柴胡屬藥用植物的分子鑒定及柴胡藥材的質(zhì)量考察
發(fā)布時(shí)間：2019-06-04 12:17
【摘要】：各種柴胡的混用濫用會(huì)導(dǎo)致柴胡質(zhì)量的不可控制以及相應(yīng)的用藥安全的問題,對(duì)不同種柴胡屬藥用植物的鑒定就顯得尤為重要,建立一種快速準(zhǔn)確的新鑒定方法迫在眉睫。本論文試圖通過DNAbarcoding技術(shù)建立對(duì)柴胡屬常見藥用植物的分子鑒定新方法,并以此為基礎(chǔ),通過HPLC手段考察不同種不同產(chǎn)地柴胡的皂苷含量,從而達(dá)到質(zhì)量評(píng)價(jià)的作用。本文首先通過小樣本擴(kuò)增不同種柴胡的4種候選DNA條形碼序列,ITS、psbA-trnH、matK、rbcL,比較這4種DNA條形碼的鑒定效率,從而篩選出最佳DNA條形碼。進(jìn)而大樣本量的擴(kuò)增該最佳DNA條形碼序列,建立起序列數(shù)據(jù)庫,對(duì)這些數(shù)據(jù)進(jìn)行變異位點(diǎn)、遺傳距離、聚類、"單倍型-物種"4個(gè)層次的分析,以建立起柴胡屬常見藥用植物的分子鑒定方法。在對(duì)柴胡藥材的質(zhì)量評(píng)價(jià)方面,本文通過HPLC技術(shù)分析柴胡藥材的柴胡皂苷含量,并通過SAS 8.0進(jìn)行統(tǒng)計(jì)學(xué)分析,從而評(píng)價(jià)不同種柴胡的優(yōu)劣,不同產(chǎn)地柴胡的優(yōu)劣。最終將建立起來的分子鑒定及質(zhì)量評(píng)價(jià)方法用于對(duì)市售柴胡藥材的評(píng)價(jià),從而了解目前柴胡市場(chǎng)信息,給出合理的建議。本文取得了如下研究結(jié)果:(1)描述并總結(jié)了 7種柴胡屬常見藥用植物的性狀及粉末顯微特征,總結(jié)了柴胡屬內(nèi)常見的藥用植物的特點(diǎn)。(2)分析比較了各種DNA條形碼對(duì)不同種柴胡的序列擴(kuò)增效率、測(cè)序成功率、遺傳距離分析、聚類分析、"單倍型-物種"對(duì)應(yīng)關(guān)系,篩選出了適合柴胡屬常見藥用植物鑒定的最佳DNA條形碼,即:ITS序列。(3)成功建立了用于鑒定常見柴胡屬藥用植物的分子鑒定方法。本實(shí)驗(yàn)通過對(duì)大樣本量的柴胡屬藥用植物的ITS序列進(jìn)行擴(kuò)增,獲得了 7種常見藥用柴胡的19種ITS序列單倍型,統(tǒng)計(jì)得到了 86個(gè)變異位點(diǎn)。以此為基礎(chǔ),進(jìn)行了變異位點(diǎn)、遺傳距離、聚類、"單倍型-物種"四個(gè)層次的分析,從而確定了 ITS序列能夠成功對(duì)柴胡屬常見藥用植物進(jìn)行鑒定,并建立了基于19種ITS序列單倍型檢索的"單倍型-物種"的鑒定方法。(4)以柴胡皂苷含量為標(biāo)準(zhǔn)評(píng)價(jià)了各類柴胡的質(zhì)量。柴胡樣品中的三種柴胡皂苷SSa、SSd、SSc的含量有著密切的正相關(guān)關(guān)系。按照柴胡皂苷的含量高低,不同種柴胡的優(yōu)劣順序?yàn)?窄竹葉柴胡銀州柴胡北柴胡錐葉柴胡狹葉柴胡。此外,本實(shí)驗(yàn)亦評(píng)價(jià)了不同產(chǎn)地柴胡的皂苷含量,發(fā)現(xiàn)山西與甘肅所產(chǎn)柴胡質(zhì)量較優(yōu),而寧夏所產(chǎn)次之。(5)對(duì)市售柴胡藥材進(jìn)行了質(zhì)量評(píng)價(jià)。本論文收集了河北安國、河南禹州、安徽亳州、廣東廣州、四川荷花池五大藥材市場(chǎng)的柴胡藥材,利用(3)中建立的分子鑒定方法,對(duì)市售柴胡藥材進(jìn)行了物種鑒定,明確了目前市場(chǎng)上柴胡以窄竹葉柴胡和北柴胡為主,銀州柴胡亦有一定份額,狹葉柴胡、錐葉柴胡、三島柴胡較少。以《中國藥典》規(guī)定的柴胡皂苷a、d含量為評(píng)價(jià)標(biāo)準(zhǔn),這五大藥材市場(chǎng)柴胡藥材合格率為100%,但不同種柴胡的皂苷含量差異較大,窄竹葉柴胡能達(dá)到4%以上,而錐葉柴胡僅為0.4%左右。
[Abstract]:The misuse of the various bupleurum can lead to the incontrol of the quality of the bupleurum and the corresponding safety of the medication. The identification of the medicinal plants of the genus Bupleurum is of particular importance, and the establishment of a rapid and accurate new method of identification is urgent. This paper attempts to establish a new method for molecular identification of the common medicinal plants of the genus Bupleurum by means of DNA barcoding, and on the basis of which, the content of the soap in different places of origin is investigated by means of HPLC, so as to achieve the effect of quality evaluation. In this paper, four candidate DNA bar code sequences, ITS, psbA-trnH, matK and rbcL of different kinds of Bupleurum chinense were amplified by small sample, and the identification efficiency of the four DNA barcodes was compared, and the optimal DNA barcode was selected. And then a large sample size is used for amplifying the optimal DNA barcode sequence, a sequence database is established, the data is subjected to analysis of the mutation sites, the genetic distance, the cluster and the "haplotype-species", so as to establish a molecular identification method for the common medicinal plants of the genus Bupleurum. In the aspect of quality evaluation of Bupleurum chinense, this paper analyzes the content of Chaihu in Bupleurum chinense by HPLC, and makes a statistical analysis by SAS 8.0, so as to evaluate the advantages and disadvantages of the different kinds of bupleurum, and the quality of the bupleurum in different places. And finally, the established molecular identification and quality evaluation method is used for evaluating the commercial radix bupleuri medicinal materials, so as to obtain the reasonable proposal of the present radix bupleuri market information. The following results were obtained: (1) The characters and the characteristics of the common medicinal plants of the