本文選題：HPLC　切入點：ITS　出處：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：甘草是我國最常用的大宗藥材之一,始載于《神農(nóng)本草經(jīng)》中,被列為上品,在臨床方劑中使用頻率極高,素有"十方九草"之稱,有補脾益氣,清熱解毒,祛痰止咳,緩急止痛,調(diào)和諸藥的功效。2015版《中國藥典》規(guī)定,烏拉爾甘草Glycyrrhiza uralensis Fisch.、光果甘草 Glycyrrhiza glabra L.及脹果甘草Glycyrrhiza inflata Bat.的干燥根及根莖可作為甘草入藥。有研究表明,不同基原甘草中活性成分含量存在顯著區(qū)別,并且存在明顯的藥效差異。然而,3種基原甘草為同屬植物,入藥部位均為根部,其顯微特征極為相似,因此難以通過傳統(tǒng)的性狀及顯微鑒定方法對其進行有效區(qū)分。因此,亟需建立一種快速準(zhǔn)確的方法對3種基原甘草進行鑒定。近些年來,分子鑒定作為傳統(tǒng)鑒定的有力補充,被廣泛運用于藥用動植物的鑒定中。2011年,中國條形碼工作組通過對中國種子植物中75科141屬1757種共約6286樣本的4個DNA候選條形碼片段(rbcL、matK、psbA-trnH和ITS)進行分析,認(rèn)為ITS序列和ITS2序列的物種分辨效率顯著高于rbcL + matK,而psbA-trnH序列被證明鑒定能力優(yōu)于其他葉綠體基因序列。因此,基于前人的研究基礎(chǔ),本文將ITS序列和pstA-trnH序列作為鑒定3種不同基原甘草的備選DNA條形碼。《中國藥典》規(guī)定,中藥甘草的指標(biāo)性成分為甘草酸和甘草苷。隨著現(xiàn)代研究的不斷深入,從甘草中分離得到了大量其他的活性成分,包括異甘草酸、異甘草苷、甘草素等,發(fā)揮著抗癌、抗病毒、抗炎、保肝等多種藥理作用。其中,三萜類化合物異甘草酸是甘草酸的差向異構(gòu)體,較甘草酸而言具有更好的活性和安全性,目前關(guān)于甘草中有效成分含量分析的研究報道均未對異甘草酸與甘草酸進行區(qū)分。同時,甘草中黃酮類化合物的含量對于確定甘草的質(zhì)量同樣十分重要。三種不同基原甘草中甘草苷、異甘草苷、甘草素、異甘草素等主要黃酮成分的含量差異鮮有報道。綜上,本論文試圖從分子層面和藥效成分含量層面對三種基原甘草進行鑒別和比較。首先,大量收集植物材料,建立種質(zhì)資源庫,利用DNA條形碼技術(shù),通過對三種不同基原甘草中核基因ITS序列和葉綠體基因psbA-trnH序列進行擴增,從而從分子水平對三種不同基原甘草進行鑒別。在明確基原的基礎(chǔ)上,以來自同一產(chǎn)地的兩年生烏拉爾甘草、光果甘草及脹果甘草作為實驗材料,排除產(chǎn)地環(huán)境及栽培條件對于次生代謝產(chǎn)物含量的影響,建立同時測定甘草酸與異甘草酸含量的HPLC方法,以及同時測定四種黃酮類成分——甘草苷、異甘草苷、甘草素與異甘草素含量的HPLC方法,通過探究三萜類及黃酮類化合物含量的差異,從而為藥用甘草的質(zhì)量控制及以不同化合物為目標(biāo)的優(yōu)質(zhì)甘草篩選與定向育種提供科學(xué)依據(jù)。本論文共取得了如下研究結(jié)果:(1)從全國7省份21居群采集了 238株甘草材料。利用PCR擴增獲得了長度為616 bp的ITS序列以及389 bp的psbA-trnH序列;通過DNAMAN比對分析,在ITS序列中找到4個變異位點,并確定了 2種ITS單倍型,在psbA-trnH序列中找到3個變異位點,并確定了 4種psbA-trnH單倍型;結(jié)合ITS及psbA-trnH序列分析,確定了 3種基原甘草的分子鑒定方案。(2)建立了同時測定甘草中2種三萜類活性成分——甘草酸與異甘草酸含量的HPLC分析方法,并對中國藥典規(guī)定的3種不同基原甘草樣品中甘草酸與異甘草酸的含量進行分析比較。結(jié)果顯示:甘草酸和異甘草酸達到了良好的分離;線性范圍分別為0.010 70～0.214 0μg和0.216 4～4.328μg;檢測限分別為3.210 ng和4.330 ng;定量限分別為11.26 ng和12.65 ng;平均回收率(n=3)分別為99.2%～100.1%和99.8%～99.9%。在3種基原的甘草樣品中,光果甘草的甘草酸和異甘草酸含量最高,分別為(2.650±0.06421)mg·g-1 和(37.18±0.844 3)mg·g-1(n=25);烏拉爾甘草的含量次之,分別為(1.975±0.05712)mg·g-1和(29.41±0.7412)mg·g-1(n=25);脹果甘草的含量最低,分別為(1.604±0.04372)mg·g-1和(17.81±0.5021)mg·g-1(n=25)。甘草酸和異甘草酸的含量存在顯著相關(guān)關(guān)系。本文方法可用于不同基原甘草中甘草酸和異甘草酸的含量分析,并為以異甘草酸為目標(biāo)的優(yōu)質(zhì)甘草篩選及定向育種奠定基礎(chǔ)。(3)建立了 3種不同基原甘草樣品中甘草苷、異甘草苷、甘草素和異甘草素含量的HPLC分析方法,并對中國藥典規(guī)定的3種不同基原甘草樣品中4種黃酮類成分的含量進行分析比較。結(jié)果顯示:甘草苷、異甘草苷、甘草素和異甘草素分離良好,線性范圍分別為 1.15×10-2～0.230μg、4.45×10-3～8.90×10-2 μg、1.15×10-3～2.30×10-2 μg、2.27×10-3～4.54×10-2μ g,檢測限和定量限依次為 1.13 ng 和 3.42 ng、0.896 ng 和 2.70 ng、0.463 ng和1.39 ng、0.454 ng和1.38 ng。本方法靈敏度、精密度、準(zhǔn)確性、重復(fù)性、回收率、耐用性均良好。在3種基原的甘草樣品中,4個黃酮類成分的含量存在極顯著差異(P0.01),甘草苷、異甘草苷、甘草素、異甘草素在烏拉爾甘草中的含量均最高,分別為(0.75±0.524)mg.g-1、(4.453±0.057)mg·g-1、(0.610±0.019)mg·g-1 和(0.272±0.008)mg·g-1,光果甘草次之,含量分別為(6.623±0.405)mg·g-1、(1.562±0.053)mg·g-1、(0.325±0.036)mg·g-1 和(0.180±0.012)mg·g-1,脹果甘草最低,含量分別為(2.700±0.232)mg·g-1、(0.821±0.042)mg·g-1、(0.153±0.006)mg·g-1 和(0.115±0.005)mg·g-1;甘草苷與異甘草苷、甘草素與異甘草素的含量在3種不同基原甘草樣品中均存在極顯著相關(guān)關(guān)系(P0.01)。本文建立的HPLC方法可用于甘草中甘草苷、異甘草苷、甘草素和異甘草素的含量分析,并為甘草的質(zhì)量控制及以黃酮類化合物為目標(biāo)的優(yōu)質(zhì)甘草篩選與定向育種提供科學(xué)依據(jù)。(4)利用(1)中建立的分子鑒定方案對來自全國4個主要中藥材市場的40份甘草藥材進行了準(zhǔn)確鑒定,并進一步利用(2)和(3)中建立的HPLC方法測定了各藥材中2種三萜類有效成分及4種黃酮類有效成分的含量,應(yīng)用SPSS 21.0對HPLC結(jié)果進行統(tǒng)計學(xué)分析,從而對市售甘草藥材的質(zhì)量進行評價。
