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Article(id=1308840500224951184, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840287129141340, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-0659, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1749657600000, receivedDateStr=2025-06-12, revisedDate=1768147200000, revisedDateStr=2026-01-12, acceptedDate=null, acceptedDateStr=null, onlineDate=1789981492334, onlineDateStr=2026-09-21, pubDate=1775923200000, pubDateStr=2026-04-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789981492334, onlineIssueDateStr=2026-09-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789981492334, creator=13701087609, updateTime=1789981492334, updator=13701087609, issue=Issue{id=1308840287129141340, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='4', pageStart='1001', pageEnd='1434', issueExtLink='null', onlineDate='null', pubDate='1775923200000', pubDateStr='2026-04-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789981441528, creator='13701087609', updateTime=1789984228828, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308851977992368434, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840287129141340, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308851977992368435, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840287129141340, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1398, endPage=1407, ext={EN=ArticleExt(id=1308840500443054993, articleId=1308840500224951184, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Identification of Coreopsis tinctoria Nutt. using ITS2 barcode and the quality evaluation of its medicinal materials based on HPLC, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Coreopsis tinctoria Nutt. is an important ethnomedicine traditionally used in Xinjiang, China. This study employed ITS2 barcode sequencing for species authentication of commercial C. tinctoria samples. Furthermore, a comprehensive quality evaluation system was established based on HPLC fingerprinting, quantitative analysis of multiple markers, and chemical pattern recognition. A total of 35 batches of C. tinctoria samples were collected from different growing regions in Xinjiang. Genomic DNA was extracted from leaves, and the ITS2 region was amplified by PCR followed by sequencing. The obtained sequences were assembled, proofread, and validated through BLAST analysis against the NCBI database. Subsequently, a neighbor-joining (NJ) phylogenetic tree was constructed based on the aligned sequences. Chromatographic fingerprinting was performed on a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). Similarity analysis of the full-spectrum fingerprints was conducted using the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 version)". Chemometric approaches including cluster analysis (CA), principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were employed. Furthermore, a validated HPLC method was developed for simultaneous quantification of four characteristic constituents. The results showed that the main haplotypes of ITS2 sequences in 35 C. tinctoria samples were H1/H2, with a length of 225 bp. The intraspecific variation was small, and the sequences were relatively conservative, indicating that ITS2 sequences can be used as a reliable DNA barcode for the identification of C. tinctoria. The HPLC fingerprinting showed 16 common peaks, and chlorogenic acid, flavanomarein, marein, and isochlorogenic acid A were successfully identified. The similarity of the samples ranged from 0.936 to 0.998. CA classified the samples into three groups, PCA obtained five principal components with a cumulative variance contribution rate of 84.953%, and PLS-DA identified eight components, including chlorogenic acid, flavanomarein, marein, as markers for regional differences. Quantitative analysis showed that the mass fractions of the four components were as follows: Chlorogenic acid 0.860-7.504 mg·g-1, flavanomarein 3.368-19.182 mg·g-1, marein 19.719-83.492 mg·g-1, and isochlorogenic acid A 2.039-12.568 mg·g-1. The DNA barcoding technique and integrated HPLC fingerprinting/multi-component method demonstrated high reliability and stability. This study provides a scientific foundation for quality control and further research on C. tinctoria.

, authors=Hairula MANERHABA, Bo CHENG, Tuerxunjiang DILINUER, Jiang HE*, Wei-jun YANG*, authorsList=Hairula MANERHABA, Bo CHENG, Tuerxunjiang DILINUER, Jiang HE, Wei-jun YANG, authorCompany=null, correspAuthors=Jiang HE, Wei-jun YANG, 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=1308840504033379226, articleId=1308840500224951184, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=雪菊ITS2序列鉴定及药材质量评价研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

