本文關(guān)鍵詞：柴胡屬藥用植物資源調(diào)查及基于代謝組學(xué)的質(zhì)量評(píng)價(jià)研究，，由筆耕文化傳播整理發(fā)布。

        中藥材質(zhì)量不穩(wěn)定是當(dāng)前中藥發(fā)展面臨的一個(gè)重大問題,造成這個(gè)問題的一個(gè)主要原因是同種藥材來源復(fù)雜多樣。長期以來,多種同屬植物被當(dāng)作一種藥材使用的情況時(shí)有發(fā)生,而對(duì)其進(jìn)行的鑒別與質(zhì)量控制都大不盡如人意。中藥柴胡始載于《神農(nóng)本草經(jīng)》,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(jīng)不調(diào)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(jù)以往統(tǒng)計(jì)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(chǎn)品種和歷史使用習(xí)慣而在民間入藥或自產(chǎn)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(xué)成分在植物發(fā)育和收采加工過程中可能會(huì)發(fā)生很大的變化,使得用傳統(tǒng)的方法對(duì)柴胡進(jìn)行鑒定存在相當(dāng)大的難度。鑒于目前有的地區(qū)把當(dāng)?shù)厮a(chǎn)的5、6種柴胡屬植物混在一起當(dāng)做藥用,當(dāng)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(dāng)混亂,藥材質(zhì)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(zhì)量穩(wěn)定。代謝組學(xué)(metabolomics)是20世紀(jì)90年代中期發(fā)展起來的對(duì)某一生物或細(xì)胞所有低分子量代謝產(chǎn)物進(jìn)行定性和定量分析的一門新學(xué)科。是繼基因組學(xué)、轉(zhuǎn)錄組學(xué)和蛋白質(zhì)組學(xué)之后的又一門新興的組學(xué)技術(shù),也是系統(tǒng)生物學(xué)研究不可或缺的重要基礎(chǔ)學(xué)科之一。作為代謝組學(xué)的重要分析手段,以(超)高效液相色譜質(zhì)譜聯(lián)用分析技術(shù)為平臺(tái),對(duì)植物所含的小分子化學(xué)成分常具有較強(qiáng)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(zhì)譜聯(lián)用分析儀,因能完整給出待分析物質(zhì)的分子離子碎片,有利于質(zhì)譜分析器對(duì)物質(zhì)的準(zhǔn)確鑒別。相對(duì)其他分析方法,所產(chǎn)生的化學(xué)成分代謝譜能夠更準(zhǔn)確地反映生物體系的狀態(tài)。由此,可比較準(zhǔn)確地對(duì)生藥質(zhì)量進(jìn)行鑒別評(píng)價(jià),亦有利于建立系統(tǒng)規(guī)范的化學(xué)成分?jǐn)?shù)據(jù)庫(如柴胡藥材數(shù)據(jù)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(biāo)轉(zhuǎn)化為少數(shù)幾個(gè)綜合指標(biāo)的非監(jiān)督性多元統(tǒng)計(jì)分析方法。作為一種高維數(shù)據(jù)解析方法,其應(yīng)用著重于發(fā)掘化學(xué)數(shù)據(jù)的外在表征與物質(zhì)內(nèi)在組成之間的相互關(guān)系,在最大程度上提取原始信息的同時(shí)對(duì)數(shù)據(jù)進(jìn)行降維處理,解決了液質(zhì)聯(lián)用等現(xiàn)代分析儀器或檢測技術(shù)提供的大量而又豐富的量測信息所帶來的統(tǒng)計(jì)分析難題,是目前代謝組學(xué)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(zhì)譜分析等數(shù)據(jù)研究。目的：對(duì)當(dāng)前柴胡屬植物的品種資源及藥用狀況進(jìn)行詳細(xì)的調(diào)查,提出確保柴胡藥材準(zhǔn)確鑒定及品種合理開發(fā)的策略；應(yīng)用液質(zhì)聯(lián)用技術(shù)平臺(tái),通過主成分分析研究,建立準(zhǔn)確、快速、可靠的柴胡類藥材鑒定和質(zhì)量評(píng)價(jià)體系,同時(shí)也為研究新品種,擴(kuò)大新藥源提供合理依據(jù)。方法：1、以文獻(xiàn)資料為指導(dǎo)對(duì)柴胡屬藥用植物的分布地點(diǎn)進(jìn)行確定,對(duì)于分布較廣的種類如北柴胡,擬收集10個(gè)產(chǎn)地以上的標(biāo)本及樣品,每一產(chǎn)地采集或收集數(shù)個(gè)-10個(gè)居群的樣品；對(duì)于分布區(qū)域較為局限的種類,則盡可能獲取多個(gè)居群的個(gè)體,同時(shí)聯(lián)合利用國內(nèi)各標(biāo)本館中所藏柴胡屬植物樣本,保證標(biāo)本和樣品的準(zhǔn)確性、全面性和代表性。將所收集到的樣本進(jìn)行準(zhǔn)確的分類鑒定,并歸納品種、產(chǎn)地等信息,標(biāo)本及實(shí)驗(yàn)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(zhì)譜聯(lián)用儀(UPLC-ESI-MS)為分析平臺(tái),對(duì)柴胡屬植物的化學(xué)成分進(jìn)行代謝譜分析,并依次對(duì)質(zhì)譜儀器的檢測參數(shù)、色譜柱溫、進(jìn)樣量、流動(dòng)相組成及流速、樣品的提取工藝等進(jìn)行考察。3、用考察出來的最佳實(shí)驗(yàn)條件對(duì)柴胡屬植物的地上地下部分依次進(jìn)行化學(xué)成分代謝譜的數(shù)據(jù)及圖譜的采集,對(duì)柴胡皂苷A、C、D等成分進(jìn)行含量測定,并對(duì)獲得的數(shù)據(jù)用多元統(tǒng)計(jì)分析軟件SIMCA-P進(jìn)行主成分分析,對(duì)不同品種、產(chǎn)地的柴胡作出質(zhì)量判定。4、將所采集的化學(xué)成分代謝譜的數(shù)據(jù)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(xué)分析專屬數(shù)據(jù)庫。結(jié)果：1、對(duì)柴胡屬植物資源的調(diào)查一共采得213份柴胡屬植物標(biāo)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(yùn)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(xí)慣不一,藥用的柴胡多為地方習(xí)用品種。即使所產(chǎn)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(shù)種農(nóng)家型的柴胡混生。目前栽培產(chǎn)量已遠(yuǎn)遠(yuǎn)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(xiàn)種質(zhì)退化,抗性降低,不同地區(qū)出產(chǎn)的藥材質(zhì)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(gè)不同的商品名稱。因此出現(xiàn)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(xiàn)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(duì)提取條件的驗(yàn)證發(fā)現(xiàn)5個(gè)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(xiàn)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(zhǔn)確、靈敏可靠、專屬性強(qiáng)。對(duì)照品以峰面積對(duì)進(jìn)樣量進(jìn)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(jié)果分別表明柴胡皂苷A在4.860μg-14.580gg范圍內(nèi),柴胡皂苷C在0.3135-3.1350gg范圍內(nèi),柴胡皂苷D在2.170μg-10.850μg范圍內(nèi)線性關(guān)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(jìn)行主成分分析時(shí),由軟件自動(dòng)擬合,確定了最佳的主成分提取個(gè)數(shù)為2；同時(shí)從分析的結(jié)果出發(fā),得以對(duì)所分析的樣品中化學(xué)成分的異同作出判別,能夠較好地按化學(xué)成分的差異及品種、產(chǎn)地等信息對(duì)樣品進(jìn)行分類,初步顯示柴胡藥材質(zhì)量與品種、產(chǎn)地存在相關(guān)關(guān)系。7、建立了擁有300多張圖的柴胡屬藥用植物化學(xué)成分譜專屬數(shù)據(jù)庫,為以后的研究工作提供了參考依據(jù)。結(jié)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(yīng)不足等問題,但由于野生的藥用植物資源曾經(jīng)受各種災(zāi)害和不良環(huán)境的選擇,抗逆性較強(qiáng),保存著栽培種不具有或已經(jīng)消失的特異基因,是寶貴的物種基因庫,開發(fā)的同時(shí)應(yīng)注意保護(hù)。2、在綜合產(chǎn)量與質(zhì)量等因素的前提下,針對(duì)合適的品種,栽培地應(yīng)盡早制定藥材生產(chǎn)質(zhì)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(shù),以從源頭提高藥材的質(zhì)量。3、本研究構(gòu)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(xué)成分分析,同時(shí),更關(guān)注產(chǎn)地及品種對(duì)藥材質(zhì)量差異所引起的問題,是當(dāng)前柴胡類藥材品種鑒定和質(zhì)量評(píng)價(jià)的重要手段之一。4、構(gòu)建柴胡屬藥用植物化學(xué)成分代謝組學(xué)專屬數(shù)據(jù)庫,方便了對(duì)柴胡研究資料的利用,有助于推動(dòng)柴胡藥材的種植、加工、銷售等規(guī)范化管理,推進(jìn)柴胡類藥材的現(xiàn)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(diào)查及基于代謝組學(xué)的質(zhì)量評(píng)價(jià)研究

