本文關鍵詞：柴胡屬藥用植物資源調查及基于代謝組學的質量評價研究，，由筆耕文化傳播整理發(fā)布。

        中藥材質量不穩(wěn)定是當前中藥發(fā)展面臨的一個重大問題,造成這個問題的一個主要原因是同種藥材來源復雜多樣。長期以來,多種同屬植物被當作一種藥材使用的情況時有發(fā)生,而對其進行的鑒別與質量控制都大不盡如人意。中藥柴胡始載于《神農本草經》,位列上品,多用于感冒發(fā)熱、寒熱往來、瘧疾、肝郁氣滯、胸肋脹痛、脫肛、子宮脫落、月經不調諸癥,為中醫(yī)常用清虛熱藥。2010版中國藥典規(guī)定,作為中藥柴胡的來源為傘形科柴胡屬植物柴胡(北柴胡Bupleurum chinense DC.)和狹葉柴胡(南柴胡B. scorzonerifolium Willd.).據以往統(tǒng)計資料記載,柴胡屬植物在我國共有42種、17變種、7變型,但柴胡屬大部分植物自古已有藥用,各地區(qū)亦按其所產品種和歷史使用習慣而在民間入藥或自產自銷。然而,由于同屬植物來源的生藥形態(tài)和顯微特征極近似,加上所含的化學成分在植物發(fā)育和收采加工過程中可能會發(fā)生很大的變化,使得用傳統(tǒng)的方法對柴胡進行鑒定存在相當大的難度。鑒于目前有的地區(qū)把當地所產的5、6種柴胡屬植物混在一起當做藥用,當中還包括有毒的大葉柴胡及活性成分極低的小柴胡,造成市場上柴胡品種相當混亂,藥材質量極不穩(wěn)定。因此,有必要尋求一種可靠、有效的柴胡生藥鑒定方法,以保證柴胡的用藥安全及質量穩(wěn)定。代謝組學(metabolomics)是20世紀90年代中期發(fā)展起來的對某一生物或細胞所有低分子量代謝產物進行定性和定量分析的一門新學科。是繼基因組學、轉錄組學和蛋白質組學之后的又一門新興的組學技術,也是系統(tǒng)生物學研究不可或缺的重要基礎學科之一。作為代謝組學的重要分析手段,以(超)高效液相色譜質譜聯(lián)用分析技術為平臺,對植物所含的小分子化學成分常具有較強的分離分析能力,尤其是具備電噴霧離子源的(超)高效液相色譜質譜聯(lián)用分析儀,因能完整給出待分析物質的分子離子碎片,有利于質譜分析器對物質的準確鑒別。相對其他分析方法,所產生的化學成分代謝譜能夠更準確地反映生物體系的狀態(tài)。由此,可比較準確地對生藥質量進行鑒別評價,亦有利于建立系統(tǒng)規(guī)范的化學成分數據庫(如柴胡藥材數據庫)。主成分分析也稱主分量分析,是旨在利用降維的思想,把多指標轉化為少數幾個綜合指標的非監(jiān)督性多元統(tǒng)計分析方法。作為一種高維數據解析方法,其應用著重于發(fā)掘化學數據的外在表征與物質內在組成之間的相互關系,在最大程度上提取原始信息的同時對數據進行降維處理,解決了液質聯(lián)用等現代分析儀器或檢測技術提供的大量而又豐富的量測信息所帶來的統(tǒng)計分析難題,是目前代謝組學研究常用且重要的分析方法,有助于類似柴胡等多來源藥材質譜分析等數據研究。目的：對當前柴胡屬植物的品種資源及藥用狀況進行詳細的調查,提出確保柴胡藥材準確鑒定及品種合理開發(fā)的策略；應用液質聯(lián)用技術平臺,通過主成分分析研究,建立準確、快速、可靠的柴胡類藥材鑒定和質量評價體系,同時也為研究新品種,擴大新藥源提供合理依據。方法：1、以文獻資料為指導對柴胡屬藥用植物的分布地點進行確定,對于分布較廣的種類如北柴胡,擬收集10個產地以上的標本及樣品,每一產地采集或收集數個-10個居群的樣品；對于分布區(qū)域較為局限的種類,則盡可能獲取多個居群的個體,同時聯(lián)合利用國內各標本館中所藏柴胡屬植物樣本,保證標本和樣品的準確性、全面性和代表性。將所收集到的樣本進行準確的分類鑒定,并歸納品種、產地等信息,標本及實驗樣品均統(tǒng)一存放于陰涼干燥處備用。2、以超高效液相色譜電噴霧質譜聯(lián)用儀(UPLC-ESI-MS)為分析平臺,對柴胡屬植物的化學成分進行代謝譜分析,并依次對質譜儀器的檢測參數、色譜柱溫、進樣量、流動相組成及流速、樣品的提取工藝等進行考察。3、用考察出來的最佳實驗條件對柴胡屬植物的地上地下部分依次進行化學成分代謝譜的數據及圖譜的采集,對柴胡皂苷A、C、D等成分進行含量測定,并對獲得的數據用多元統(tǒng)計分析軟件SIMCA-P進行主成分分析,對不同品種、產地的柴胡作出質量判定。4、將所采集的化學成分代謝譜的數據及圖譜整理歸納后,以V C++及SQLserver2005等軟件,建立柴胡屬植物代謝組學分析專屬數據庫。結果：1、對柴胡屬植物資源的調查一共采得213份柴胡屬植物標本,分屬19種、7變種、4變型；柴胡屬植物資源主要集中分布于我國的東北、華北和西北等地區(qū),資源蘊藏量占全國的60%以上,品種又以柴胡、狹葉柴胡和銀州柴胡為主。2、由于各地用藥習慣不一,藥用的柴胡多為地方習用品種。即使所產為栽培的柴胡,但由于種源不純或各地區(qū)互相引種,栽培地常有數種農家型的柴胡混生。目前栽培產量已遠遠超過野生品,但由于長期的人工栽培,加上種植集中且品種單一,品種出現種質退化,抗性降低,不同地區(qū)出產的藥材質量大多參差不齊。3、作為商品用途的各種柴胡,無論來源是野生或者栽培,不同品種基原的柴胡在部分地區(qū)的購銷過程中使用相同的藥名;而同一品種基原的柴胡藥材往往又有多個不同的商品名稱。因此出現了同一基原植物在不同地區(qū)商品中歸類不同,而同一商品名可能包括不同基原的植物,存在異物同名或同物異名的混亂現象,品名與品種區(qū)分不明。4、初步考察出了適合柴胡屬植物的供試品提取條件為20倍溶媒比的5%氨水甲醇,超聲波提取5次,每次30分鐘；對提取條件的驗證發(fā)現5個平行樣品的柴胡皂苷A、D總提取率分別為：0.5176%、0.5144%、0.5181%、0.5152%、0.5167%,平均值為0.5168%,RSD值為0.409%,表明優(yōu)化后提取條件的可行性及重現性均較好。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,含量測定方法快速準確、靈敏可靠、專屬性強。對照品以峰面積對進樣量進行線性回歸得方程為柴胡皂苷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。結果分別表明柴胡皂苷A在4.860μg-14.580gg范圍內,柴胡皂苷C在0.3135-3.1350gg范圍內,柴胡皂苷D在2.170μg-10.850μg范圍內線性關系良好。柴胡皂苷A的在樣品中的含量介于0.0452%-0.3963%,柴胡皂苷C的含量介于0.0096%-0.1945%,柴胡皂苷D的含量則介于0.1082%-0.3673%。6、進行主成分分析時,由軟件自動擬合,確定了最佳的主成分提取個數為2；同時從分析的結果出發(fā),得以對所分析的樣品中化學成分的異同作出判別,能夠較好地按化學成分的差異及品種、產地等信息對樣品進行分類,初步顯示柴胡藥材質量與品種、產地存在相關關系。7、建立了擁有300多張圖的柴胡屬藥用植物化學成分譜專屬數據庫,為以后的研究工作提供了參考依據。結論：1、作為一種傳統(tǒng)用藥,柴胡屬藥用植物在華北、西北、東北等地有比較廣泛的資源分布。雖然栽培資源的開發(fā)一定程度上緩解了本品在市場上的供應不足等問題,但由于野生的藥用植物資源曾經受各種災害和不良環(huán)境的選擇,抗逆性較強,保存著栽培種不具有或已經消失的特異基因,是寶貴的物種基因庫,開發(fā)的同時應注意保護。2、在綜合產量與質量等因素的前提下,針對合適的品種,栽培地應盡早制定藥材生產質量管理規(guī)范并盡可能按要求優(yōu)選一套成熟的柴胡種植技術,以從源頭提高藥材的質量。3、本研究構建的UPLC-ESI-Q-TOF-MS分析方法,為多基原的柴胡類藥材作出了更為“全面”的化學成分分析,同時,更關注產地及品種對藥材質量差異所引起的問題,是當前柴胡類藥材品種鑒定和質量評價的重要手段之一。4、構建柴胡屬藥用植物化學成分代謝組學專屬數據庫,方便了對柴胡研究資料的利用,有助于推動柴胡藥材的種植、加工、銷售等規(guī)范化管理,推進柴胡類藥材的現代化研究。

