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Article(id=1222469877823168963, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469875008790921, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2019-0395, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1558281600000, receivedDateStr=2019-05-20, revisedDate=1561996800000, revisedDateStr=2019-07-02, acceptedDate=null, acceptedDateStr=null, onlineDate=1769389131265, onlineDateStr=2026-01-26, pubDate=1573488000000, pubDateStr=2019-11-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1769389131265, onlineIssueDateStr=2026-01-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1769389131265, creator=13701087609, updateTime=1769389131265, updator=13701087609, issue=Issue{id=1222469875008790921, tenantId=1146029695717560320, journalId=1189982191388893191, year='2019', volume='54', issue='11', pageStart='1881', pageEnd='2140', issueExtLink='null', onlineDate='null', pubDate='1573488000000', pubDateStr='2019-11-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1769389130593, creator='13701087609', updateTime=1769389577080, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1222471747778109959, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469875008790921, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1222471747778109960, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469875008790921, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2083, endPage=2088, ext={EN=ArticleExt(id=1222469880092287583, articleId=1222469877823168963, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Comparative study for morphological identification, chemical component analysis, and DNA barcode determination of three plants and their varieties in Genus Mentha, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

The morphological identification, chemical component analysis, and DNA barcode determination were investigated on Genus Mentha (including Mentha haplocalyx Briq., Mentha piperita Linn., Mentha spicata Linn. and Mentha cultivated) in order to reveal the origin of Herba Mentha as a drug, and ensure the accuracy in clinic application. The morphological characters, chemical composition analysis by GC-MS/MS and DNA content measure by polymerase chain reaction (PCR) were reported in this study for inter- or intra- species divergence. Based on the morphology, axillary verticillasters was recognized as the typical character for Mentha haplocalyx Briq. Carvone was used as an index component for chemical composition analysis of Mentha spicata Linn. Gene clustering analysis divided 22 batches of samples into two molecular groups. Mentha haplocalyx Briq. is distinguishably different from Mentha spicata Linn. Mentha piperita Linn. and other cultivated plants were distributed between these two species. The results obtained by morphological identification, chemical composition analysis, and DNA barcode determination show good correlations, but each identification method has its limit. In view of the fact that hybridization of the plants in Genus Mentha is common, identification relying on only one method is not recommended.

, authors=null, authorsList=Fei-fei WANG, Jie LIU, Feng-yan HE, Nan-ping ZHANG, Yi HE, Jing LI, Yu-mei ZHANG, Jian ZHENG, Shuang-cheng MA, authorCompany=null, correspAuthors=Jian ZHENG, Shuang-cheng MA, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2019 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1222469881119892118, articleId=1222469877823168963, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=薄荷属3种植物及变种的性状鉴别、化学成分分析及DNA条形码研究比较, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

对薄荷属植物薄荷、胡椒薄荷、留兰香及其栽培变种进行性状鉴定、化学成分鉴定和分子生物学鉴定，比较不同分析方法对薄荷属植物鉴别结果的差异，以了解药用薄荷的基源情况，确保药用薄荷在临床中的正确使用。采用性状鉴别、GC-MS/MS测定挥发性化学成分和聚合酶链式反应（PCR）对22批次薄荷属样品进行分析，确定它们的种间归属情况。在性状鉴别中，“轮伞花序腋生”被认为是薄荷典型特征之一；在化学成分鉴别中，香芹酮可作为鉴别留兰香的指标性成分；在基因聚类分析中，22批次样品主要归属为2个分枝，薄荷和留兰香分别属于2个不同分枝中，但胡椒薄荷及部分杂交种则在两个分枝中均有分布。采用性状、化学成分和基因序列分析3种方法对22批次薄荷属植物进行分析，3种不同分类方法的分类结果具有一定的相关性，但每种方法均有局限性，且薄荷杂交现象较为常见，单一方法鉴别存在一定困难。

, authors=null, authorsList=王菲菲, 刘杰, 何风艳, 张南平, 何轶, 李静, 张聿梅, 郑健, 马双成, authorCompany=null, correspAuthors=郑健, 马双成, authorNote=null, correspAuthorsNote=
*郑健, Tel: 86-10-67095776, E-mail: ;
