《中国药典》所载甘草的分子鉴定及市售甘草药材的质量评价

发布时间：2018-04-08 16:01

  本文选题：HPLC　切入点：ITS　出处：《北京中医药大学》2017年硕士论文

【摘要】：甘草是我国最常用的大宗药材之一,始载于《神农本草经》中,被列为上品,在临床方剂中使用频率极高,素有"十方九草"之称,有补脾益气,清热解毒,祛痰止咳,缓急止痛,调和诸药的功效。2015版《中国药典》规定,乌拉尔甘草Glycyrrhiza uralensis Fisch.、光果甘草 Glycyrrhiza glabra L.及胀果甘草Glycyrrhiza inflata Bat.的干燥根及根茎可作为甘草入药。有研究表明,不同基原甘草中活性成分含量存在显著区别,并且存在明显的药效差异。然而,3种基原甘草为同属植物,入药部位均为根部,其显微特征极为相似,因此难以通过传统的性状及显微鉴定方法对其进行有效区分。因此,亟需建立一种快速准确的方法对3种基原甘草进行鉴定。近些年来,分子鉴定作为传统鉴定的有力补充,被广泛运用于药用动植物的鉴定中。2011年,中国条形码工作组通过对中国种子植物中75科141属1757种共约6286样本的4个DNA候选条形码片段(rbcL、matK、psbA-trnH和ITS)进行分析,认为ITS序列和ITS2序列的物种分辨效率显著高于rbcL + matK,而psbA-trnH序列被证明鉴定能力优于其他叶绿体基因序列。因此,基于前人的研究基础,本文将ITS序列和pstA-trnH序列作为鉴定3种不同基原甘草的备选DNA条形码。《中国药典》规定,中药甘草的指标性成分为甘草酸和甘草苷。随着现代研究的不断深入,从甘草中分离得到了大量其他的活性成分,包括异甘草酸、异甘草苷、甘草素等,发挥着抗癌、抗病毒、抗炎、保肝等多种药理作用。其中,三萜类化合物异甘草酸是甘草酸的差向异构体,较甘草酸而言具有更好的活性和安全性,目前关于甘草中有效成分含量分析的研究报道均未对异甘草酸与甘草酸进行区分。同时,甘草中黄酮类化合物的含量对于确定甘草的质量同样十分重要。三种不同基原甘草中甘草苷、异甘草苷、甘草素、异甘草素等主要黄酮成分的含量差异鲜有报道。综上,本论文试图从分子层面和药效成分含量层面对三种基原甘草进行鉴别和比较。首先,大量收集植物材料,建立种质资源库,利用DNA条形码技术,通过对三种不同基原甘草中核基因ITS序列和叶绿体基因psbA-trnH序列进行扩增,从而从分子水平对三种不同基原甘草进行鉴别。在明确基原的基础上,以来自同一产地的两年生乌拉尔甘草、光果甘草及胀果甘草作为实验材料,排除产地环境及栽培条件对于次生代谢产物含量的影响,建立同时测定甘草酸与异甘草酸含量的HPLC方法,以及同时测定四种黄酮类成分——甘草苷、异甘草苷、甘草素与异甘草素含量的HPLC方法,通过探究三萜类及黄酮类化合物含量的差异,从而为药用甘草的质量控制及以不同化合物为目标的优质甘草筛选与定向育种提供科学依据。本论文共取得了如下研究结果:(1)从全国7省份21居群采集了 238株甘草材料。利用PCR扩增获得了长度为616 bp的ITS序列以及389 bp的psbA-trnH序列;通过DNAMAN比对分析,在ITS序列中找到4个变异位点,并确定了 2种ITS单倍型,在psbA-trnH序列中找到3个变异位点,并确定了 4种psbA-trnH单倍型;结合ITS及psbA-trnH序列分析,确定了 3种基原甘草的分子鉴定方案。(2)建立了同时测定甘草中2种三萜类活性成分——甘草酸与异甘草酸含量的HPLC分析方法,并对中国药典规定的3种不同基原甘草样品中甘草酸与异甘草酸的含量进行分析比较。结果显示:甘草酸和异甘草酸达到了良好的分离;线性范围分别为0.010 70～0.214 0μg和0.216 4～4.328μg;检测限分别为3.210 ng和4.330 ng;定量限分别为11.26 ng和12.65 ng;平均回收率(n=3)分别为99.2%～100.1%和99.8%～99.9%。在3种基原的甘草样品中,光果甘草的甘草酸和异甘草酸含量最高,分别为(2.650±0.06421)mg·g-1 和(37.18±0.844 3)mg·g-1(n=25);乌拉尔甘草的含量次之,分别为(1.975±0.05712)mg·g-1和(29.41±0.7412)mg·g-1(n=25);胀果甘草的含量最低,分别为(1.604±0.04372)mg·g-1和(17.81±0.5021)mg·g-1(n=25)。甘草酸和异甘草酸的含量存在显著相关关系。本文方法可用于不同基原甘草中甘草酸和异甘草酸的含量分析,并为以异甘草酸为目标的优质甘草筛选及定向育种奠定基础。(3)建立了 3种不同基原甘草样品中甘草苷、异甘草苷、甘草素和异甘草素含量的HPLC分析方法,并对中国药典规定的3种不同基原甘草样品中4种黄酮类成分的含量进行分析比较。结果显示:甘草苷、异甘草苷、甘草素和异甘草素分离良好,线性范围分别为 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,检测限和定量限依次为 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。本方法灵敏度、精密度、准确性、重复性、回收率、耐用性均良好。在3种基原的甘草样品中,4个黄酮类成分的含量存在极显著差异(P0.01),甘草苷、异甘草苷、甘草素、异甘草素在乌拉尔甘草中的含量均最高,分别为(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,含量分别为(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,胀果甘草最低,含量分别为(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;甘草苷与异甘草苷、甘草素与异甘草素的含量在3种不同基原甘草样品中均存在极显著相关关系(P0.01)。本文建立的HPLC方法可用于甘草中甘草苷、异甘草苷、甘草素和异甘草素的含量分析,并为甘草的质量控制及以黄酮类化合物为目标的优质甘草筛选与定向育种提供科学依据。(4)利用(1)中建立的分子鉴定方案对来自全国4个主要中药材市场的40份甘草药材进行了准确鉴定,并进一步利用(2)和(3)中建立的HPLC方法测定了各药材中2种三萜类有效成分及4种黄酮类有效成分的含量,应用SPSS 21.0对HPLC结果进行统计学分析,从而对市售甘草药材的质量进行评价。
[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�涓�11.26 ng鍜，

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