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Article(id=1308841227555012743, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840985296204417, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-1261, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1761235200000, receivedDateStr=2025-10-24, revisedDate=1768838400000, revisedDateStr=2026-01-20, acceptedDate=null, acceptedDateStr=null, onlineDate=1789981665743, onlineDateStr=2026-09-21, pubDate=1778515200000, pubDateStr=2026-05-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789981665743, onlineIssueDateStr=2026-09-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789981665743, creator=13701087609, updateTime=1789981665743, updator=13701087609, issue=Issue{id=1308840985296204417, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='5', pageStart='1435', pageEnd='1880', issueExtLink='null', onlineDate='null', pubDate='1778515200000', pubDateStr='2026-05-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789981607984, creator='13701087609', updateTime=1789984207975, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308851890515968861, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840985296204417, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308851890515968862, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840985296204417, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1869, endPage=1875, ext={EN=ArticleExt(id=1308841227861196936, articleId=1308841227555012743, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Molecular identification of the origin species of rhubarb medicinal materials, decoction pieces and Chinese patent medicines based on VPCR, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Rhei Radix et Rhizoma is one of the genuine medicinal materials in Sichuan. Its original species are Rheum palmatum, Rh. tanguticum, and Rh. officinale, recorded by Chinese Pharmacopoeia (2025 Edition). However, there are many cases of multi-origin mixing and adulterant incorporation in the current commercially available Rhei Radix et Rhizoma, and the variety origin needs to be clarified urgently. In this study, the ndhF-rpl32 region of chloroplast genomes, as specific DNA barcode reported previously, was selected and confirmed. The VPCR method was established to identify the authenticity and origin of rhubarb medicinal materials, decoction pieces and Chinese patent medicines, in the process of optimizing DNA extraction procedure, amplification system and amplification procedure. Compared with the traditional PCR method, the amplification procedure has been refined through a VPCR heating model, curtailing the detection from 1.5 h to 30 min. The 85 batches of samples collected from the circulation area were tested by VPCR method, including 30 batches of medicinal materials, 40 batches of decoction pieces and 15 batches of Chinese patent medicines which contain Rhei Radix et Rhizoma. The experimental results showed that 31 batches are Rh. tanguticum (including 18 batches of medicinal materials, 10 batches of decoction pieces and 3 batches of Chinese patent medicines), 36 batches are Rh. officinale (including 1 batches of medicinal materials, 25 batches of decoction pieces and 10 batches of Chinese patent medicines) and 13 batches are Rh. palmatum (including 8 batches of medicinal materials and 5 batches of decoction pieces). The pass rate is 94.1%. Three batches of rhubarb medicinal materials (No. CD1, CD2, CD3) and two batches of Chinese patent medicines (No.Z4 tablet, No.Z12 granule) were determined to be Rh. franzenbachii Munt, as one of counterfeit Rhei Radix et Rhizoma varieties. The VPCR method established in this study can be used as supplement to rhapontin TLC inspection test of Rhei Radix et Rhizoma in Chinese Pharmacopoeia (2025 Edition) and standardize the market for this clinical medication as well as improve the safety of Rhei Radix et Rhizoma.

