柴胡属药用植物资源调查及基于代谢组学的质量评价研究

  本文关键词：柴胡属药用植物资源调查及基于代谢组学的质量评价研究，，由笔耕文化传播整理发布。

        中药材质量不稳定是当前中药发展面临的一个重大问题,造成这个问题的一个主要原因是同种药材来源复杂多样。长期以来,多种同属植物被当作一种药材使用的情况时有发生,而对其进行的鉴别与质量控制都大不尽如人意。中药柴胡始载于《神农本草经》,位列上品,多用于感冒发热、寒热往来、疟疾、肝郁气滞、胸肋胀痛、脱肛、子宫脱落、月经不调诸症,为中医常用清虚热药。2010版中国药典规定,作为中药柴胡的来源为伞形科柴胡属植物柴胡(北柴胡Bupleurum chinense DC.)和狭叶柴胡(南柴胡B. scorzonerifolium Willd.).据以往统计资料记载,柴胡属植物在我国共有42种、17变种、7变型,但柴胡属大部分植物自古已有药用,各地区亦按其所产品种和历史使用习惯而在民间入药或自产自销。然而,由于同属植物来源的生药形态和显微特征极近似,加上所含的化学成分在植物发育和收采加工过程中可能会发生很大的变化,使得用传统的方法对柴胡进行鉴定存在相当大的难度。鉴于目前有的地区把当地所产的5、6种柴胡属植物混在一起当做药用,当中还包括有毒的大叶柴胡及活性成分极低的小柴胡,造成市场上柴胡品种相当混乱,药材质量极不稳定。因此,有必要寻求一种可靠、有效的柴胡生药鉴定方法,以保证柴胡的用药安全及质量稳定。代谢组学(metabolomics)是20世纪90年代中期发展起来的对某一生物或细胞所有低分子量代谢产物进行定性和定量分析的一门新学科。是继基因组学、转录组学和蛋白质组学之后的又一门新兴的组学技术,也是系统生物学研究不可或缺的重要基础学科之一。作为代谢组学的重要分析手段,以(超)高效液相色谱质谱联用分析技术为平台,对植物所含的小分子化学成分常具有较强的分离分析能力,尤其是具备电喷雾离子源的(超)高效液相色谱质谱联用分析仪,因能完整给出待分析物质的分子离子碎片,有利于质谱分析器对物质的准确鉴别。相对其他分析方法,所产生的化学成分代谢谱能够更准确地反映生物体系的状态。由此,可比较准确地对生药质量进行鉴别评价,亦有利于建立系统规范的化学成分数据库(如柴胡药材数据库)。主成分分析也称主分量分析,是旨在利用降维的思想,把多指标转化为少数几个综合指标的非监督性多元统计分析方法。作为一种高维数据解析方法,其应用着重于发掘化学数据的外在表征与物质内在组成之间的相互关系,在最大程度上提取原始信息的同时对数据进行降维处理,解决了液质联用等现代分析仪器或检测技术提供的大量而又丰富的量测信息所带来的统计分析难题,是目前代谢组学研究常用且重要的分析方法,有助于类似柴胡等多来源药材质谱分析等数据研究。目的：对当前柴胡属植物的品种资源及药用状况进行详细的调查,提出确保柴胡药材准确鉴定及品种合理开发的策略；应用液质联用技术平台,通过主成分分析研究,建立准确、快速、可靠的柴胡类药材鉴定和质量评价体系,同时也为研究新品种,扩大新药源提供合理依据。方法：1、以文献资料为指导对柴胡属药用植物的分布地点进行确定,对于分布较广的种类如北柴胡,拟收集10个产地以上的标本及样品,每一产地采集或收集数个-10个居群的样品；对于分布区域较为局限的种类,则尽可能获取多个居群的个体,同时联合利用国内各标本馆中所藏柴胡属植物样本,保证标本和样品的准确性、全面性和代表性。将所收集到的样本进行准确的分类鉴定,并归纳品种、产地等信息,标本及实验样品均统一存放于阴凉干燥处备用。2、以超高效液相色谱电喷雾质谱联用仪(UPLC-ESI-MS)为分析平台,对柴胡属植物的化学成分进行代谢谱分析,并依次对质谱仪器的检测参数、色谱柱温、进样量、流动相组成及流速、样品的提取工艺等进行考察。3、用考察出来的最佳实验条件对柴胡属植物的地上地下部分依次进行化学成分代谢谱的数据及图谱的采集,对柴胡皂苷A、C、D等成分进行含量测定,并对获得的数据用多元统计分析软件SIMCA-P进行主成分分析,对不同品种、产地的柴胡作出质量判定。4、将所采集的化学成分代谢谱的数据及图谱整理归纳后,以V C++及SQLserver2005等软件,建立柴胡属植物代谢组学分析专属数据库。结果：1、对柴胡属植物资源的调查一共采得213份柴胡属植物标本,分属19种、7变种、4变型；柴胡属植物资源主要集中分布于我国的东北、华北和西北等地区,资源蕴藏量占全国的60%以上,品种又以柴胡、狭叶柴胡和银州柴胡为主。2、由于各地用药习惯不一,药用的柴胡多为地方习用品种。即使所产为栽培的柴胡,但由于种源不纯或各地区互相引种,栽培地常有数种农家型的柴胡混生。目前栽培产量已远远超过野生品,但由于长期的人工栽培,加上种植集中且品种单一,品种出现种质退化,抗性降低,不同地区出产的药材质量大多参差不齐。3、作为商品用途的各种柴胡,无论来源是野生或者栽培,不同品种基原的柴胡在部分地区的购销过程中使用相同的药名;而同一品种基原的柴胡药材往往又有多个不同的商品名称。因此出现了同一基原植物在不同地区商品中归类不同,而同一商品名可能包括不同基原的植物,存在异物同名或同物异名的混乱现象,品名与品种区分不明。4、初步考察出了适合柴胡属植物的供试品提取条件为20倍溶媒比的5%氨水甲醇,超声波提取5次,每次30分钟；对提取条件的验证发现5个平行样品的柴胡皂苷A、D总提取率分别为：0.5176%、0.5144%、0.5181%、0.5152%、0.5167%,平均值为0.5168%,RSD值为0.409%,表明优化后提取条件的可行性及重现性均较好。5、利用UPLC-ESI-Q-TOF-MS分析柴胡中所含柴胡皂苷A、C、D等成分,含量测定方法快速准确、灵敏可靠、专属性强。对照品以峰面积对进样量进行线性回归得方程为柴胡皂苷A：Y=3.144X+37.08,r=0.9994；柴胡皂苷C：Y=18.40X+39.18,r=0.9989；柴胡皂苷D：Y=3.225X+161.8,r=0.9989。结果分别表明柴胡皂苷A在4.860μg-14.580gg范围内,柴胡皂苷C在0.3135-3.1350gg范围内,柴胡皂苷D在2.170μg-10.850μg范围内线性关系良好。柴胡皂苷A的在样品中的含量介于0.0452%-0.3963%,柴胡皂苷C的含量介于0.0096%-0.1945%,柴胡皂苷D的含量则介于0.1082%-0.3673%。6、进行主成分分析时,由软件自动拟合,确定了最佳的主成分提取个数为2；同时从分析的结果出发,得以对所分析的样品中化学成分的异同作出判别,能够较好地按化学成分的差异及品种、产地等信息对样品进行分类,初步显示柴胡药材质量与品种、产地存在相关关系。7、建立了拥有300多张图的柴胡属药用植物化学成分谱专属数据库,为以后的研究工作提供了参考依据。结论：1、作为一种传统用药,柴胡属药用植物在华北、西北、东北等地有比较广泛的资源分布。虽然栽培资源的开发一定程度上缓解了本品在市场上的供应不足等问题,但由于野生的药用植物资源曾经受各种灾害和不良环境的选择,抗逆性较强,保存着栽培种不具有或已经消失的特异基因,是宝贵的物种基因库,开发的同时应注意保护。2、在综合产量与质量等因素的前提下,针对合适的品种,栽培地应尽早制定药材生产质量管理规范并尽可能按要求优选一套成熟的柴胡种植技术,以从源头提高药材的质量。3、本研究构建的UPLC-ESI-Q-TOF-MS分析方法,为多基原的柴胡类药材作出了更为“全面”的化学成分分析,同时,更关注产地及品种对药材质量差异所引起的问题,是当前柴胡类药材品种鉴定和质量评价的重要手段之一。4、构建柴胡属药用植物化学成分代谢组学专属数据库,方便了对柴胡研究资料的利用,有助于推动柴胡药材的种植、加工、销售等规范化管理,推进柴胡类药材的现代化研究。