马双成, Tel: 86-10-67095272, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2019, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=hGTNS41gGLyh9vkrtILIUA==, magXml=merrstSA2vvlwbvpfRWbWg==, pdfUrl=null, pdf=7/tr8lDNnEPD06DS/UJZHg==, pdfFileSize=369937, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=At34Jp5eTa8HYSmxMGFJAQ==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=UniFOE9I2ybwnPZ0uqYlOw==, mapNumber=null, fund=null)}, authors=[Author(id=1222469881539322547, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1222469881673540282, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, 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Acta Pharm Sin (药学学报), 1981, 16: 849-859., articleTitle=A prelinminary study on species of genus Mentha cultivated in China, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1222469881426076329, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, xref=null, ext=[AuthorCompanyExt(id=1222469881434464937, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, companyId=1222469881426076329, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=National Institutes for Food and Drug Control, Beijing 100050, China), AuthorCompanyExt(id=1222469881447047850, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, companyId=1222469881426076329, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=中国食品药品检定研究院, 北京 100050)])], figs=[ArticleFig(id=1222469888527032418, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, language=EN, label=null, caption=null, figureFileSmall=ZHtoOCQxEQynhBBBx3wLBw==, figureFileBig=At34Jp5eTa8HYSmxMGFJAQ==, tableContent=null), ArticleFig(id=1222469888623501420, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, language=CN, label=Figure 1, caption= Phylogenetic tree of Genus <i>Mentha</i> with the ITS2 sequences using NJ method. 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No. Classification Location Reference materials intercomparison TIC result DNA barcode Morphological identification
1 Mentha cultivated Funan county, Fuyang city, Anhui province α-Pinene, cinene, 1, 8-oxido- p-menthane, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, isomenthone, piperitoneoxide Mentha haplocalyx Briq. Mentha haplocalyx Briq. (axillary verticillasters, round leaf)
2 Mentha piperita Linn. Pharmacy from Beijing α-Pinene, cinene, 1, 8-oxido-p-menthane, menthone, menthol, pulegone, carvone, menthyl acetate Isomenthone, sabinene, 3-octanol, isomenthone, piperitoneoxide Mentha haplocalyx Briq. decoction pieces
3 Mentha haplocalyx Briq. Self-culivation α-Pinene, cinene, 1, 8-oxido-p-menthane, menthone, menthol β-Pinene, sabinene, myrcene, 3-octanol, Mentha haplocalyx Briq. Mentha haplocalyx Briq.
4 Mentha haplocalyx Briq. Yangqu county, Taiyuan city, Shanxi province α-Pinene, cinene, 1, 8-oxido-p-menthane, menthone, menthol, menthyl acetate Sabinene, Mentha haplocalyx Briq. Mentha haplocalyx Briq. (axillary verticillasters)
5 Mentha haplocalyx Briq. Pharmacy from Beijing α-Pinene, cinene, linalool, menthone, menthol, pulegone Sabinene, 3-octanol, isomenthone, piperitoneoxide Mentha haplocalyx Briq. decoction pieces
6 Mentha haplocalyx Briq. Reference crud drug 1209162005038 α-Pinene, cinene, linalool, menthone, menthol, menthyl acetate Sabinene, 3-octanol, isomenthone, Mentha haplocalyx Briq. decoction pieces
7 Mentha haplocalyx Briq. Reference crud drug 120916200401 α-Pinene, cinene, linalool, menthone, menthol Sabinene, myrcene Mentha haplocalyx Briq. decoction pieces
8 Mentha haplocalyx Briq. Reference crud drug 1209162015 Isomenthone, menthone, menthol, menthyl acetate Isomenthone, piperitoneoxide Mentha haplocalyx Briq. decoction pieces
9 Mentha piperita Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido-p-menthane, linalool, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
10 Mentha spicata Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene, 3-octanol Mentha spicata Linn. Mentha piperita Linn.
11 Mentha spicata Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene Mentha spicata Linn. Mentha piperita Linn.
12 Mentha spicata Linn. Online shopping α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene Mentha spicata Linn. Mentha piperita Linn.
13 Mentha spicata Linn. Online shopping α-Pinene, cinene, carvone Sabinene, myrcene Mentha spicata Linn. Mentha piperita Linn.
14 Mentha haplocalyx Briq. Eighteen Li town, Qiao district, Bozhou city, Anhui province α-Pinene, cinene Sabinene, 3-octanol, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
15 Mentha haplocalyx Briq. Chouji town, Xuyi county, Huai'an city, Jiangsu province α-Pinene, cinene, menthone, pulegone β-Pinene, sabinene, myrcene, isomenthone, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
16 Mentha piperita Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido-p-menthane, carvone, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
17 Mentha spicata Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene Mentha haplocalyx Briq. Mentha piperita Linn.
18 Mentha haplocalyx Briq. Shanghai chongming island α-Pinene, cinene, menthone, menthol Sabinene Mentha haplocalyx Briq. Mentha haplocalyx Briq.
