善色谱峰的峰形和分离度，经过反复摸索确定了流动相洗脱梯度，使药材中的成分在取得良好分离度的基础上保持稳定，经过制备、分析、校验，建立了磐安元胡的HPLC指纹图谱，并且比较了不同加工方法、不同产地出产的元胡之间的差异。结果表明：元胡药材指纹图谱能够反映药材之间的相似性，省内不同地区产的元胡经同一方法加工处理后的HPLC指纹图谱无明显差异，但浙产元胡与外省产元胡存在一定差异，通过比较确定了磐安元胡的特征区域：在9min、45min左右，浙产元胡有明显的小峰，江苏和陕西的样品却不明显或无；70min左右，两个外省产元胡有个小峰，浙产元胡却无，为鉴别磐安产道地药材元胡提供了科学依据。同时，本文也对市场上元胡的不同加工方法进行比较，结果表明：硫磺熏蒸法使元胡HPLC图谱出峰数减少，可能是部分成分发生变化或丧失，不推荐使用；未加工过的生药材与经沸水煮过的药材在化学成分上发生了明显变化，从HPLC指纹图谱上可以直接辨认，通过聚类分析法和主成分分析法也能很好地区分三种不同加工方法处理的元胡。根据以上分析结果，选择典型的HPLC图谱，建立了元胡HPLC指纹图谱的共有模式，并给出了共有峰的相对保留时间和相对保留峰面积的波动范围。除了建立HPLC指纹图谱，还得到不同产地元胡的FTIR谱图，并进行相似度分析，结果表明不同产地元胡的相似度基本在0.90以上，很难将其区分，但是不同加工方法处理的元胡FTIR图谱相似度存在明显差异，用该方法可以区分。2．浙贝母化学指纹图谱的建立浙贝母为百合科植物浙贝母(Fritillaria thunbergii Miq．)的干燥鳞茎。主产浙江、江苏、安徽等地，主要成分为生物碱。不同贝母所含生物碱种类不尽相同，本文根据中国药典(2005版一部)中的浙贝母生物碱成分的提取方法稍加改变，用超声波提取法代替加热回流法制备供试样品，考察了不同流动相，通过反复摸索确定了流动相的梯度。由于贝母中大部分生物碱类成分无紫外吸收，因此用蒸发光散射(ELSD)检测代替紫外检测，建立了5个磐安浙贝、1个宁波浙贝、1个江苏省产浙贝以及川贝、皖贝、平贝的HPLC-ELSD图谱，通过比较发现皖贝、川贝、东贝、浙贝之间存在明显差异，确定了皖贝所特有的峰：6号、12号、13号、15号峰；东贝虽然是浙贝的变种，但是从HPLC图谱上可以看出两者的明显差异，找出了东贝的特征峰：2号、7号、9号、18号峰；川贝样品有2种，为同一种川贝不同生长年限的鳞茎，生长年限较长的川贝与浙贝差异明显，而生长年限较短的川贝与浙贝相近，说明其二次代谢产物的累积是个动态变化的过程；浙江省磐安及宁波所产浙贝之间无显著差异，聚类分析和主成分分析都是分布在一起的，但是江苏南通产浙贝与浙产浙贝在聚类图上分离，可能是栽培环境不同所致，也表明了磐安产浙贝的道地性。与此同时，本研究也采用FTIR技术，得到不同种贝母的红外光谱图谱，基于化学计量学通过提取图谱中的有效指纹信息，采用多元统计学中的聚类分析后，发现与HPLC聚类图结果在大的格局上是一致的，而在HPLC聚类图中不能区分的东贝和平贝在FTIR聚类图谱上却可以区分，可是FTIR方法是否能鉴别贝母有待进一步研究。更多还原

【Abstract】 Rhizoma Coridalis and Bulbus Fritillariae Thunbergii are two famous traditional Chinese medicines from Pan’ an county of Zhejiang province, meanwhile they are included "eight sapor of Zhejiang". Because of the environment and climate there in Zhejiang province is special, the quality of these two traditional Chinese medicine is much better and much more steady than any other regions’. At present, some production bases in certain scale have formed. Though these medicine materials are partly from these production bases, most of them are purchased from farmers. Due to the nonstandard production, the quality is quite different, and different species may mix together. So it is necessary to identify the genuine medicine materials and build chemical fingerprints for them in order to guide good agriculture practice (GAP). This article builds High Performance Liquid Chromatography (HPLC) fingerprints and Fourier Transform Infrared Radiation (FTIR) fingerprints for Rhizome Coridalis and Bulbus Fritillaria thunbergii. At the same time, it analyzes the differences between genuine medicine materials grown in Pan’ an and materials from other places, In the end, we build integrated chemical fingerprints of the two medicine materials from Zhejiang province.1. Fingerprints of genuine Corydalis yanhusuo W. T. WangThe traditional Chinese material Rhizoma Coridalis is the dry bulb of plant Corydalis yanhusuo W. T. Wang et Z. Y. Su et C.Y. Wu which belongs to family of Papaveraceae. It mainly grows in Zhejiang province and has been used to promote blood circulation, reinforce vital energy, and alleviate pain. The modern scientific study has proved that its major active components are alkaloids, especially tetrahydropalmatine which can alleviate pain effectively. In this article, the experiment used tetrahydropalmatine as control, then we compared different extract methods and different mobile phase systems. For the purpose of optimize the mobile phase