, authors=Cong-cong ZHANG, Qing-qing AI, Xiao LUO, Hong CHEN, Xin-ran WU, Teng-xiao MAO*, authorsList=Cong-cong ZHANG, Qing-qing AI, Xiao LUO, Hong CHEN, Xin-ran WU, Teng-xiao MAO, authorCompany=null, correspAuthors=Teng-xiao MAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2026 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=1308841229169819799, articleId=1308841227555012743, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=利用VPCR对大黄药材饮片及中成药中大黄成分基原鉴定的研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

大黄作为川产道地药材之一, 中国药典(2025年版)收载的基原物种为掌叶大黄、唐古特大黄及药用大黄, 然而当前市售大黄存在多基原混杂及伪品掺入等现象, 品种基原亟待理清。本研究采用文献报道叶绿体ndhF-rpl32片段作为特异条形码并进行验证, 通过优化DNA提取步骤、扩增体系和扩增程序, 建立一种可用于大黄药材、饮片及中成药中大黄成分真伪及基原鉴定的VPCR方法。相较于传统的PCR方法, VPCR (Ⅴ-shaped polymerase chain reaction)法将扩增时间由1.5 h缩短至30 min。采用建立的VPCR方法检测市售流通领域收集到的85批大黄样品, 包括30批药材、40批饮片及15批含大黄的中成药。结果表明: 样品中31批为唐古特大黄(药材18批, 饮片10批, 中成药3批), 36批为药用大黄(药材1批, 饮片25批, 中成药10批), 13批为掌叶大黄(药材8批, 饮片5批), 合格率为94.1%。3批药材(编号CD1、CD2、CD3)、2批中成药(编号Z4为片剂、Z12为颗粒剂)判定为大黄伪品中的华北大黄。本研究建立的VPCR方法可作为对中国药典(2025年版)大黄项下薄层色谱检查土大黄苷方法的补充, 对规范大黄药材市场、保证大黄临床用药安全提供依据。

, authors=张聪聪, 艾青青, 罗霄, 陈红, 伍欣然, 毛腾霄*, authorsList=张聪聪, 艾青青, 罗霄, 陈红, 伍欣然, 毛腾霄, authorCompany=null, correspAuthors=毛腾霄, authorNote=

作者贡献:

张聪聪负责实验设计、数据分析、文章撰写及修改; 艾青青负责样本收集; 罗霄负责药材性状鉴定; 陈红负责稿件修改; 伍欣然责数据管理、稿件修改; 毛腾霄负责实验设计、总体把关和稿件修改等工作。

, correspAuthorsNote=
毛腾霄, Tel: 86-28-85366371, E-mail:
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Mod Chin Med (中国现代中药), 2025, 27: 1183-1189., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1308841229417283736, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, xref=null, ext=[AuthorCompanyExt(id=1308841229425672345, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, companyId=1308841229417283736, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Chengdu Institute for Drug Control, NMPA Key Laboratory for Quality Monitoring and Evaluation of Traditional Chinese Medicine, Chengdu 610046, China), AuthorCompanyExt(id=1308841229438255258, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, companyId=1308841229417283736, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=成都市药品检验研究院, 国家药品监督管理局中药材质量监测评价重点实验室, 四川 成都 610046)])], figs=[ArticleFig(id=1308841231606710465, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=EN, label=null, caption=null, figureFileSmall=aHbDUzsEpwlBS/DqsvLUAg==, figureFileBig=dsFxIJUBym85wJmw628Zkg==, tableContent=null), ArticleFig(id=1308841231669625026, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=CN, label=Fig.1, caption=Optimization of primer concentration. M: DL 2000 Marker; Lane 1 and 7: Negative control; Lanes 2-6: Primer concentration of 0.2, 0.4, 0.6, 0.8, 1.0 μmol·L-1 from Rheum palmatum; Lanes 8-12: Primer concentration of 0.2, 0.4, 0.6, 0.8, 1.0 μmol·L-1 from