雪菊(Coreopsis tinctoria Nutt.)是我国新疆地区重要的民族传统习用药材。本研究基于ITS2基因序列对市售雪菊药材进行物种鉴定以明确其真伪; 同时, 通过建立HPLC指纹图谱、多指标成分含量测定结合化学模式识别的方法, 构建雪菊药材的质量评价体系。从新疆不同产区共收集35批样品, 提取基因组DNA, 经PCR扩增获得ITS2基因序列并进行拼接, 通过NCBI数据库BLAST比对后构建邻接法(neighbor joining, NJ)系统进化树。采用ZORBAX SB-C18色谱柱(250 mm × 4.6 mm, 5 μm)建立药材指纹图谱, 结合《中药色谱指纹图谱相似度评价系统(2012版)》进行全谱分析; 采用聚类分析(cluster analysis, CA)、主成分分析(principal component analysis, PCA)和偏最小二乘法-判别分析(partial least squares-discriminant analysis, PLS-DA)进行化学计量学研究, 并建立HPLC法同时测定4个特征成分含量。结果显示: 35批次雪菊药材ITS2序列主体单倍型为H1/H2, 长度均为225 bp, 种内发生变异较小且序列较为保守, 表明ITS2序列可作为雪菊药材鉴定的可靠DNA条形码。HPLC指纹图谱得到16个共有峰, 成功指认出绿原酸、黄诺玛苷、马里苷和异绿原酸A, 样品相似度为0.936~0.998。CA将样品分为3类, PCA得到5个主成分的累积方差贡献率为84.953%, PLS-DA确定绿原酸、黄诺玛苷和马里苷等8个成分为产地差异标志物。定量分析显示4种成分质量分数分别为: 绿原酸0.860~7.504、黄诺玛苷3.368~19.182、马里苷19.719~83.492和异绿原酸A 2.039~12.568 mg·g-1。本研究建立的DNA条形码鉴定技术及HPLC指纹图谱-多指标含量测定方法稳定可靠, 为雪菊药材质量控制和深入研究提供了科学依据。

, authors=满尔哈巴·海如拉, 程波, 地力努尔·吐尔逊江, 何江*, 杨伟俊*, authorsList=满尔哈巴·海如拉, 程波, 地力努尔·吐尔逊江, 何江, 杨伟俊, authorCompany=null, correspAuthors=何江, 杨伟俊, authorNote=

#

共同第一作者.

作者贡献:

满尔哈巴·海如拉设计实验、撰写及修改论文; 程波设计实验、提供学术指导及修改论文; 地力努尔·吐尔逊江负责实验样本及数据的采集; 何江、杨伟俊提供学术指导及基金支持。所有的作者都对原稿的最终版本做出了贡献。