systems; we added macromolecule organic acid, such as acetic acid and diethylamide to improve the peak shape and resolution. Finally, we ascertained the mobile phase systems and elution grade. Through the process of preparation, development analysis, evaluation and verification, we established the HPLC fingerprint of C. yanhusuo and compared the ones yielded from different places and different disposed methods. The consequence indicates that the fingerprint can reflect the resemblance of the medical material and provide reliable evidence to control the quality of C. yanhusuo. At the same time, we compared the distinction of the fingerprint of C. yanhusuo which yielded from different places and found that there is no significant difference in Zhejiang samples, but quite different between Zhejiang province and other province. The characteristic areas in the fingerprints of Pan’an areas are: 9min, 45min when there is a small peak in Pan’an samples but other samples don’t have, and 70min, two samples of other provinces have an obvious peak, but Pan’an samples don’t have. At the same time, we compare different disposed methods, the result showed: the fingerprint of the sample dealt with sulfur had less peaks than other samples, may be some componets lost or changed. The fresh materials and boiled ones have obvious differences which can identify from fingerprints and cluster analysis as well as PCA method also. Besides, we constructed FTIR spectrum fingerprints of C. yanhusuo yielded from difference places, and analyzed their similarity. The result indicated the similarity of the spectrum fingerprints is above 0.90, which is hard to be distinguished.2. Fingerprints of genuine Fritillaria thunbergii Miq.The traditional Chinese material Bulbus Fritillariae Thunbergii is the dry bulb of plant Fritillaria thunbergii Miq. which belongs to the family of Liliaceae. It mainly grows in Zhejiang province, Jiangsu province and An’hui province. Its active components are alkaloids, which are varied in different species. This article used the method from Pharmacopoeia of People’s Republic of China (2005 Edition I) and made a little change which was using ultrasonic extraction method instead of distilling method recroded in Pharmacopoeia to extract their alkaloid components. We ascertained the mobile phase systems and elution grade through repeated process. Because most alkaloids have no ultraviolet absorption, we used Evaporative Light Scattering Detector (ELSD) to detect their components. We constructed five Fritillaria thunbergii Miq. fingerprints from Pan’an , one from Ningbo, one from Jiangsu and other three species. Through comparation, we ascertain the characteristic peaks of WanBei, they are: peak6, peak12, peak13, peakl5; the characteristic peaks of F. thunbergii Miq. var. chekiangensis Hsiao et K.O. Hsia are peak2, peak7, peak9, peak18; there are two samples from Sichuan province, they are belong to the same specie, just different in growth years, but the fingerprints of them are different, this might caused by the environment. Furthermore, we established the FTIR fingerprints of different Fritillaria species and chose the representational community associating with stechiometry, we can distinguish different species of Fritillaria by cluster analysis.更多还原