19 Mentha haplocalyx Briq. Funan county, Fuyang city, Anhui province α-Pinene, cinene, DL-menthol, menthone Sabinene, myrcene, isomenthone, piperitoneoxide Mentha haplocalyx Briq. Mentha haplocalyx Briq. (axillary verticillasters)
20 Mentha spicata Linn. Changzhou county, Suzhou city, Jiangsu province α-Pinene, cinene, carvone β-Pinene, sabinene, myrcene, 3-octanol Mentha spicata Linn. Mentha spicata Linn. (axillary verticillasters)
21 Mentha piperita Linn. Funan county, Fuyang city, Anhui province α-Pinene, cinene, 1, 8-oxido- p-menthane, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol Mentha spicata Linn. Mentha piperita Linn.
22 Mentha cultivated Yangqu county, Taiyuan city, Shanxi province α-Pinene, cinene, 1, 8-oxido- p-menthane, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, isomenthone, piperitoneoxide Mentha spicata Linn. Mentha piperita Linn.
), ArticleFig(id=1222469888975822984, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469877823168963, language=CN, label=Table 1, caption=

The morphological identification, chemical compound analysis, and DNA barcodes for Genus Mentha

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Classification Location Reference materials intercomparison TIC result DNA barcode Morphological identification
1 Mentha cultivated Funan county, Fuyang city, Anhui province α-Pinene, cinene, 1, 8-oxido- p-menthane, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, isomenthone, piperitoneoxide Mentha haplocalyx Briq. Mentha haplocalyx Briq. (axillary verticillasters, round leaf)
2 Mentha piperita Linn. Pharmacy from Beijing α-Pinene, cinene, 1, 8-oxido-p-menthane, menthone, menthol, pulegone, carvone, menthyl acetate Isomenthone, sabinene, 3-octanol, isomenthone, piperitoneoxide Mentha haplocalyx Briq. decoction pieces
3 Mentha haplocalyx Briq. Self-culivation α-Pinene, cinene, 1, 8-oxido-p-menthane, menthone, menthol β-Pinene, sabinene, myrcene, 3-octanol, Mentha haplocalyx Briq. Mentha haplocalyx Briq.
4 Mentha haplocalyx Briq. Yangqu county, Taiyuan city, Shanxi province α-Pinene, cinene, 1, 8-oxido-p-menthane, menthone, menthol, menthyl acetate Sabinene, Mentha haplocalyx Briq. Mentha haplocalyx Briq. (axillary verticillasters)
5 Mentha haplocalyx Briq. Pharmacy from Beijing α-Pinene, cinene, linalool, menthone, menthol, pulegone Sabinene, 3-octanol, isomenthone, piperitoneoxide Mentha haplocalyx Briq. decoction pieces
6 Mentha haplocalyx Briq. Reference crud drug 1209162005038 α-Pinene, cinene, linalool, menthone, menthol, menthyl acetate Sabinene, 3-octanol, isomenthone, Mentha haplocalyx Briq. decoction pieces
7 Mentha haplocalyx Briq. Reference crud drug 120916200401 α-Pinene, cinene, linalool, menthone, menthol Sabinene, myrcene Mentha haplocalyx Briq. decoction pieces
8 Mentha haplocalyx Briq. Reference crud drug 1209162015 Isomenthone, menthone, menthol, menthyl acetate Isomenthone, piperitoneoxide Mentha haplocalyx Briq. decoction pieces
9 Mentha piperita Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido-p-menthane, linalool, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
10 Mentha spicata Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene, 3-octanol Mentha spicata Linn. Mentha piperita Linn.
11 Mentha spicata Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene Mentha spicata Linn. Mentha piperita Linn.
12 Mentha spicata Linn. Online shopping α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene Mentha spicata Linn. Mentha piperita Linn.
13 Mentha spicata Linn. Online shopping α-Pinene, cinene, carvone Sabinene, myrcene Mentha spicata Linn. Mentha piperita Linn.
14 Mentha haplocalyx Briq. Eighteen Li town, Qiao district, Bozhou city, Anhui province α-Pinene, cinene Sabinene, 3-octanol, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
15 Mentha haplocalyx Briq. Chouji town, Xuyi county, Huai'an city, Jiangsu province α-Pinene, cinene, menthone, pulegone β-Pinene, sabinene, myrcene, isomenthone, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
16 Mentha piperita Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido-p-menthane, carvone, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, piperitoneoxide Mentha haplocalyx Briq. Mentha piperita Linn.
17 Mentha spicata Linn. Beijing flower market α-Pinene, cinene, 1, 8-oxido- p-menthane, carvone β-Pinene, sabinene, myrcene Mentha haplocalyx Briq. Mentha piperita Linn.