genus Bupleurum were described and summarized, and the characteristics of the common medicinal plants in the genus Bupleurum were summarized. (2) The sequence amplification efficiency, the success rate of the sequencing, the genetic distance analysis, the cluster analysis and the "haplotype-species"-corresponding relation of the various DNA barcodes on the different kinds of bupleurum are compared, and the optimal DNA barcode suitable for the identification of the common medicinal plants of the genus Bupleurum is selected, that is, the ITS sequence. (3) The molecular identification method for identifying the medicinal plants of the genus Bupleurum is successfully established. In this experiment, the ITS sequence of the medicinal plants of Bupleurum belonging to the genus Bupleurum was amplified, and 19 kinds of ITS sequence haplotypes of 7 common medicinal bupleurum were obtained, and 86 mutation sites were obtained. On the basis of this, the analysis of four levels, such as mutation site, genetic distance, cluster and "haplotype-species", was carried out, so that it was determined that the ITS sequence could successfully identify the common medicinal plants of the genus Bupleurum, and established the identification method of the "haplotype-species" based on the haplotype search of the 19 kinds of ITS sequences. And (4) the quality of the various bupleurum is evaluated by using the content of the chaihu soap as the standard. There is a close positive correlation between the contents of three kinds of the Chinese Chaihu soap, SSa, SSd and SSc in the Bupleurum chinense sample. According to the content of the chaihu, the advantages and disadvantages of the different kinds of bupleurum are as follows: In addition, the experiment also evaluated the soap content of Bupleurum in different places, and found that the quality of the bupleurum produced by Shanxi and Gansu was better, and the second in Ningxia. And (5) carrying out quality evaluation on the commercial radix bupleuri medicinal materials. In this paper, the Chinese medicinal materials of the five medicinal materials of Anguo, Henan, Anhui, Guangdong, Guangzhou and Sichuan were collected, and the molecular identification methods established in (3) were used to identify the medicinal materials of Bupleurum chinense. It is clear that the radix bupleuri on the market is mainly composed of the narrow bamboo leaves, the bupleurum root and the northern bupleurum, and the radix bupleuri of the silver state also has a certain share, and the radix bupleuri, the bupleurum root and the three islands of the bupleurum are less. The content of Chaihu was 100%, but the content of Chaihu was more than 4%, while the root of Chaihu was only about 0.4%.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R284.1
[Abstract]:The misuse of the various bupleurum can lead to the incontrol of the quality of the bupleurum and the corresponding safety of the medication. The identification of the medicinal plants of the genus Bupleurum is of particular importance, and the establishment of a rapid and accurate new method of identification is urgent. This paper attempts to establish a new method for molecular identification of the common medicinal plants of the genus Bupleurum by means of DNA barcoding, and on the basis of which, the content of the soap in different places of origin is investigated by means of HPLC, so as to achieve the effect of quality evaluation. In this paper, four candidate DNA bar code sequences, ITS, psbA-trnH, matK and rbcL of different kinds of Bupleurum chinense were amplified by small sample, and the identification efficiency of the four DNA barcodes was compared, and the optimal DNA barcode was selected. And then a large sample size is used for amplifying the optimal DNA barcode