Rumex, figureFileSmall=aHbDUzsEpwlBS/DqsvLUAg==, figureFileBig=dsFxIJUBym85wJmw628Zkg==, tableContent=null), ArticleFig(id=1308841231837397187, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=EN, label=null, caption=null, figureFileSmall=gOurCLsx7r/fGqcBPaLf8w==, figureFileBig=7Gnh+hZR9hBMZ/uXtlVgWQ==, tableContent=null), ArticleFig(id=1308841231904506052, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=CN, label=Fig.2, caption=Optimization of reaction temperature. M: DL2000 Marker; Lane 1 and 7: Negative control; Lanes 2-6: Reaction temperature of 51, 53, 55, 57, 59 ℃ from Rheum palmatum; Lanes 8-12: Reaction temperature of 51, 53, 55, 57, 59 ℃ from Rumex, figureFileSmall=gOurCLsx7r/fGqcBPaLf8w==, figureFileBig=7Gnh+hZR9hBMZ/uXtlVgWQ==, tableContent=null), ArticleFig(id=1308841231967420613, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=EN, label=null, caption=null, figureFileSmall=YRWSod7CsjMkfJIguSukLA==, figureFileBig=7fIEXC0rHfyp4EEpDTBKjg==, tableContent=null), ArticleFig(id=1308841232034529478, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=CN, label=Fig.3, caption=Gel electrophoresis visualization of reference and confusable medicinal materials amplification. M: DL2000 Marker; Lane 1: Negative control; Lanes 2 Rheum tanguticum; Lane 3: Rheum officinale; Lane 4: Rheum palmatum; Lane 5: Rheumfranzenbachii; Lane 6: Rumex; Lane 7: Rumex nepalensis; Lane 8: Rumex japonicus; Lane 9: Rumex obtusifolius, figureFileSmall=YRWSod7CsjMkfJIguSukLA==, figureFileBig=7fIEXC0rHfyp4EEpDTBKjg==, tableContent=null), ArticleFig(id=1308841232114221255, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=EN, label=null, caption=null, figureFileSmall=TtZY9PSTrUl5Rsokpc3Sdg==, figureFileBig=2NS1fBDMR54OPMnzeoK78g==, tableContent=null), ArticleFig(id=1308841232198107336, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841227555012743, language=CN, label=Fig.4, caption=Sequencing peak diagram of forward and reverse primers for VPCR products. 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利用VPCR对大黄药材饮片及中成药中大黄成分基原鉴定的研究
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张聪聪 , 艾青青 , 罗霄 , 陈红 , 伍欣然 , 毛腾霄 *
药学学报 | 研究论文 2026,61(5): 1869-1875
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药学学报 |研究论文 2026 , 61 (5) : 1869 -1875
利用VPCR对大黄药材饮片及中成药中大黄成分基原鉴定的研究
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张聪聪, 艾青青, 罗霄, 陈红, 伍欣然, 毛腾霄*
作者信息
  • 成都市药品检验研究院, 国家药品监督管理局中药材质量监测评价重点实验室, 四川 成都 610046
通讯作者:
毛腾霄, Tel: 86-28-85366371, E-mail:
作者简介:

作者贡献:

张聪聪负责实验设计、数据分析、文章撰写及修改; 艾青青负责样本收集; 罗霄负责药材性状鉴定; 陈红负责稿件修改; 伍欣然责数据管理、稿件修改; 毛腾霄负责实验设计、总体把关和稿件修改等工作。

Molecular identification of the origin species of rhubarb medicinal materials, decoction pieces and Chinese patent medicines based on VPCR
Cong-cong ZHANG, Qing-qing AI, Xiao LUO, Hong CHEN, Xin-ran WU, Teng-xiao MAO*
Affiliations
  • Chengdu Institute for Drug Control, NMPA Key Laboratory for Quality Monitoring and Evaluation of Traditional Chinese Medicine, Chengdu 610046, China
出版时间: 2026-05-12 doi: 10.16438/j.0513-4870.2025-1261