18 Mentha haplocalyx Briq. Shanghai chongming island α-Pinene, cinene, menthone, menthol Sabinene Mentha haplocalyx Briq. Mentha haplocalyx Briq.
19 Mentha haplocalyx Briq. Funan county, Fuyang city, Anhui province α-Pinene, cinene, DL-menthol, menthone Sabinene, myrcene, isomenthone, piperitoneoxide Mentha haplocalyx Briq. Mentha haplocalyx Briq. (axillary verticillasters)
20 Mentha spicata Linn. Changzhou county, Suzhou city, Jiangsu province α-Pinene, cinene, carvone β-Pinene, sabinene, myrcene, 3-octanol Mentha spicata Linn. Mentha spicata Linn. (axillary verticillasters)
21 Mentha piperita Linn. Funan county, Fuyang city, Anhui province α-Pinene, cinene, 1, 8-oxido- p-menthane, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol Mentha spicata Linn. Mentha piperita Linn.
22 Mentha cultivated Yangqu county, Taiyuan city, Shanxi province α-Pinene, cinene, 1, 8-oxido- p-menthane, menthone, menthol, menthyl acetate β-Pinene, sabinene, myrcene, 3-octanol, isomenthone, piperitoneoxide Mentha spicata Linn. Mentha piperita Linn.
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薄荷属3种植物及变种的性状鉴别、化学成分分析及DNA条形码研究比较
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王菲菲 , 刘杰 , 何风艳 , 张南平 , 何轶 , 李静 , 张聿梅 , 郑健 * , 马双成 *
药学学报 | 研究论文 2019,54(11): 2083-2088
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药学学报 |研究论文 2019 , 54 (11) : 2083 -2088
薄荷属3种植物及变种的性状鉴别、化学成分分析及DNA条形码研究比较
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王菲菲, 刘杰, 何风艳, 张南平, 何轶, 李静, 张聿梅, 郑健* , 马双成*
作者信息
  • 中国食品药品检定研究院, 北京 100050
通讯作者:
*郑健, Tel: 86-10-67095776, E-mail: ;
马双成, Tel: 86-10-67095272, E-mail:
Comparative study for morphological identification, chemical component analysis, and DNA barcode determination of three plants and their varieties in Genus Mentha
Fei-fei WANG, Jie LIU, Feng-yan HE, Nan-ping ZHANG, Yi HE, Jing LI, Yu-mei ZHANG, Jian ZHENG* , Shuang-cheng MA*
Affiliations
  • National Institutes for Food and Drug Control, Beijing 100050, China
出版时间: 2019-11-12 doi: 10.16438/j.0513-4870.2019-0395
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对薄荷属植物薄荷、胡椒薄荷、留兰香及其栽培变种进行性状鉴定、化学成分鉴定和分子生物学鉴定，比较不同分析方法对薄荷属植物鉴别结果的差异，以了解药用薄荷的基源情况，确保药用薄荷在临床中的正确使用。采用性状鉴别、GC-MS/MS测定挥发性化学成分和聚合酶链式反应（PCR）对22批次薄荷属样品进行分析，确定它们的种间归属情况。在性状鉴别中，“轮伞花序腋生”被认为是薄荷典型特征之一；在化学成分鉴别中，香芹酮可作为鉴别留兰香的指标性成分；在基因聚类分析中，22批次样品主要归属为2个分枝，薄荷和留兰香分别属于2个不同分枝中，但胡椒薄荷及部分杂交种则在两个分枝中均有分布。采用性状、化学成分和基因序列分析3种方法对22批次薄荷属植物进行分析，3种不同分类方法的分类结果具有一定的相关性，但每种方法均有局限性，且薄荷杂交现象较为常见，单一方法鉴别存在一定困难。

薄荷  /  留兰香  /  胡椒薄荷  /  性状鉴别  /  化学成分分析  /  DNA条形码

The morphological identification, chemical component analysis, and DNA barcode determination were investigated on Genus Mentha (including Mentha haplocalyx Briq., Mentha piperita Linn., Mentha spicata Linn. and Mentha cultivated) in order to reveal the origin of Herba Mentha as a drug, and ensure the accuracy in clinic application. The morphological characters, chemical composition analysis by GC-MS/MS and DNA content measure by polymerase chain reaction (PCR) were reported in this study for inter- or intra- species divergence. Based on the morphology, axillary verticillasters was recognized as the typical character for Mentha haplocalyx Briq. Carvone was used as an index component for chemical composition analysis of Mentha spicata Linn. Gene clustering analysis divided 22 batches of samples into two molecular groups. Mentha haplocalyx Briq. is distinguishably different from Mentha spicata Linn. Mentha piperita Linn. and other cultivated plants were distributed between these two species. The results obtained by morphological identification, chemical composition analysis, and DNA barcode determination show good correlations, but each identification method has its limit. In view of the fact that hybridization of the plants in Genus Mentha is common, identification relying on only one method is not recommended.