, correspAuthorsNote=
何江, Tel: 86-991-2320292, E-mail:
杨伟俊, E-mail:
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Suzhou: Soochow University, 2016., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1308840504234705819, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, xref=null, ext=[AuthorCompanyExt(id=1308840504243094428, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, companyId=1308840504234705819, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Key Laboratory of Uyghur Medicine, Xinjiang Institute of Materia Medica, Urumqi 830010, China), AuthorCompanyExt(id=1308840504247288733, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, companyId=1308840504234705819, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=新疆维吾尔自治区药物研究院, 维吾尔药重点实验室, 新疆 乌鲁木齐 830010)])], figs=[ArticleFig(id=1308840506084393921, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=EN, label=null, caption=null, figureFileSmall=HmuDJspsPRK52rqUbfM22Q==, figureFileBig=J0H9E0RwG+4F2pmOthrqRQ==, tableContent=null), ArticleFig(id=1308840506164085698, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=CN, label=Fig.1, caption=Construction of evolutionary tree of closely related species of Coreopsis tinctoria Nutt., figureFileSmall=HmuDJspsPRK52rqUbfM22Q==, figureFileBig=J0H9E0RwG+4F2pmOthrqRQ==, tableContent=null), ArticleFig(id=1308840506336052163, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=EN, label=null, caption=null, figureFileSmall=xXhnsp1PKf4qKfg+cKU+cA==, figureFileBig=r/VWYI1u+VwDBVFtjjRC6w==, tableContent=null), ArticleFig(id=1308840506407355332, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=CN, label=Fig.2, caption=Fingerprints of 35 batches of C. tinctoria. A: HPLC fingerprint of 15 batches of C. tinctoria and reference; B: HPLC chromatogram of mixed standards. 5: Chlorogenic acid; 8: Flavanomarein; 11: Marein; 12: Isochlorogenic acid A; 1-4, 6-7, 9, 10, 13-16: Unkonwn, figureFileSmall=xXhnsp1PKf4qKfg+cKU+cA==, figureFileBig=r/VWYI1u+VwDBVFtjjRC6w==, tableContent=null), ArticleFig(id=1308840506596099013, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=EN, label=null, caption=null, figureFileSmall=o1596Zp1aV9kzdSvHe4SAQ==, figureFileBig=VgA9Iaj9eseSixkve97CLg==, tableContent=null), ArticleFig(id=1308840506650624966, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=CN, label=Fig.3, caption=Integrated cluster heatmap and principal component analysis plot of C. tinctoria. A: Cluster heat map analysis of C. tinctoria; B: PCA lithotripsy diagram of C. tinctoria; C: Plot of PCA scores of C. tinctoria. 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PLS-DA: Partial least squares-discriminant analysis; VIP: Variable importance in projection, figureFileSmall=Cg5E/yUqf7UCpwm6WSrjUg==, figureFileBig=coujaSrB8sqyfVdFlGj6JA==, tableContent=null), ArticleFig(id=1308840508483535817, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Sample No.OriginSample No.Origin
S1Qapqal Xibe Autonomous County, Ili PrefectureS19Dabancheng District, Ürümqi City
S2Altay City, Altay PrefectureS20Gongliu County, Ili Prefecture
S3Habahe County, Altay PrefectureS21Zhaosu County, Ili Prefecture
S4Taxkorgan Tajik Autonomous County, Kashgar PrefectureS22Shaya County, Aksu Prefecture
S5Pishan County, Hotan PrefectureS23Shaya County, Aksu Prefecture
S6Turpan City, Turpan PrefectureS24Shaya County, Aksu Prefecture
S7Dabancheng District, Ürümqi CityS25Shaya County, Aksu Prefecture
S8Pishan County, Hotan PrefectureS26Korla City, Bayingolin Mongol Autonomous Prefecture
S9Pishan County, Hotan PrefectureS27Qapqal Xibe Autonomous County, Ili Prefecture
S10Pishan County, Hotan PrefectureS28Qapqal Xibe Autonomous County, Ili Prefecture
S11Pishan County, Hotan PrefectureS29Taxkorgan Tajik Autonomous County, Kashgar Prefecture
S12Ürümqi CityS30Yopurga County, Kashgar Prefecture
S13Altay City, Altay PrefectureS31Taxkorgan Tajik Autonomous County, Kashgar Prefecture
S14Dabancheng District, Ürümqi CityS32Kashgar City, Kashgar Prefecture
S15Dabancheng District, Ürümqi CityS33Kashgar City, Kashgar Prefecture
S16Dabancheng District, Ürümqi CityS34Kashgar City, Kashgar Prefecture
S17Qapqal Xibe Autonomous County, Ili PrefectureS35Kashgar City, Kashgar Prefecture
S18Altay City, Altay Prefecture
), ArticleFig(id=1308840508546450378, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=CN, label=Tab.1, caption=

Soure of samples of Coreopsis tinctoria Nutt.

