基于DNA条形码和红外光谱（FTIR）技术构建中药材青蒿快速鉴定体系

There are several critical problems existing not conducive to use Artemisia annua L. as a Chinese medicinal material, such as serious confusion and counterfeit, large differences of artemisinin content, poor quality now. Aiming to resolving these problems, we applied multi-fragment DNA barcoding identification strategy. Through the measurement and analysis of multi-loci combination, including nuclear gene ITS and chloroplast DNA regions rbcL, matK and trnH-psbA, combined with morphological evidence, universal DNA barcodes for A. annua were screened. At the same time, two methods, FTIR and HPLC, were adopted to determine the content of main chemical components in A. annua from different sources. Taking the artemisinin content determined by high performance liquid chromatographic method as dependent variable, using multiple regression equation, finding characteristic adsorption peak in infrared spectrogram, combined with curve fitting equation and Fourier self-deconvolution (FSD), characteristic adsorption peak area difference was calculated. And chemical recognition mathematical model for authentic output of A. annua was also established using SIMCA method. We will establish rapid identification and quality system of A.annua. This research will provide dependable standards and technology platforms for the identification and utilization of medical herbs A. annua.

针对中药材青蒿（Artemisia annua L.）存在严重的混伪、青蒿素含量差异大、质量难于保证的状况，采用多片段DNA条形码鉴定策略，通过对多基因位点组合（包括核基因的ITS和叶绿体基因的rbcL、matK以及trnH-psbA 等DNA序列）进行测定和分析，结合形态学证据，筛选出药材青蒿通用的DNA条形码序列。同时，采用FTIR技术和HPLC两种方法测定不同来源药材青蒿的主要化学成分的含量。以高效液相色谱方法测定的青蒿素含量为因变量，运用多元回归方程，在红外光谱图中找到与青蒿素含量的特征吸收峰，结合曲线拟合方程和傅里叶去自卷积计算出其特征吸收子峰的峰面积差异，同时利用SIMCA方法建立道地产出青蒿药材化学识别数学模型。构建中药材青蒿快速鉴定体系。为青蒿药材的鉴定和资源利用等提供可靠标准和技术平台。

生产中黄花蒿（Artemisia annua L.）与其近缘种种类混杂严重，影响了黄花蒿的药材质量。项目采用FTIR和DNA条形码技术对黄花蒿及其近缘种进行鉴定比较，以期为黄花蒿药材青蒿的快速鉴定提供快速、准确的方法。. (1)采用DNA条形码对黄花蒿及近缘种进行了鉴定研究。候选的4条序列（ITS、ITS2、rbcL、psbK-psbI）中，ITS2序列具有较好的PCR扩增成功率和测序成功率，在所考察的候选序列中种间变异大于种内变异，种间变异和种内变异也较高。ITS2序列的种间和种内遗传变异能较好的区分开，形成了较为明显的barcoding gap区。ITS2条形码可准确的鉴别中药材黄花蒿.. (2)利用FTIR技术和GC-MS分别对不同种黄花蒿及近缘种叶片原药材及挥发油成分进行检测和鉴定。挥发油以黄花蒿含量最高（1.86%），其次是南牡蒿、茵陈蒿、青蒿、牡蒿和艾蒿。FTIR分析结果表明，黄花蒿及近缘种一维图谱相似，酰胺类、芳香类以及萜类化合物种类较多且含量丰富；二阶导数图谱中，黄花蒿青蒿素成分振动吸收明显增强，可以明显将黄花蒿与其混淆中区分开。GC-MS分析显示，黄花蒿与其近缘种的挥发油成分中共检测出17个共有峰，28种化学成分，均含有较高樟脑、á-杜松烯、Crocetane、植烷、2,4-二叔丁基苯酚，但不同种间成分含量差异很大，植烷在黄花蒿中含量明显高于其它近缘种，龙脑成分只能在黄花蒿叶片中检测出，然而，á-雪松烯在青蒿、南牡蒿、茵陈蒿均较高，而在黄花蒿，艾蒿，牡蒿中含量均较低。最后通过聚类分析探讨了黄花蒿与其近缘种挥发油成分差异性，6 种材料明显聚为2类，其中，黄花蒿与牡蒿、艾蒿聚为一类，青蒿与茵陈蒿和南牡蒿聚为一类。本研究结果为黄花蒿药材的真伪鉴别及其药材质量评价提供了快速而有效的分析手段。. (3) 采用ISSR分子标记技术，对黄花蒿野生种群及其子代的遗传多样性水平和群体遗传结构进行检测和比较。. 黄花蒿具有较高的遗传多样性水平，遗传分化系数（Gst）为0.1749，遗传变异有82.51%发生在种群内的不同个体间，17.49%发生在不同种群间。广西玉林（YL）和浙江杭州（HZ）野生种群的遗传多样性变异较大，应作为重点保护种群。黄花蒿野生种群的遗传多样性参数值高于子代种群。