Mentha haplocalyx Briq.  /  Mentha spicata Linn.  /  Mentha piperita Linn.  /  morphological identification  /  chemical compound analysis  /  DNA barcode
王菲菲, 刘杰, 何风艳, 张南平, 何轶, 李静, 张聿梅, 郑健, 马双成. 薄荷属3种植物及变种的性状鉴别、化学成分分析及DNA条形码研究比较. 药学学报, 2019 , 54 (11) : 2083 -2088 . DOI: 10.16438/j.0513-4870.2019-0395
Fei-fei WANG, Jie LIU, Feng-yan HE, Nan-ping ZHANG, Yi HE, Jing LI, Yu-mei ZHANG, Jian ZHENG, Shuang-cheng MA. Comparative study for morphological identification, chemical component analysis, and DNA barcode determination of three plants and their varieties in Genus Mentha[J]. Acta Pharmaceutica Sinica, 2019 , 54 (11) : 2083 -2088 . DOI: 10.16438/j.0513-4870.2019-0395
正文
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薄荷为唇形科植物薄荷Mentha haplocalyx Briq.的干燥地上部分, 具有疏散风热、清利头目、利咽、透疹、疏肝行气等功效[1]。薄荷最早记载于唐代孙思邈《千金·食治》中, 名为蕃荷菜, “味苦、辛、温、无毒。可久食, 却肾气, 令人口气香。主辟邪毒, 除劳弊。形瘦疲倦者不可久食, 动消渴病”[2]。《本草纲目》记载:薄荷, 辛能发散, 凉能清利, 专于清风散热。故头痛、头风、眼目、咽喉、口齿诸病为要药[3]。
据报道, 世界上薄荷属植物有30个种, 其中变种有140多种。现今在园艺栽培上已有600多个品种[4]。薄荷原产我国东北各省以及库页岛、日本、琉球、朝鲜等地[5]。古代本草中记载了薄荷、石薄荷和胡薄荷3种不同的植物, 药用薄荷一直是唇形科植物薄荷(Mentha haplocalyx Briq.), 而石薄荷和胡薄荷不作药用[2, 6]。留兰香原产于南欧地区, 自20世纪中后期开始在我国大规模引种[7]。据报道, 我国薄荷属栽培种类, 共有11种、1个亚种、2个变种及2个变型。胡椒薄荷是其中常见的栽培品种, 原产欧洲及地中海沿岸一带, 1959年从国外引进, 其精油广泛应用于食品、化妆品和医药卫生等方面[8]。有研究[9]将薄荷属植物分为3类:薄荷醇化学型(包括薄荷和胡椒薄荷)、香芹酮化学型(包括留兰香和皱叶留兰香)和辣薄荷烯酮氧化物化学型(包括灰薄荷、兴安薄荷、圆叶薄荷和假薄荷等)。其中, 薄荷(Mentha haplocalyx Briq.)、胡椒薄荷(Mentha piperita Linn.)、留兰香(Mentha spicata Linn.)和皱叶留兰香(Mentha crispata Schrad. ex Willd.)等常作为芳香及药用植物栽培[10, 11]。
在临床功效方面, 薄荷可疏散风热、清利头目、利咽、透疹、疏肝行气, 主要用于风热感冒、头痛、目赤和风疹麻疹等症。留兰香味辛、甘, 性微温, 具有和中理气、祛风散寒、消炎、止血、收敛之功效, 用于治疗伤风感冒、胃气痛、鼻衄、目赤和疔疮热疥等症。目前研究认为, 留兰香对重症痔疮和慢性及溃疡性结肠炎等疾病具有良好的药效[12-14]。可见, 薄荷属不同种植物的药效存在差异, 但是由于它们种间易杂交具有多型性的特点, 使得不同种间的关系确定有一定难度。近年来, 由于薄荷属植物作为香料大量种植, 使其他种或其他杂交种薄荷混入药用薄荷的风险大大增加, 对薄荷属植物的鉴别方法[15]进行研究, 可保证薄荷临床用药的安全有效。
