基于ITS2条形码的玛咖药材鉴别及9种元素的测定

Identification of Lepidium meyenii Walp.based on ITS2 sequence analysis and determination of 9 elements

目的:利用DNA分子鉴别技术,从分子水平快速、准确鉴别玛咖药材及其同科混伪品植物;采用石墨炉原子吸收分光光度法(GFASS)和火焰原子吸收分光光度法(FASS)测定不同产地玛咖中镉(^(112)Cd)、铜(^(63)Cu)、铅(^(207)Pb)、铬(^(52)Cr)4种有害重金属元素以及钙(^(40)Ca)、镁(^(24)Mg)、钠(^(23)Na)、钾(^(39)K)、铁(^(56)Fe)5种元素的含量,并进行安全性评价。方法:利用DNA试剂盒提取玛咖及其混伪品(萝卜、芫根)样本共9个,PCR扩增其提出的DNA并进行双向测序,应用Codon Code Aligner V 6.0.2软件对峰图进行校对拼接,比较其内转录间隔区2(ITS2)的GC含量,结合在Gen Bank上下载的7条序列,采用MEGA 6.0软件计算种内、种间Kimura 2-parameter(K2P)遗传距离,最后构建邻接树(NJ tree)进行系统聚类分析。采用微波消解进行前处理,使用石墨原则吸收法和火焰原子吸收法分别对不同产地玛咖中的9种元素进行测定,对比《药用植物及制剂进出口绿色行业标准》,对玛咖的安全性进行评价。结果:玛咖的ITS2序列长度为190 bp,其中GC含量为54.7%,同时其二级结构明显与其混伪品存在差异,可以根据NJ tree将其与其他混伪品区分开来。微量元素标准曲线线性相关度均大于0.999 0,Cd、Cu、Pb、Cr、Ca、Mg、Na、K、Fe元素的回收率分别为93.2%、96.7%、95.4%、102.7%、94.3%、95.7%、105.9%、96.4%、98.1%。参照《药用植物及制剂进出口绿色行业标准》中的重金属元素限量标准,部分产地玛咖中的Cd元素含量超标,并且不同产地玛咖中各个元素的含量有一定的差异。结论:ITS2可以作为DAN条形码对玛咖与其混伪品进行准确、快速的鉴别,为玛咖的鉴定提供准确的分子依据。GFASS法快速、准确,灵敏度高,无背景影响,成本低廉,可较好地用于中药材中重金属残留量的测定,并且通过对玛咖中元素的研究,对确保其质量和安全性有着重要的意义。

Objective： To identify Lepidium meyenii and its adulterants rapidly and precisely using DNA molecular identification technology. To determine the harmful heavy metals including cadmium （ ^112Cd ）, copper （ ^63Cu ）, lead （ ^207Pb ） and chromium （ ^52Cr ） and elements including calcium （ ^40Ca ） ,magnesmm （ ^24Mg ）, sodium （ ^23Na ）, potassium （ ^39K ） and iron （ ^56Fe ） by graphite furnace atomic absorption spectrophotometry （ GFASS ） and flame atomic absorption spectrometry （ FASS ） and make safety evaluation for the results. Methods： The total DNA was extracted from Lepidium meyenii and its adulterants （ radish and yuenkanin ） using the DNA kit. The DNA samples were amplified and sequenced bi-directionally. The obtained sequences were put together by Aligner V 6.0.2 CodonCode software, and GC content was compared. The intraspecies and interspecific genetic distances of the samples were downloaded from the GenBank and were calculated based on Kimura 2-parameter model by using MEGA 6.0. At last, the neighbor-joining （ NJ ） tree phylogenetic trees were constructed for cluster analysis. By using microwave digestion pretreatment, GFASS and FASS, 9 elements in Lepidium meyenii from different habitats were determined of and their safety was evaluated. Results： The length of the internal transcribed spacer 2 （ ITS2 ） sequence of Lepidium meyenii was 190 bp and the content of GC was 54.7%. The secondary structure of Lepidium meyenii was obviously different from its adulterants and they could be distinguished according to the NJ tree. The standard curve of the elements was linear and the linear correlation was greater than 0. 999 0. The recoveries （ n=9 ） of Cd, Cu, Pb, Cr, Ca, Mg, Na, K, Fe were 93.2%, 96.7%, 95.4%, 102.7%, 94.3%, 95.7%, 105.9%, 96.4% and 98. l%, respectively. Cadmium in Lepidium meyenii from some of habitats exceeded the limits regulated in ＂Green Trade Standards of Importing Exporting Medicinal plants Preparations＂. And there were differences in content of various elements in Lepidium meyenii from different habitats. Conclusion： ITS2 could be used to identify Lepidium meyenii and its adulterants, and it provided a molecular basis for accurate identification of Lepidium meyenii. The GFASS method was simple, fast and accurate, which had a high sensitivity with no influential background. It was also a low-cost technology suitable for the determination of heavy metals in Chinese herbs. To ensure quality and safety of Lepidium meyenii, it was significant to investigate its elements including heavy metals.