    Inconsistent quality of Chinese medicinal materials is currently one of the major problems in the development of traditional Chinese medicine. A major cause of this problem is the same medicine has varied sources. For a long time, though several kinds of plants from the same genus had been used as one kind of crude drug, its identification and quality control are not satisfactory.As a traditional Chinese medicine, chaihu (Radix Bupleuri), at the earliest, was ranked the top grade in the "Shen Nong’s Herbal Classic". It was a medicine of deficient heat clearing which was used for influenza, fever, alternate chills and fever, malaria, stagnation of liver-Qi, chest rib swelling, anal prolapse, uterus prolapse, menoxenia, and so on. Its officially authorized origins in2010Chinese Pharmacopoeia are Bupleurum chinense and B.scorzonerifolium. According to the past statistical records, the genus Bupleurum represents42species,17varieties and7forma in China. But most of the plants since ancient times had been utilized as Radix bupleuri. In addition, citizens in some regions sell the plants of Bupleurum theirselves according to the offerings and historical usage habits. However, the phytomorph and microscopic characteristics of Bupleurum medicinal plants are fairly approximate, and the chemical constituents contained in plants would change a lot in the process of growth and harvesting. So, there is a considerable difficulty in distinguishing these species with traditional identification methods. At present,5or6kinds of local Bupleurum plants were jumbled together to be used, even including toxic (B.longiradiatum) or little active constituents contained materials (B.tenue). This lead to confusion in the market and uneven quality of the drug. Therefore, it is necessary to establish a reliable and effective identification method for improving the quality standard for Radix Bupleuri.Metabolomics, a new science and technology developed in the mid of1990s (after genomics, transcriptomics and proteomics) was an indispensable basic disciplines for systems biology research. It refers to a holistic analytical approach to all the low molecular weight metabolites in an organism or cells. As an important approach of metabolomics,(ultra) high performance liquid chromatography tandem mass spectrometry provide an analysis platform on small molecules with strong separation and analysis of chemical composition in organism. Especially with the help of electrospray ion source chromatography mass spectrometry analyzer, the molecular ion fragments can be accurately detected and identified. The generated metabolic spectrum would more accurately reflect the biological system state compare to other analytical methods. There will benefit for quality identification of crude drug, as well as establishing a systematic standardized chemical composition database (like Radix Bupleuri).Principal component analysis is a kind of non-supervised multivariate statistical method which was designed to transform multi-index into several comprehensive index using dimension reduction. As a high-dimensional data analysis method, PCA focuses on discovering the inner relationship between exterior characterization of chemical data and internal composition of materials. It is a commonly used and important analysis method for metabolomics which can be dealt with abundant organisms metabolic information (Bupleurum or other multi-source herbs) from mass spectrometry analytical instruments.Objective:To make a detailed investigation of natural resource and medicinal situation of Bupleurum and to raise a strategy which the crude drugs of Radix Bupleuri cultivars would attain an accurate identification and reasonable exploitation. To