本文收集了薄荷属药材及园艺样品共计22批次样品。实验拟通过性状、化学成分分析和聚合酶链式反应(PCR)分析, 对22批次样品进行分类鉴定。实验中选择了候选序列(ITS2)进行扩增, 通过种内、种间遗传变异分析和barcoding gap分析与其性状和化学成分进行比较, 分析比较薄荷、胡椒薄荷、留兰香及疑似杂交品种的异同, 比较这3种方法在鉴别薄荷属样品的优势或不足。通过实验, 可以较为系统的分析和探讨目前市场常见的薄荷属样品市场混乱现象的解决方案, 为薄荷药材的鉴定和作为饮片入药提供了技术支持。
材料与方法
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仪器  Agilent 7890B气相色谱-7000C三重四级杆质谱仪(美国Agilent公司), 万分之一电子天平AE240和十万分之一天平电子天平XS105DU (瑞士Mettler公司), KQ-300DA型医用数控超声波清洗器(昆山市超声仪器有限公司), 球磨仪MM400 (德国莱驰公司), ABI Veriti梯度PCR仪和ABI 3730XL测序仪(美国ABI公司), Drict-Q5超纯水机(美国Millipore公司), EPS-300电泳仪(上海朗赋实业有限公司), 全自动凝胶成像系统(美国BIORAD公司)。
材料  植物基因组DNA提取试剂盒(DP305, 天根生化科技有限公司); Ex taq (RR001B)和dNTP Mixture (4030) (日本TaKaRa公司); GelRed (41003, 美国Biotium公司); 琼脂糖(法国Biowest公司); Tris-base (美国Sigma公司); 冰醋酸(分析纯, 500 mL)、EDTANa2·2H2O (国药集团化学试剂有限公司); 引物ITS2由上海捷瑞生物工程有限公司合成。
薄荷脑对照品(批号: 110728-200506)、(-)-薄荷酮对照品(批号: 111705-201105)、胡薄荷酮对照品(批号: 111706-201606)、α-蒎烯对照品(批号: 110897-200502)、柠檬烯对照品(批号: 100470-201503)、芳樟醇对照品(批号: 111503-201603)、桉油精对照品(批号: 110788-201506)和乙酰薄荷酯对照品(批号: 190044-201001)购自中国食品药品检定研究院; 香芹酮对照品(批号: C110521000-00610)购自德国Dr. Ehrenstorfer公司。
薄荷对照药材3批次(批号: 120916-8903; 120916-200401和120916-201511)购自中国食品药品检定研究院; 市售薄荷药材6批, 购自北京、太原、阜阳、江苏、上海和安徽等市各药店, 留兰香新鲜样品4批, 购自北京市各花卉市场, 上述样品均经中国食品药品检定研究院张南平主任药师鉴定分别为Mentha haplocalyx Briq.和Mentha spicata Linn.。另从产地收集样品9批次, 其中6批次由性状判断为胡椒薄荷, 其余为杂交品种。
薄荷及留兰香主要挥发性成分分析
对照品溶液制备  精密称取薄荷脑、(-)-薄荷酮、胡薄荷酮、α-蒎烯、柠檬烯、芳樟醇、桉油精、香芹酮、乙酰薄荷酯对照品适量, 分别加乙酸乙酯制成每1 mL含2.8、2.6、2.6、3.4、2.0、5.0、3.0、5.6和4.2 μg的混合溶液, 作为混合对照品溶液。
供试品溶液制备  精密称取待测样品0.5 g, 置具塞锥形瓶中, 精密加入乙酸乙酯溶液10 mL, 密塞, 称量, 冰浴下超声处理(功率300 W, 频率40 kHz) 20 min, 放冷, 再称量, 乙酸乙酯补足减失的量, 摇匀, 0.45 μm滤膜滤过, 取续滤液, 即得。
色谱及质谱条件  采用HP-5ms毛细管柱(30 m×0.25 mm, 0.25 μm); 载气为氦气; 程序升温, 初始温度50 ℃, 5 ℃·min-1升至150 ℃, 20 ℃·min-1升至270 ℃, 保持10 min; 分流比10:1;进样口温度220 ℃; 进样体积1 μL。质谱接口温度300 ℃; 离子源温度230 ℃; 电子轰击电离(EI)模式; 轰击能量70 eV; 扫描方式:一级质谱全扫描(TIC)。
TIC检测结果  取22批次样品, 按“色谱及质谱条件”项下条件进行TIC检测。
DNA提取及测定
样品制备  分别将薄荷叶样品用75%乙醇浸湿的棉球擦拭干净, 晾干后用球磨仪粉碎成超细粉, 称取10~20 mg备用。按照植物基因组DNA提取试剂盒操作步骤进行DNA提取。利用Nano Vue Plus (GE)测定A260 nm/280 nm、A260 nm/230 nm和DNA浓度。