[Abstract]:Licorice is one of the most commonly used medicinal materials in China, was contained in the "Shen Nong's herbal classic", was listed as the top grade, frequently used in clinical prescription, known as "ten to nine grass", spleen qi, detoxification, cough expectorant, relieving pain, to reconcile various drug efficacy of.2015 < > version of Chinese Pharmacopoeia provisions, Ural Glycyrrhiza uralensis Fisch. light fruit licorice, dried root and rhizome of Glycyrrhiza glabra L. and licorice Glycyrrhiza inflata Glycyrrhiza inflata Bat. can be used as licorice. Studies have shown that there are significant differences between the content of active components in different raw licorice base, and there are obvious differences in efficacy. However, the 3 base the original licorice for both plants, medicinal parts are the root of the microscopic characteristics are very similar, so it is difficult to through the traditional characters and microscopic identification method to distinguish it. Therefore, it is urgent to build a rapid and accurate method for 3 kinds of raw licorice were identified. In recent years, molecular identification as a powerful supplement to the traditional identification, is widely used in the identification of medicinal plants in.2011, China bar working group by 4 DNA candidate bar code fragments on the China seed plants 75 families, 141 genera and 1757 species of a total of about 6286 samples (rbcL matK, psbA-trnH, and ITS) were analyzed, ITS sequences and ITS2 sequences of species resolution rate was significantly higher than that of rbcL + matK and psbA-trnH sequences were shown to sequence chloroplast gene identification ability is better than the other. Therefore, based on previous research, the ITS and pstA-trnH sequences as the identification of 3 different raw licorice alternative DNA bar code. China Pharmacopoeia > regulations, the index components of licorice for glycyrrhizic acid and liquiritin. With the further research, get a lot of other active constituents isolated from Glycyrrhiza, including ISO Glycyrrhizic acid, isoliquiritin, isoliquiritigenin, play anticancer, antiviral, anti-inflammatory, hepatoprotective and other pharmacological effects. Among them, three terpene compound isoglycyrrhizinate is epimers of glycyrrhizic acid is sweet, activity and safety of oxalic acid has better, the present study reports on the analysis of the content