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Sample No.OriginSample No.Origin
S1Qapqal Xibe Autonomous County, Ili PrefectureS19Dabancheng District, Ürümqi City
S2Altay City, Altay PrefectureS20Gongliu County, Ili Prefecture
S3Habahe County, Altay PrefectureS21Zhaosu County, Ili Prefecture
S4Taxkorgan Tajik Autonomous County, Kashgar PrefectureS22Shaya County, Aksu Prefecture
S5Pishan County, Hotan PrefectureS23Shaya County, Aksu Prefecture
S6Turpan City, Turpan PrefectureS24Shaya County, Aksu Prefecture
S7Dabancheng District, Ürümqi CityS25Shaya County, Aksu Prefecture
S8Pishan County, Hotan PrefectureS26Korla City, Bayingolin Mongol Autonomous Prefecture
S9Pishan County, Hotan PrefectureS27Qapqal Xibe Autonomous County, Ili Prefecture
S10Pishan County, Hotan PrefectureS28Qapqal Xibe Autonomous County, Ili Prefecture
S11Pishan County, Hotan PrefectureS29Taxkorgan Tajik Autonomous County, Kashgar Prefecture
S12Ürümqi CityS30Yopurga County, Kashgar Prefecture
S13Altay City, Altay PrefectureS31Taxkorgan Tajik Autonomous County, Kashgar Prefecture
S14Dabancheng District, Ürümqi CityS32Kashgar City, Kashgar Prefecture
S15Dabancheng District, Ürümqi CityS33Kashgar City, Kashgar Prefecture
S16Dabancheng District, Ürümqi CityS34Kashgar City, Kashgar Prefecture
S17Qapqal Xibe Autonomous County, Ili PrefectureS35Kashgar City, Kashgar Prefecture
S18Altay City, Altay Prefecture
), ArticleFig(id=1308840508642919371, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
PCEigenvalueVariance contribution rate/%Cumulative variance contribution rate/%
15.91536.97036.970
23.16319.76756.737
31.96812.29769.034
41.5059.40478.438
51.0426.51584.953
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Principal component (PC) eigenvalues and contribution

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PCEigenvalueVariance contribution rate/%Cumulative variance contribution rate/%
15.91536.97036.970
23.16319.76756.737
31.96812.29769.034
41.5059.40478.438
51.0426.51584.953
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Peak numberVIPPeak numberVIP
161.363 370.995 212
151.355 6390.969 622
41.161 430.875 233
51.153 120.781 712
101.142 18140.759 834
81.091 2210.676 328
111.089 6460.636 806
121.043 13130.326 196
), ArticleFig(id=1308840508844245966, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=CN, label=Tab.3, caption=

VIP scores of PLS-DA of 16 chromatographic peaks. *The peak numbers are arranged in descending order of VIP values. Variables with VIP values greater than 1 are generally recognized as having a significant contribution to the classification and discrimination of the model

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Peak numberVIPPeak numberVIP
161.363 370.995 212
151.355 6390.969 622
41.161 430.875 233
51.153 120.781 712
101.142 18140.759 834
81.091 2210.676 328
111.089 6460.636 806
121.043 13130.326 196
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CompoundEquationR2Range/μg·mL-1
Chlorogenic acidY = 364.6X - 134.270.999 430.080-180.480
FlavanomareinY = 445.84X - 45.6690.999 525.120-150.720
MareinY = 238.22X - 31.1921.000 032.960-197.760
Isochlorogenic acid AY = 133.23X - 72.7990.999 38.160-48.960
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Results of a linear relationship investigation of 4 index components of C. tinctoria

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CompoundEquationR2Range/μg·mL-1
Chlorogenic acidY = 364.6X - 134.270.999 430.080-180.480
FlavanomareinY = 445.84X - 45.6690.999 525.120-150.720
MareinY = 238.22X - 31.1921.000 032.960-197.760
Isochlorogenic acid AY = 133.23X - 72.7990.999 38.160-48.960
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Sample No.Mass fraction/mg·g-1
Chlorogenic acidFlavanomareinMareinIsochlorogenic acid A
S11.0269.13952.6962.135
S25.29310.62260.8288.937
S35.18811.28952.8637.450
S44.10119.18283.4924.644
S55.9268.84952.7509.287
S61.7529.67338.7236.937
S73.0909.22345.7477.683
S83.1369.59850.86512.568
S93.9598.85539.0439.188
S101.7148.58644.8786.597
S111.5676.32234.2686.926
S121.0904.87825.2204.646
S132.7844.79229.5077.443
S144.11611.06250.37410.304
S152.2649.14051.1188.008
S163.21311.22254.7747.702
S171.8357.68744.6384.742
S181.7559.24245.9083.429
S193.5477.49736.8489.645
S201.0263.63819.7193.636
S212.6618.01738.8876.881
S220.8605.92624.9523.936
S231.38711.47647.1356.612
S242.5559.57141.8207.591
S252.5097.16744.2714.172
S261.7803.36823.9897.092
S270.8737.83646.3352.039
S282.5075.82833.4528.860
S292.2067.14350.6952.076
S304.53511.91949.95410.671
S317.5049.19351.6475.355
S323.4709.62654.1669.221
S331.31213.52038.3974.535
S341.98711.32854.5005.105
S354.7055.85129.2009.757
), ArticleFig(id=1308840509154624466, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840500224951184, language=CN, label=Tab.5, caption=