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摘要
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大黄作为川产道地药材之一, 中国药典(2025年版)收载的基原物种为掌叶大黄、唐古特大黄及药用大黄, 然而当前市售大黄存在多基原混杂及伪品掺入等现象, 品种基原亟待理清。本研究采用文献报道叶绿体ndhF-rpl32片段作为特异条形码并进行验证, 通过优化DNA提取步骤、扩增体系和扩增程序, 建立一种可用于大黄药材、饮片及中成药中大黄成分真伪及基原鉴定的VPCR方法。相较于传统的PCR方法, VPCR (Ⅴ-shaped polymerase chain reaction)法将扩增时间由1.5 h缩短至30 min。采用建立的VPCR方法检测市售流通领域收集到的85批大黄样品, 包括30批药材、40批饮片及15批含大黄的中成药。结果表明: 样品中31批为唐古特大黄(药材18批, 饮片10批, 中成药3批), 36批为药用大黄(药材1批, 饮片25批, 中成药10批), 13批为掌叶大黄(药材8批, 饮片5批), 合格率为94.1%。3批药材(编号CD1、CD2、CD3)、2批中成药(编号Z4为片剂、Z12为颗粒剂)判定为大黄伪品中的华北大黄。本研究建立的VPCR方法可作为对中国药典(2025年版)大黄项下薄层色谱检查土大黄苷方法的补充, 对规范大黄药材市场、保证大黄临床用药安全提供依据。

大黄  /  VPCR  /  真伪鉴定  /  基原鉴定

Rhei Radix et Rhizoma is one of the genuine medicinal materials in Sichuan. Its original species are Rheum palmatum, Rh. tanguticum, and Rh. officinale, recorded by Chinese Pharmacopoeia (2025 Edition). However, there are many cases of multi-origin mixing and adulterant incorporation in the current commercially available Rhei Radix et Rhizoma, and the variety origin needs to be clarified urgently. In this study, the ndhF-rpl32 region of chloroplast genomes, as specific DNA barcode reported previously, was selected and confirmed. The VPCR method was established to identify the authenticity and origin of rhubarb medicinal materials, decoction pieces and Chinese patent medicines, in the process of optimizing DNA extraction procedure, amplification system and amplification procedure. Compared with the traditional PCR method, the amplification procedure has been refined through a VPCR heating model, curtailing the detection from 1.5 h to 30 min. The 85 batches of samples collected from the circulation area were tested by VPCR method, including 30 batches of medicinal materials, 40 batches of decoction pieces and 15 batches of Chinese patent medicines which contain Rhei Radix et Rhizoma. The experimental results showed that 31 batches are Rh. tanguticum (including 18 batches of medicinal materials, 10 batches of decoction pieces and 3 batches of Chinese patent medicines), 36 batches are Rh. officinale (including 1 batches of medicinal materials, 25 batches of decoction pieces and 10 batches of Chinese patent medicines) and 13 batches are Rh. palmatum (including 8 batches of medicinal materials and 5 batches of decoction pieces). The pass rate is 94.1%. Three batches of rhubarb medicinal materials (No. CD1, CD2, CD3) and two batches of Chinese patent medicines (No.Z4 tablet, No.Z12 granule) were determined to be Rh. franzenbachii Munt, as one of counterfeit Rhei Radix et Rhizoma varieties. The VPCR method established in this study can be used as supplement to rhapontin TLC inspection test of Rhei Radix et Rhizoma in Chinese Pharmacopoeia (2025 Edition) and standardize the market for this clinical medication as well as improve the safety of Rhei Radix et Rhizoma.

