不同基原甘草的分子鉴定及市售甘草药材的质量评价

The molecular identification of licorice species and the quality evaluation of licorice slices

甘草是我国最常用的大宗中药材之一,在《神农本草经》中被列为上品。2015版《中华人民共和国药典》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。然而不同基原甘草药效差异显著,而通过传统方法对其进行鉴别十分困难。为了快速、准确地对不同基原甘草进行鉴定,本文从全国7省份21居群采集了240株甘草。利用PCR扩增获得了长度为616 bp的ITS序列以及389 bp的psb A-trn H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psb A-trn H序列中找到3个变异位点,并确定了4种psb A-trn H单倍型;结合ITS及psb A-trn H序列分析,确定了3种基原甘草的分子鉴定方案。利用该方案对来自全国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HPLC法测定各药材中2种三萜类有效成分(甘草酸及异甘草酸)及4种黄酮类有效成分(甘草素、异甘草素、甘草苷、异甘草苷)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。本文为不同基原甘草的准确鉴定提供了分子鉴定方案,并且对甘草药材的流通现状及质量评价具有指导意义。

Licorice is one of the most common herbs in traditional Chinese medicine,and classified as top grade in Shen Nong Ben Cao Jing. There are three different original plants of licorice stipulated in Chinese Pharmacopeia,Glycyrrhiza uralensis Fisch.,Glycyrrhiza glabra L.,and Glycyrrhiza inflata Bat. However,previous investigation showed that the pharmacodynamic effects of the three licorices were quite different. It is very difficult to identify them by the classical identification methods. In order to establish a fast and effective identification method,we collected 240 licorice plants from 21 populations of 7 provinces,and amplified their ITS and psb A-trn H sequences. ITS sequences with a full length of 616 bp and psb A-trn H sequences with a full length of 389 bp were obtained separately. Using DNAMAN to analyze these sequences,4 variable sites were found in ITS sequences and 2 ITS haplotypes were determined,and 3 variable sites were found in psb A-trn H sequences and 4 psb A-trn H haplotypes were determined. With the combination analysis of ITS and psb A-trn H sequences,the molecular identification method of original licorice was established. Using this method,40 samples of licorice slices collected from 4 main herbal material markets in China were identified successfully. Furthermore,the contents of 2 triterpenes,18α-glycyrrhizic acid and 18β-glycyrrhizic acid,and 4 flavonoids,liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin in these licorice pieces were examined by HPLC and the results were analyzed using SPSS 21.0. This study provides a new method in identification of licorice,which may serve as a guideline for quality control of licorice slices.