聚合酶链式反应及测序  引物名称: ITS2F (ATG CGATACTTGGTGTGAAT)和ITS3R (GACGCTTCTC CAGACTACAAT), PCR扩增的反应条件: 94 ℃, 5 min; 94 ℃, 30 s, 56 ℃, 30 s; 72 ℃, 45 s; 35循环; 72 ℃, 10 min。PCR反应体系25 μL: dNTP (2.5 mmol·L-1) 2 μL, 反应缓冲液2.5 μL, 上下游引物各1 μL, Taq DNA聚合酶0.2 μL, DNA模板1 μL, 高压蒸汽灭菌去离子水17.3 μL。PCR产物经纯化后使用ABI 3730XL测序仪进行双向测序。
数据处理  测序峰图使用CodonCode Aligner 3.7.1 (美国Codon Code公司)校对拼接, 去除引物区段。将所有序列利用MEGA 5.0 (molecular evolutionary genetics analysis)分析比对。基于K2P模型进行遗传距离分析, 用NJ法(Neighbor-Joining Tree)建立聚类树, 通过Bootstrap (1 000次重复)对各分支进行支持率检验。
结果
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1 薄荷属植物形态及性状分析
根据《中国植物志》和《中国药典》的性状描述, 本研究的22份样品的主要性状可划分为3种, 分别为薄荷、胡椒薄荷和留兰香[16, 17]。通过对实验中薄荷、留兰香和杂交品种的性状观察, 认为“轮伞花序腋生”是确定样品是否为薄荷的主要依据。样品20 (表 1)符合薄荷性状特征, 却具有留兰香特征气味, 可能是一种栽培变种。
2 样品主要成分分析
通过对照品比对和NIST谱库检索, 共鉴定出15个化合物(α-蒎烯、β-蒎烯、桧烯、月桂烯、3-辛醇、柠檬烯、桉油精、芳樟醇、薄荷酮、异薄荷酮、薄荷脑、胡薄荷酮、香芹酮、氧化胡椒酮和乙酰薄荷酯), 其中9个化合物经对照品比对确证。各化合物的相对含量见表 1。薄荷样品的TIC图谱中主要为薄荷脑的色谱峰, 部分薄荷样品TIC图谱中较明显的色谱峰还有薄荷酮、胡薄荷酮、氧化胡椒酮或乙酰薄荷酯的色谱峰。留兰香样品的TIC图谱中主要为香芹酮和柠檬烯的色谱峰, 其中香芹酮色谱峰最高。
一般地, 薄荷样品的TIC图谱中主要为薄荷脑的色谱峰, 部分薄荷样品TIC图谱中较明显的色谱峰, 还有薄荷酮、胡薄荷酮、氧化胡椒酮或乙酰薄荷酯的色谱峰。留兰香样品的TIC图谱中主要为香芹酮和柠檬烯的色谱峰, 其中香芹酮色谱峰最高。在对疑似杂交样品进行检测中发现, 每个样品中所含成分差异很大, 有些样品未检出薄荷酮、薄荷脑或香芹酮等指标成分, 见表 1所示。胡椒薄荷中, 色谱峰检出化学成分较为多样。一般认为, 品质较好的胡椒薄荷其化学成分中主要含有薄荷酮、薄荷呋喃、乙酰薄荷酯和薄荷脑等成分, 且含量合计占组分总量的65%以上[18]。
3 样品DNA条形码序列信息及种内变异分析
对22批次样品的序列特征进行分析, ITS2序列均在250 bp左右, 测序成功率为100 %。根据以上数据分析, 基于ITS2序列建立薄荷、留兰香等样品的NJ树(图 1)。结果表明, ITS2序列能将薄荷和留兰香样品进行区分, 并呈现较好的单系性。全部样品主体分布于两枝, 15种样品分类于薄荷枝, 7种样品分类于留兰香枝。
4 检测结果比较
实验通过对22批次薄荷样品的性状、化学成分和测序比对结果进行分析。结果发现, 3批次薄荷对照药材被划归为薄荷, 4批次留兰香样品被划归为留兰香, 表明样品测序结果与已知样品一致。在对疑似杂交样品进行分析时, 2、9、14、15和16号被认为是胡椒薄荷, 归为薄荷分支, 仅21号被归为留兰香分支。20号样品性状与薄荷一致, 但香型为留兰香香型, 在化学成分分析中检出香芹酮成分, 被认为是一类栽培变种, 其基因测序分类中也被分为留兰香分枝。
在前期研究中, 为了进一步考察香芹酮作为留兰香检查指标的专属性, 实验中采用MRM法对13批次归属清楚的样品进行测定。结果表明9批次薄荷样品中均无香芹酮色谱峰检出, 4批留兰香样品中有明显香芹酮色谱峰检出。
讨论
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薄荷属植物种内杂交情况普遍, 由于多型性、雄花两性花同株或异株现象、杂交与无性繁殖等原因, 种数极不确切。通过性状鉴别、化学成分分析和基因序列聚类的方法所得出的结论在杂交品种中存在较多争议。