    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.

        柴胡屬藥用植物資源調查及基于代謝組學的質量評價研究

摘要3-8ABSTRACT8-14第一章 前言17-21    1.1 柴胡藥材質量評價研究的意義17-18    1.2 代謝組學在藥材質量評價的應用與意義18-19    1.3 本課題研究思路19    1.4 本課題的研究意義19-21第二章 柴胡屬藥用植物資源及藥用狀況調查21-32    2.1 柴胡屬藥用植物資源調查21-28    2.2 柴胡商品藥材的使用情況28-30    2.3 討論30-31    2.4 小結31-32第三章 基于UPLC-ESI-Q-TOF-MS的柴胡屬藥用植物代謝組學分析32-60    3.1 材料和方法32-43    3.2 柴胡樣品皂苷A、C、D的含量測定43-51    3.3 代謝組學數據分析51-57    3.4 討論57-60第四章 柴胡屬藥用植物化學成分代謝譜數據庫的構建60-67    4.1 數據庫編輯語言的選擇60-61    4.2 數據庫的設計61-62    4.3 數據庫的特點及功能62-65    4.4 數據庫的運行管理與維護65    4.5 結語65-67第五章 結論67-68參考文獻68-71攻讀學位期間成果71-72綜述72-85    參考文獻82-85附錄85-88    一、中英文(符號)縮寫對照表85-86    二、柴胡屬植物形態(tài)86-88致謝88-90統(tǒng)計學審稿證明90

  本文關鍵詞：柴胡屬藥用植物資源調查及基于代謝組學的質量評價研究，由筆耕文化傳播整理發(fā)布。