Rhei Radix et Rhizoma  /  Ⅴ-shaped polymerase chain reaction  /  authenticity verification  /  origin identification
张聪聪, 艾青青, 罗霄, 陈红, 伍欣然, 毛腾霄. 利用VPCR对大黄药材饮片及中成药中大黄成分基原鉴定的研究. 药学学报, 2026 , 61 (5) : 1869 -1875 . DOI: 10.16438/j.0513-4870.2025-1261
Cong-cong ZHANG, Qing-qing AI, Xiao LUO, Hong CHEN, Xin-ran WU, Teng-xiao MAO. Molecular identification of the origin species of rhubarb medicinal materials, decoction pieces and Chinese patent medicines based on VPCR[J]. Acta Pharmaceutica Sinica, 2026 , 61 (5) : 1869 -1875 . DOI: 10.16438/j.0513-4870.2025-1261
正文
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大黄(Rhei Radix et Rhizoma)是我国传统常用多基原中药, 始载于《神农本草经》列为下品[1]。中国药典(2025年版)一部收载大黄为蓼科植物掌叶大黄(Rheum palmatum L.)、唐古特大黄(Rh. tanguticum Maxim. ex Balf.)及药用大黄(Rh. officinale Baill.)的干燥根和根茎[2]。大黄作为川产道地药材之一, 可通过多成分-靶点-通路的途径, 发挥抗病毒、抗炎、抗氧化应激、抗细胞凋亡和调节机体免疫等功能[3, 4]。调查显示, 近年来大黄药材需求量较大, 过度采挖使野生大黄蕴藏量严重下降, 部分地区野生资源面临枯竭[5], 而人工栽培技术较为粗放, 导致大黄种质资源混乱, 种质质量难以保证[6]。大黄基原植物与同属和酸模属多种植物的性状及显微特征较相似, 致使当前市场大黄品种混乱, “以次充好”现象严重。常见伪品有大黄属植物华北大黄(Rh. franzenbachii Munt.)、河套大黄(Rh. hotaoense C. Y. Cheng et Kao)、藏边大黄(Rh. australe D. Don)、天山大黄(Rh. Wittrockii Lundstr.)和土大黄(Rumex)等。土大黄为蓼科植物酸模属植物: 钝叶酸模(Ru. obtusifolius L.)、羊蹄(Ru. japonicas Houtt.)、尼泊尔酸模(Ru. nepalensis Spreng.)、网果酸模(Ru. chalepensis Mill.)、巴天酸模(Ru. patientia L.)等的根及根茎[7]。目前市售大黄饮片质量参差不齐、煎煮工艺各异, 进而影响大黄汤剂及其中成药临床疗效[8-10]。正品大黄有很好的导泻效果而伪品则无明显导泻作用, 因此大黄药材真伪直接关系到临床用药安全性和有效性[11]。另一方面, 大黄为典型的多基原、多功效药材; 有报道表明掌叶大黄、唐古特大黄和药用大黄3种正品大黄的化学成分有一定的差别, 尤其是蒽醌类成分含量相差较大[12], 并且三者在药理活性上也存在不同程度的差异[13]。为了确保大黄更为精确的对症用药, 有必要针对不同功效用途, 选取不同基原大黄入药。因此, 在鉴定大黄真伪的前提下, 进一步鉴定大黄基原物种也具有重要的意义。
与传统中药鉴定方法相比, 基于DNA序列标记的中药材分子鉴定技术具有不依赖于药材形态、无需鉴定人长年经验积累、重复性好、准确度高等优点。特别是在鉴定近缘种药材上, 可以很好地解决因药材外观形状极其相似而导致的鉴定困难。因此, 近年来分子鉴定技术在中药鉴定领域得到了广泛的应用[14-16]。传统的聚合酶链式反应(polymerase chain reaction, PCR)方法包括变性、退火、延伸三个步骤, 每个步骤都需要一定的时间来确保核酸的有效扩增。已有研究发现, PCR反应在一个动态的变温过程中即可实现核酸的扩增, 即反应体系的温度只需要在高低两个温度点之间来回变化, 不需要在固定的温度点停留, 就可以实现核酸的快速扩增。该扩增方法可以将核酸扩增时间从原来的1 h以上缩短至30 min左右, 由于体系的温度-时间曲线图呈现多个重复的Ⅴ型, 因此该方法命名为VPCR (Ⅴ-shaped polymerase chain reaction)。本课题组前期已将该方法用于鉴定半夏、虎掌南星和水半夏[17]。