establish an accurate, fast and reliable identification and quality evaluation system by means of principal component analysis on LC-MS data of Bupleurum so as to provide a reasonable study basis for expansion of new drug source from different Bupleurum species.Methods:1. Geographical distribution of Bupleurum medicinal plants were confirmed based on the documentary guidance. Several-10groups of specimens of more than10origins were intended to collect for widely distributed species such as Bupleurum chinense. But species of limited distribution were intended to collect as many groups as possible. In addition, take full advantage of collections from various herbaria to ensure those Bupleurum samples would be comprehensive and representative. Along with accurate classification and identification, samples information of origins and species should be summed up before unified store in a cool dry place.2. Metabolic spectrum analysis of Bupleurum plants were carried out by means of ultra-high performance liquid chromatography tandem electrospray ionization mass spectrometry (UPLC-ESI-MS). Device parameters such as chromatographic column temperature, injection quantity, mobile phase, velocity, sample preparation would be successively inspected.3. Metabolic spectrum data of both above and below ground parts of Bupleurum plants were acquired under the best sifted experimental conditions. The content of saikosaponin A, C, D was respectively assayed. The samples quality of different species and origins were exclusive determined by PCA with SIMCA-P.4. Metabolic spectrum data of the samples were collected and summed up to establish a metabolomics database of Bupleurum medicianl plants by software of VC++and SQL Server2005.Results:1. There were a total of213specimens which belonging to19species,7varieties and4forma were collected during the resources survey. Survey result shows that over60%of the whole Bupleurum resources reserves were mainly distributed in china’s northeast, north and the northwest, and most of them were Bupleurum chinense, B.scorzonerifolium and B.yinchowense.2. Due to the different customs, Bupleurum medicinal plants were mostly conventional varieties. Because of impure provenance or mixed varieties planted in different region, there were still several cultivars grow in cultivated land. At present, the yield of cultivation production has far more than the wild products. Coupled with a concentrated single species planting for a long time, germplasm degradation and resistance reduction came out. As a result, most of the quality of the materials from different regions was uneven.3. As a commodity, no matter the source was wild or cultivated, different Bupleurum medicinal plants using a same name during buying and selling process. In addition, Bupleurum herbs of the same species often have several different trade names in some regions. So, there was a homonym or synonym confusion which the same base source of plants classified in different parts of goods and the same product name may include a different source plants. As a result, the name confuses the variety.4. The sample extraction condition was20times5%ammonia methanol extract for five times,30minutes each time. The total extraction rate of saikosaponin A and D in demonstration test with five parallel samples were0.5176%,0.5144%,0.5181%,0.5152%,0.5167%, an average of0.5168%, RSD is0.409%. The result shows that the optimized extraction condition is feasibility and reproducibility.5. Assay of