Content determination results of 4 components in 35 batches of C. tinctoria

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample No.Mass fraction/mg·g-1
Chlorogenic acidFlavanomareinMareinIsochlorogenic acid A
S11.0269.13952.6962.135
S25.29310.62260.8288.937
S35.18811.28952.8637.450
S44.10119.18283.4924.644
S55.9268.84952.7509.287
S61.7529.67338.7236.937
S73.0909.22345.7477.683
S83.1369.59850.86512.568
S93.9598.85539.0439.188
S101.7148.58644.8786.597
S111.5676.32234.2686.926
S121.0904.87825.2204.646
S132.7844.79229.5077.443
S144.11611.06250.37410.304
S152.2649.14051.1188.008
S163.21311.22254.7747.702
S171.8357.68744.6384.742
S181.7559.24245.9083.429
S193.5477.49736.8489.645
S201.0263.63819.7193.636
S212.6618.01738.8876.881
S220.8605.92624.9523.936
S231.38711.47647.1356.612
S242.5559.57141.8207.591
S252.5097.16744.2714.172
S261.7803.36823.9897.092
S270.8737.83646.3352.039
S282.5075.82833.4528.860
S292.2067.14350.6952.076
S304.53511.91949.95410.671
S317.5049.19351.6475.355
S323.4709.62654.1669.221
S331.31213.52038.3974.535
S341.98711.32854.5005.105
S354.7055.85129.2009.757
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雪菊ITS2序列鉴定及药材质量评价研究
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满尔哈巴·海如拉 , 程波 , 地力努尔·吐尔逊江 , 何江 * , 杨伟俊 *
药学学报 | 研究论文 2026,61(4): 1398-1407
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药学学报 |研究论文 2026 , 61 (4) : 1398 -1407
雪菊ITS2序列鉴定及药材质量评价研究
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满尔哈巴·海如拉, 程波, 地力努尔·吐尔逊江, 何江* , 杨伟俊*
作者信息
  • 新疆维吾尔自治区药物研究院, 维吾尔药重点实验室, 新疆 乌鲁木齐 830010
通讯作者:
何江, Tel: 86-991-2320292, E-mail:
杨伟俊, E-mail:
作者简介:

#

共同第一作者.

作者贡献:

满尔哈巴·海如拉设计实验、撰写及修改论文; 程波设计实验、提供学术指导及修改论文; 地力努尔·吐尔逊江负责实验样本及数据的采集; 何江、杨伟俊提供学术指导及基金支持。所有的作者都对原稿的最终版本做出了贡献。