在薄荷属植物的性状鉴别中, 主要根据:轮伞花序腋生, 茎、叶、花萼有毛等特点, 判断薄荷样品。在生长习性观察中, 留兰香植物根茎分布在土壤内较浅(约6 cm), 挖起时不易折断, 匍匐茎多而长, 有时茎露出地面; 而薄荷或胡椒薄荷等分布在土壤内较深(7~13 cm), 挖起时则易折断, 匍匐茎少而短[18]。从芳香油的气味区别留兰香和薄荷也有很大差别, 早春出苗时, 揉留兰香叶嗅之有浓烈的气味, 而薄荷气味甚淡, 不同气味表明所含化学成分具有差异[19]。在化学成分分析中, 薄荷样品中含有薄荷脑和薄荷酮等主要成分, 留兰香样品中主要含有香芹酮等成分。胡椒薄荷主要含有乙酰薄荷酯或胡薄荷酮等成分。
一直以来, 中药的真伪和优劣是中药检定的主要目的。传统检定中, 真伪鉴别主要依赖于中药的性状和化学成分鉴别。近年来, DNA条形码技术得到了广泛的应用, 特别在中药的真伪鉴别中, 与传统的性状和化学鉴别方法相互补充, 为中药的真伪鉴别提供了新的手段。在样品优劣评价时, 化学成分分析可以针对特定指标性成分进行定性和定量检测, 准确客观, 具有其他方法所不具有的优越性; 性状、显微鉴别对检验人员的经验要求较高, 短时间较难掌握; 分子生物学手段目前尚不能针对中药材优劣的评价。针对不同中药材的鉴定, 这3种方法也是各有优势。例如, 分子生物学目前解决不了同种中药材不同药用部位投料的问题。而在同属的药用植物和非药用植物鉴别中, 存在药材饮片(或细粉)性状特征和化学成分相似的问题, 而采用DNA条形码可以快速地将近缘属植物进行区分。针对分子生物学方法在中药鉴定中的应用, 目前仍存在一些难以解决的问题:第一, 中药一般为干燥的植物或动物体, 实验无法分析样品所处的生物学状态, 即无法辨别是否为药典规定的药用部位或者是否在规定的采收期采收。第二, 在近缘种的归属中, 应根据不同药材的基因组特点设计对应的引物进行分析, 目前常采用的通用引物(ITS、CO1等)在近缘种区分中具有很大的局限性, 不同的引物可能导致不同的结果。第三, 由于药材中DNA的降解情况难以控制, 所以在对样品中目的药材含量的检测存在较大难度, 绝对定量和相对定量方法都有待摸索。
薄荷属植物的鉴别, 本文推荐首先采用化学方法测定挥发油成分进行确证。一般认为, 留兰香挥发油中含有香芹酮。中药志、中华本草等各中药典籍以及中国药典均认为薄荷脑是薄荷中的主要成分[1, 12]。本文建议在中国药典薄荷检测中, 增加香芹酮的检查项以及薄荷脑含量测定项, 从而确保药用薄荷的临床应用的有效性。在GAP栽培中, 应注意薄荷品种资源的保护, 减少串种的情况, 建议培育中尽量采用无性繁殖, 确保种植资源正确。经过多年研究, 研究人员发现留兰香对重症痔疮和慢性及溃疡性结肠炎等疾病具有良好的药效, 并不能单纯将其作为薄荷伪品进行检测或处罚[12-14]。同时应注重对留兰香的药效进行开发, 为新药的研发提供可能的研究方向。
致谢 山西省食品药品检验所提供部分薄荷药材标本。

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2019年第54卷第11期
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doi: 10.16438/j.0513-4870.2019-0395
  • 接收时间：2019-05-20
  • 首发时间：2026-01-26
  • 出版时间：2019-11-12
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  • 收稿日期：2019-05-20
  • 修回日期：2019-07-02
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    中国食品药品检定研究院, 北京 100050

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*郑健, Tel: 86-10-67095776, E-mail: ;
马双成, Tel: 86-10-67095272, E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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