sequence, a sequence database is established, the data is subjected to analysis of the mutation sites, the genetic distance, the cluster and the "haplotype-species", so as to establish a molecular identification method for the common medicinal plants of the genus Bupleurum. In the aspect of quality evaluation of Bupleurum chinense, this paper analyzes the content of Chaihu in Bupleurum chinense by HPLC, and makes a statistical analysis by SAS 8.0, so as to evaluate the advantages and disadvantages of the different kinds of bupleurum, and the quality of the bupleurum in different places. And finally, the established molecular identification and quality evaluation method is used for evaluating the commercial radix bupleuri medicinal materials, so as to obtain the reasonable proposal of the present radix bupleuri market information. The following results were obtained: (1) The characters and the characteristics of the common medicinal plants of the genus Bupleurum were described and summarized, and the characteristics of the common medicinal plants in the genus Bupleurum were summarized. (2) The sequence amplification efficiency, the success rate of the sequencing, the genetic distance analysis, the cluster analysis and the "haplotype-species"-corresponding relation of the various DNA barcodes on the different kinds of bupleurum are compared, and the optimal DNA barcode suitable for the identification of the common medicinal plants of the genus Bupleurum is selected, that is, the ITS sequence. (3) The molecular identification method for identifying the medicinal plants of the genus Bupleurum is successfully established. In this experiment, the ITS sequence of the medicinal plants of Bupleurum belonging to the genus Bupleurum was amplified, and 19 kinds of ITS sequence haplotypes of 7 common medicinal bupleurum were obtained, and 86 mutation sites were obtained. On the basis of this, the analysis of four levels, such as mutation site, genetic distance, cluster and "haplotype-species", was carried out, so that it was determined that the ITS sequence could successfully identify the common medicinal plants of the genus Bupleurum, and established the identification method of the "haplotype-species" based on the haplotype search of the 19 kinds of ITS sequences. And (4) the quality of the various bupleurum is evaluated by using the content of the chaihu soap as the standard. There is a close positive correlation between the contents of three kinds of the Chinese Chaihu soap, SSa, SSd and SSc in the Bupleurum chinense sample. According to the content of the chaihu, the advantages and disadvantages of the different kinds of bupleurum are as follows: In addition, the experiment also evaluated the soap content of Bupleurum in different places, and found that the quality of the bupleurum produced by Shanxi and Gansu was better, and the second in Ningxia. And (5) carrying out quality evaluation on the commercial radix bupleuri medicinal materials. In this paper, the Chinese medicinal materials of the five medicinal materials of Anguo, Henan, Anhui, Guangdong, Guangzhou and Sichuan were collected, and the molecular identification methods established in (3) were used to identify the medicinal materials of Bupleurum chinense. It is clear that the radix bupleuri on the market is mainly composed of the narrow bamboo leaves, the bupleurum root and the northern bupleurum, and the radix bupleuri of the silver state also has a certain share, and the radix bupleuri, the bupleurum root and the three islands of the bupleurum are less. The content of Chaihu was 100%, but the content of Chaihu was more than 4%, while the root of Chaihu was only about 0.4%.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R284.1
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