本研究选用文献[18]报道的叶绿体高变区ndhF-rpl32基因片段作为特异条形码, 并对该片段的鉴定能力及其引物4F、4R的特异性进行确认, 通过优化VPCR反应体系, 建立了一种用于大黄药材、饮片及中成药中大黄成分真伪及基原鉴定的VPCR技术, 为规范大黄药材市场、保证大黄临床用药安全提供依据。
材料与方法
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对照药材、易混药材与样品唐古特大黄(批号: 120902-201912)、药用大黄(批号: 120984-202203)、掌叶大黄(批号: 121249-202105)、华北大黄(批号: 121676-201201)和土大黄(批号: 121291-201803) 5种对照药材均购自中国食品药品检定研究院。尼泊尔酸模、羊蹄、钝叶酸模等大黄基原易混药材由本院中药室提供, 并由罗霄副主任中药师鉴定无误。85批样品均购于市售流通环节, 包括30批药材、40批饮片及15批含有大黄成分的中成药。
试剂 2×TaqMan Master Mix (批号29623)购自康为世纪生物科技股份有限公司; DP305植物DNA提取试剂盒(批号A1204A)购自天根生化科技(北京)有限公司; DL2000 DNA Maker (批号: 20B109Z)、GoldView核酸染料Ⅰ (批号: 240G04RI)、上样缓冲液6×Loading Buffer (批号: 70G01C)、1 mol·L-1 Tris-HCl (pH 8.0, 批号: 23AD12Y)、0.5 mol·L-1 EDTA (pH 8.0, 批号: 240C09Y)、微柱浓缩DNA凝胶回收试剂盒(批号: 240E11GW)、聚乙烯吡咯烷酮(PVP, 批号: 202401)购自北京庄盟国际生物基因科技有限公司; 50×TAE电泳缓冲液(批号: K4113735)购自生工生物工程(上海)股份有限公司; 其余化学试剂均为国产分析纯。引物(PAGE纯化)由北京擎科生物科技股份有限公司合成。DNA测序由北京擎科生物科技股份有限公司完成。核分离缓冲液的配置[19]: PVP10g, 1 mol·L-1 Tris-HCl (pH = 8.0) 50 mL, 0.5 mol·L-1 EDTA (pH 8.0) 20 mL, 氯化钠20.454 g, 充分溶解后定容至500 mL, 灭菌后加入β-巯基乙醇10 mL。
仪器 CP225D型天平(德国赛多利斯公司); 5810R型冷冻离心机(德国Eppendorf公司); MM400型球磨仪(德国莱驰公司); QuantStudioTM 5荧光定量PCR仪(美国ABI公司); Nanodrop One微量分光光度计(美国赛默飞公司); PowerPacTM Basic电泳仪(美国伯乐公司); 200型凝胶成像仪(美国Azure Biosystems公司); TU-100型恒温金属浴(上海一恒科学仪器有限公司)。
基因组DNA的提取   采取商品化的植物DNA提取试剂盒(柱式)对样本的DNA进行提取: 取样时采用一次性刀片切去药材及饮片的表面部分, 刮取中间部分约30 mg (对照药材直接称取)置于2.0 mL离心管中, 加入钢珠, 在球磨仪内研磨使样品呈粉末状, 按照植物DNA提取试剂盒说明书操作步骤进行基因组DNA提取。对于饮片和中成药处理细节作如下改动: 将饮片粉末加入1 mL核分离缓冲液充分混匀, 在冷冻高速离心机中12 000 r·min-1离心5 min, 弃上清液, 重复该步骤2次; 中成药称取约2 g, 丸剂切成小块, 散剂直接称取, 片剂刮去包衣切碎, 置于50 mL离心管中, 加入35 mL核分离液并涡旋充分混匀后, 7 500 r·min-1离心3 min, 弃上清液, 重复此步骤直至上清几乎无色。将经核分离缓冲液处理后的样品按提取试剂盒说明书操作。