Identification of Coreopsis tinctoria Nutt. using ITS2 barcode and the quality evaluation of its medicinal materials based on HPLC
Hairula MANERHABA, Bo CHENG, Tuerxunjiang DILINUER, Jiang HE* , Wei-jun YANG*
Affiliations
  • Key Laboratory of Uyghur Medicine, Xinjiang Institute of Materia Medica, Urumqi 830010, China
出版时间: 2026-04-12 doi: 10.16438/j.0513-4870.2025-0659
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摘要
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雪菊(Coreopsis tinctoria Nutt.)是我国新疆地区重要的民族传统习用药材。本研究基于ITS2基因序列对市售雪菊药材进行物种鉴定以明确其真伪; 同时, 通过建立HPLC指纹图谱、多指标成分含量测定结合化学模式识别的方法, 构建雪菊药材的质量评价体系。从新疆不同产区共收集35批样品, 提取基因组DNA, 经PCR扩增获得ITS2基因序列并进行拼接, 通过NCBI数据库BLAST比对后构建邻接法(neighbor joining, NJ)系统进化树。采用ZORBAX SB-C18色谱柱(250 mm × 4.6 mm, 5 μm)建立药材指纹图谱, 结合《中药色谱指纹图谱相似度评价系统(2012版)》进行全谱分析; 采用聚类分析(cluster analysis, CA)、主成分分析(principal component analysis, PCA)和偏最小二乘法-判别分析(partial least squares-discriminant analysis, PLS-DA)进行化学计量学研究, 并建立HPLC法同时测定4个特征成分含量。结果显示: 35批次雪菊药材ITS2序列主体单倍型为H1/H2, 长度均为225 bp, 种内发生变异较小且序列较为保守, 表明ITS2序列可作为雪菊药材鉴定的可靠DNA条形码。HPLC指纹图谱得到16个共有峰, 成功指认出绿原酸、黄诺玛苷、马里苷和异绿原酸A, 样品相似度为0.936~0.998。CA将样品分为3类, PCA得到5个主成分的累积方差贡献率为84.953%, PLS-DA确定绿原酸、黄诺玛苷和马里苷等8个成分为产地差异标志物。定量分析显示4种成分质量分数分别为: 绿原酸0.860~7.504、黄诺玛苷3.368~19.182、马里苷19.719~83.492和异绿原酸A 2.039~12.568 mg·g-1。本研究建立的DNA条形码鉴定技术及HPLC指纹图谱-多指标含量测定方法稳定可靠, 为雪菊药材质量控制和深入研究提供了科学依据。

雪菊  /  DNA条形码  /  HPLC指纹图谱  /  化学计量学  /  含量测定

Coreopsis tinctoria Nutt. is an important ethnomedicine traditionally used in Xinjiang, China. This study employed ITS2 barcode sequencing for species authentication of commercial C. tinctoria samples. Furthermore, a comprehensive quality evaluation system was established based on HPLC fingerprinting, quantitative analysis of multiple markers, and chemical pattern recognition. A total of 35 batches of C. tinctoria samples were collected from different growing regions in Xinjiang. Genomic DNA was extracted from leaves, and the ITS2 region was amplified by PCR followed by sequencing. The obtained sequences were assembled, proofread, and validated through BLAST analysis against the NCBI database. Subsequently, a neighbor-joining (NJ) phylogenetic tree was constructed based on the aligned sequences. Chromatographic fingerprinting was performed on a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). Similarity analysis of the full-spectrum fingerprints was conducted using the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 version)". Chemometric approaches including cluster analysis (CA), principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were employed. Furthermore, a validated HPLC method was developed for simultaneous quantification of four characteristic constituents. The results showed that the main haplotypes of ITS2 sequences in 35 C. tinctoria samples were H1/H2, with a length of 225 bp. The intraspecific variation was small, and the sequences were relatively conservative, indicating that ITS2 sequences can be used as a reliable DNA barcode for the identification of C. tinctoria. The HPLC fingerprinting showed 16 common peaks, and chlorogenic acid, flavanomarein, marein, and isochlorogenic acid A were successfully identified. The similarity of the samples ranged from 0.936 to 0.998. CA classified the samples into three groups, PCA obtained five principal components with a cumulative variance contribution rate of 84.953%, and PLS-DA identified eight components, including chlorogenic acid, flavanomarein, marein, as markers for regional differences. Quantitative analysis showed that the mass fractions of the four components were as follows: Chlorogenic acid 0.860-7.504 mg·g-1, flavanomarein 3.368-19.182 mg·g-1, marein 19.719-83.492 mg·g-1, and isochlorogenic acid A 2.039-12.568 mg·g-1. The DNA barcoding technique and integrated HPLC fingerprinting/multi-component method demonstrated high reliability and stability. This study provides a scientific foundation for quality control and further research on C. tinctoria.