of active ingredients of licorice in none of the Glycyrrhizic acid and glycyrrhizic acid were distinguished. At the same time, the content of flavonoids in licorice is also important in determining the quality of licorice. Three kinds of different medium raw licorice licorice glycosides, isoliquiritin, isoliquiritigenin, rarely reported differences in the content of isoliquiritigenin mainly flavonoids. To sum up, this paper attempts to the original three kinds of licorice were identified and compared from the molecular level and the content of effective composition of layers. First of all, a large collection of plant materials, the establishment of germplasm resources, the use of DNA barcode technology based on three different Ji Yuangan The grass of nuclear gene ITS sequence and chloroplast psbA-trnH gene sequences were amplified, and from the molecular level of three kinds of raw licorice were identified. Based on the original clear, from the same origin of the Ural biennial licorice, Glycyrrhiza glabra and Glycyrrhiza inflata as experimental material, eliminate influence of environment and cultivation conditions of origin the content of secondary metabolites, the determination method of HPLC content of glycyrrhizic acid and glycyrrhizic acid, and simultaneous determination of four flavonoids, liquiritin, isoliquiritin, HPLC liquiritigenin and isoliquiritigenin content, the content of compound three explore differences of terpenoids and flavonoids, which provide the scientific the basis for the quality control of medicinal licorice and compounds with different target screening and breeding of high quality licorice. The results obtained are as follows: (1) from the country's 7 provinces in 21 populations Collected 238 strains of licorice materials. The ITS sequence was obtained by PCR amplification of 616 BP in length and psbA-trnH sequence of 389 BP DNAMAN; by comparison analysis, find 4 mutation sites in ITS sequences, and identified 2 ITS haplotypes, found 3 mutation sites in psbA-trnH sequences, and identified 4 the combination of ITS and psbA-trnH haplotypes; psbA-trnH sequence analysis, to determine the molecular identification scheme of 3 Glycyrrhizic group. (2) was established for the simultaneous determination of 2 kinds of licorice three terpene active ingredient, glycyrrhizic acid and glycyrrhizic acid content of the HPLC analysis method, the content and the Chinese Pharmacopoeia of 3 different origin licorice samples of glycyrrhizic acid and glycyrrhizic acid are analyzed and compared. Results show: glycyrrhizic acid and glycyrrhizic acid to achieve good separation; the linear range was 0.01070 ~ 0.2140 g and 0.2164 ~ 4.328 g; the detection limit was 3.210 ng and 4.33 0 ng;瀹氶噺闄愬垎鍒負(fù)11.26 ng鍜，

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