特异DNA条形码及引物确认为确认ndhF-rpl32序列对大黄基原物种的鉴定能力及其引物的特异性, 以对照药材唐古特大黄、药用大黄、掌叶大黄、华北大黄和土大黄的基因组DNA为模板序列, 使用引物4F (5′-TCGATTCACCGGCTCTTACC-3′)、4R (5′-TGAAA TGAGACTGGTTATTTAG-3′)扩增ndhF-rpl32基因序列, 产物经纯化后外送测序。利用MEGA12软件进行序列比对, 并基于K2P (Kimura 2-parameter)模型进行遗传距离分析, 构建NJ (neighbor-joining algorithm)系统进化树, 设置Bootstrap重复值为1 000, 计算遗传距离。
VPCR反应体系优化   以掌叶大黄和土大黄为代表样品, 分别对不同引物浓度和退火温度进行优化, 分别设置引物浓度为0.2、0.4、0.6、0.8、1.0 μmol·L-1, 退火温度为51、53、55、57、59 ℃。VPCR反应体系为: 2×TaqMan MasterMix 12.5 μL, 上下游引物(10 μm)各0.5~2.5 μL, DNA模板约50 ng, 加灭菌双蒸水补足体积至25 μL。VPCR扩增条件为: 95 ℃, 30 s; 94 ℃, 1 s, 51~59 ℃, 1 s, 35个循环。
琼脂糖凝胶电泳   以对照药材、易混药材及样品的基因组DNA为模板, 分别使用引物4F、4R, 采用优化的VPCR程序扩增后, 取扩增产物5 μL与上样缓冲液1 μL进行混合, 将混合液加至经GoldView核酸染料Ⅰ染色的琼脂糖凝胶(1.5%)进行电泳分离, 分离电压110 V, 电泳时间30 min; 电泳结束后, 将该凝胶置于琼脂糖凝胶成像系统进行观察并拍照。
VPCR产物纯化、测序及NJ进化树的构建   按照DNA凝胶回收试剂盒对待测样品的VPCR产物进行纯化, 外送生物公司进行双向测序, 利用SnapGene软件对测序所得峰图文件进行序列拼接, 利用MEGA 12软件进行Clustal W多序列比对、计算K2P距离和构建NJ系统进化树, Bootstrap 1 000次重复检验分支系统发育的可靠性。
结果
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1　DNA质检及对照药材基原确认
所提取的基因组DNA经微量分光光度计测量纯度A260/280均为1.7~1.9, 浓度均大于或等于30 ng·μL-1, 满足后续实验的要求。对照药材及易混药材的matK序列经通用引物扩增并测序后在GenBank BLAST (https://blastncbi.nlm.nih.gov/Blast.cgi)网站进行比对分析, 对物种基原进行再次确认。
2　特异条形码确认
大黄基原物种ndhF-rpl32序列的种内遗传距离均为0, 种间遗传距离为0.005~0.015 (平均遗传距离为0.01), 种内、种间存在显著的条形码间隙(barcoding gap), 表明所筛选片段符合DNA条形码的要求。
3　VPCR反应体系优化
掌叶大黄和土大黄VPCR产物的亮度除在0.2 μmol·L-1引物浓度下较暗, 在0.4~0.8 μmol·L-1引物浓度下亮度基本一致。由于过高的引物浓度会导致非特异性扩增物的产生, 所以优化的引物浓度为0.6 μmol·L-1。掌叶大黄在退火温度为53、55、57 ℃时, VPCR产物最亮; 土大黄在退火温度为53、55 ℃时, VPCR产物最亮, 所以优化的退火温度为55 ℃。结果见图1、2。
优化后的VPCR反应体系为: 2×Taqman MasterMix 12.5 μL, 上下游引物终浓度各0.6 μmol·L-1, DNA模板约50 ng, 加灭菌双蒸水补足体积至25 μL。VPCR扩增条件为: 95 ℃, 30 s; 94 ℃, 1 s, 55 ℃ 1 s, 35个循环。
4　扩增体系确认
采用优化后的VPCR程序扩增得到长度约为600 bp的单一条带, 表明引物具有较高的特异性。对照药材及易混药材的电泳结果见图3。在紫外灯下切取目的基因条带, 经DNA凝胶回收试剂盒纯化后, 外送生物公司双向测序, 正反向引物测序峰图见图4。