saikosaponin A, C, D, and other ingredients contained in Bupleurum medicinal plants by the analysis method of UPLC-ESI-Q-TOF-MS is rapid, accurate, sensitive, reliable and specific. Linear regression equation of peak area corresponding the injection volume of saikosaponin A, saikosaponin C, saikosaponin D, respectively was Y=3.144X+37.08, r=0.9994; Y=18.40X+39.18, r=0.9989; Y=3.225X+161.8, r=0.9989. The result shows that saikosaponin A in the range of4.860μg-14.580μg, saikosaponin C in the range of0.3135-3.1350μg, saikosaponin D in the range of2.170μg-10.850μg respectively has a good linear relationship. The content of saikosaponin A in the samples was ranged from0.0452%-0.3963%, the content of saikosaponin C was ranged from0.0096%-0.1945%, the content of saikosaponin D was ranged from0.1082%-0.3673%.6. Two principal components were determined by the software of SIMCA-P during the analysis. By the help of PCA, similarities and differences of chemical composition in samples can be discriminated. Samples were able to classify according to the difference of chemical composition and species. A correlated relationship was initially performed among Bupleurum medicinal materials quality with variety and origin.7. A proprietary database of chemical composition of Bupleurum medicinal plants with over300spectrums was established to provide a reference for future research work.Conclusion:1. As a kind of traditional herbs, Bupleurum medicinal plants were widely distributed in the north, northwest northeast of China. Although developments of Bupleurum medicinal plants cultivation, to a certain extent alleviate the inadequate supply of Radix Bupleuri in the market. However, wild resources of medicinal plants had live through a variety of disasters and adverse environmental choice. It is a valuable species gene pool should be protected which the cultivars didn’t possess or had disappeared.2. According to a comprehensive consider premise on the yield and quality of Radix Bupleuri, a standardized management and efficient planting technology of medicinal materials cultivation should be put into practice as early as possible for suitable Bupleurum medicinal plants, so as to improve the quality of the crude drug from the beginning.3. UPLC-ESI-Q-TOF-MS offers a more comprehensive approach for chemical composition of multi-source medicinal plants of Bupleurum. Meanwhile, the analysis approach also is an alternative method of medicinal materials quality evaluation and identification, which pays more attention to the quality differences related to different varieties and origins.4. With the help of chemical composition database of Bupleurum medicinal plants, research data would be fully utilized to promote a standardized management on cultivation, processing, sales and modernization process of Radix Bupleuri.

        柴胡属药用植物资源调查及基于代谢组学的质量评价研究

摘要3-8ABSTRACT8-14第一章 前言17-21    1.1 柴胡药材质量评价研究的意义17-18    1.2 代谢组学在药材质量评价的应用与意义18-19    1.3 本课题研究思路19    1.4 本课题的研究意义19-21第二章 柴胡属药用植物资源及药用状况调查21-32    2.1 柴胡属药用植物资源调查21-28    2.2 柴胡商品药材的使用情况28-30    2.3 讨论30-31    2.4 小结31-32第三章 基于UPLC-ESI-Q-TOF-MS的柴胡属药用植物代谢组学分析32-60    3.1 材料和方法32-43    3.2 柴胡样品皂苷A、C、D的含量测定43-51    3.3 代谢组学数据分析51-57    3.4 讨论57-60第四章 柴胡属药用植物化学成分代谢谱数据库的构建60-67    4.1 数据库编辑语言的选择60-61    4.2 数据库的设计61-62    4.3 数据库的特点及功能62-65    4.4 数据库的运行管理与维护65    4.5 结语65-67第五章 结论67-68参考文献68-71攻读学位期间成果71-72综述72-85    参考文献82-85附录85-88    一、中英文(符号)缩写对照表85-86    二、柴胡属植物形态86-88致谢88-90统计学审稿证明90

  本文关键词：柴胡属药用植物资源调查及基于代谢组学的质量评价研究，由笔耕文化传播整理发布。