摘要3-8ABSTRACT8-14第一章 前言17-21    1.1 柴胡藥材質(zhì)量評(píng)價(jià)研究的意義17-18    1.2 代謝組學(xué)在藥材質(zhì)量評(píng)價(jià)的應(yīng)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(diào)查21-32    2.1 柴胡屬藥用植物資源調(diào)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(jié)31-32第三章 基于UPLC-ESI-Q-TOF-MS的柴胡屬藥用植物代謝組學(xué)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(xué)數(shù)據(jù)分析51-57    3.4 討論57-60第四章 柴胡屬藥用植物化學(xué)成分代謝譜數(shù)據(jù)庫的構(gòu)建60-67    4.1 數(shù)據(jù)庫編輯語言的選擇60-61    4.2 數(shù)據(jù)庫的設(shè)計(jì)61-62    4.3 數(shù)據(jù)庫的特點(diǎn)及功能62-65    4.4 數(shù)據(jù)庫的運(yùn)行管理與維護(hù)65    4.5 結(jié)語65-67第五章 結(jié)論67-68參考文獻(xiàn)68-71攻讀學(xué)位期間成果71-72綜述72-85    參考文獻(xiàn)82-85附錄85-88    一、中英文(符號(hào))縮寫對(duì)照表85-86    二、柴胡屬植物形態(tài)86-88致謝88-90統(tǒng)計(jì)學(xué)審稿證明90

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