5　样品检测
采用本研究所建立的VPCR方法, 对85批样品包括30批药材、40批饮片及15批中成药进行扩增并外送测序, 序列经比对分析, 所得结果见图5。其中唐古特大黄31批(药材18批, 饮片10批, 中成药3批), 药用大黄36批(药材1批, 饮片25批, 中成药10批), 掌叶大黄13批(药材8批, 饮片5批)。3批大黄药材(编号: CD1、CD2、CD3)和2批中成药(编号Z4为片剂、Z12为颗粒剂)与华北大黄分为一簇, 判定为大黄伪品。图6详细展示了以上5批伪品与对照药材和易混药材的聚类情况。
讨论
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本研究利用建立的VPCR法对市售的85批大黄药材、饮片及中成药进行检测, 检出5批华北大黄, 阳性样品率为5.9%。2018年四川省中药材(饮片)专项抽检对大黄进行了全面的质量考核, 在105批大黄样品中有3批检查出土大黄苷[20]。中国药典(2025年版)一部中收载的150余个大黄复方制剂中, 大黄的质量控制多采用显微、薄层色谱、高效液相色谱法; 土大黄苷作为伪品大黄中的特征性成分, 仅明确了5个处方(九味肝泰胶囊、三黄片、致康胶囊、痔疮胶囊和通窍耳聋丸)中大黄类药材要进行土大黄苷定性检测。已有文献[21-23]报道在清肺抑火制剂、十滴水和复方龙胆碳酸氢钠片中发现存在伪品大黄代替大黄投料的情况。
本研究在使用商品化试剂盒提取基因组DNA时, 根据大黄药材性质优化了提取步骤, 以保证高质量的DNA用于后续基因扩增。中成药大多为复方制剂, 在生产过程中经历了复杂的加工, 其DNA降解程度较中药材及饮片而言更高, 检测难度加大[24]。对于大黄饮片和以大黄原粉入药的中成药, 前处理时使用核分离缓冲液能够显著提高DNA的提取质量; 而对于大黄在处方中占比较低、以提取液入药的中成药品种, 其DNA富集质量、扩增测序的结果均不太理想, 采用VPCR法较难确定其基原物种。本课题组前期建立了半夏易混伪品的多重实时荧光VPCR系统, 根据ITS序列设计特异性的引物和探针, 通过单管反应同时检测半夏、虎掌南星、水半夏3种物质, 并结合直接裂解提取法与超快速VPCR扩增, 使整个实验流程缩短至32 min以内。本研究采用ndhF-rpl32作为特异条形码, VPCR产物经纯化后测序, 将序列通过软件分析生成系统进化树, 以聚类图判断大黄真伪及基原。本研究虽然已将PCR扩增过程缩短至30 min, 并优化商品化试剂盒的提取步骤, 但DNA提取步骤仍需花费较长时间。下一步可开发大黄DNA直接裂解提取法, 减少DNA提取所花费的时间; 并收集更多大黄基原的易混药材品种, 进一步扩大本方法可鉴定的品种范围。
本研究基于特异条形码建立的VPCR法能够实现对大黄药材、饮片及中成药中的大黄成分进行真伪及基原鉴定, 可作为中国药典(2025年版)大黄项下薄层色谱检查土大黄苷方法的补充, 从而完善大黄的鉴定标准, 并用以控制复方制剂中大黄的准确投料, 以保障此类药物的用药安全。

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doi: 10.16438/j.0513-4870.2025-1261
  • 接收时间：2025-10-24
  • 首发时间：2026-09-21
  • 出版时间：2026-05-12
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  • 收稿日期：2025-10-24
  • 修回日期：2026-01-20
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    成都市药品检验研究院, 国家药品监督管理局中药材质量监测评价重点实验室, 四川 成都 610046

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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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