Coreopsis tinctoria Nutt.  /  DNA barcoding  /  HPLC fingerprint  /  chemometrics  /  content determination
满尔哈巴·海如拉, 程波, 地力努尔·吐尔逊江, 何江, 杨伟俊. 雪菊ITS2序列鉴定及药材质量评价研究. 药学学报, 2026 , 61 (4) : 1398 -1407 . DOI: 10.16438/j.0513-4870.2025-0659
Hairula MANERHABA, Bo CHENG, Tuerxunjiang DILINUER, Jiang HE, Wei-jun YANG. Identification of Coreopsis tinctoria Nutt. using ITS2 barcode and the quality evaluation of its medicinal materials based on HPLC[J]. Acta Pharmaceutica Sinica, 2026 , 61 (4) : 1398 -1407 . DOI: 10.16438/j.0513-4870.2025-0659
正文
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雪菊(Coreopsis tinctoria), 学名两色金鸡菊(Coreopsis tinctoria Nutt), 为菊科金鸡菊属蛇目菊种一年生草本植物, 原产于北美洲[1], 在中国各地均有栽培, 新疆是主要产区之一, 尤其和田地区皮山县克里阳乡产的雪菊品质最佳[2]。因其能够生长在新疆昆仑山北麓海拔1 500~3 000 m的高寒山区, 故被称为雪菊或昆仑雪菊[3], 当地居民常采集新鲜头状花序浸泡当花茶饮用。雪菊中含有氨基酸类、多糖类、多酚类、有机酸类、黄酮类、挥发油类、甾类和香豆素等多种化学成分[4], 具有调节血糖、抗氧化、抗肿瘤等多种药理活性[5]。
古扎力努尔等[6]研究结果表明不同产地雪菊中绿原酸及总黄酮含量有差异, 其含量普遍高于其他种类菊花中绿原酸的含量, 总黄酮平均含量大于10%, 其中克里阳高海拔地区生长的雪菊中总黄酮含量最高为18.34%。徐赛等[7]通过DPPH法和ABTS法评估了雪菊胎菊与全菊的体外抗氧化能力, 结果表明, 雪菊中富含绿原酸和异绿原酸A (3,5-O-二咖啡酰基奎宁酸)等酚酸类化合物具有显著的抗氧化作用。周新宇等[8]通过UV-Vis检测出雪菊总黄酮含量为168.44 ± 1.27~289.42 ± 0.25 mg·g-1, 结果显示新疆克里阳雪菊总黄酮含量高且抗氧化活性较好。木合塔尔·吐尔洪等[9]通过RP-HPLC法测定昆仑雪菊中绿原酸, 含量平均值为2.456 mg·g-1。程伟等[10]基于HPLC方法对不同产地雪菊中黄诺玛苷进行测定, 其含量为7.44~19.82 mg·g-1。目前对雪菊中异绿原酸A的含量测定的相关报道较少。高娟娟[11]经不同方式干燥的雪菊, 其提取物均具有不同程度的抗氧化活性, 采用40 ℃烘干时雪菊提取物中总黄酮及异奥卡宁、马里苷、芦丁、奥卡宁含量较高, 其提取物的体外抗氧化活性最好。Ma等[12]的从雪菊中分离、鉴定并制备得到马里苷等11种多酚化合物, 都具有不同程度的抗氧化活性。Han等[13]研究发现马里苷在大鼠体内大部分转化可水解代谢生成奥卡宁发挥抗氧化活性。于凡等[14]采用UPLC-DAD建立了雪菊总黄酮部位中异奥卡宁、马里苷、奥卡宁等10种成分含量测定方法, 选用285与378 nm作为检测波长。姚新成等[15]优化超声波提取雪菊总黄酮, 获得最佳提取条件为超声提取30 min, 液料比21∶1, 乙醇浓度60%, 可获得质量稳定、含量较高的总黄酮提取物。
本研究拟通过DNA条形码及HPLC指纹图谱整合化学模式识别技术与多指标含量测定方法, 构建中药材“分子-化学”多维质量控制体系。DNA条形码技术对雪菊药材及其近缘物种进行物种特异性基因片段扩增与测序, 通过GenBank数据库进行Blast比对, 从分子层面解决传统形态学鉴别难以区分的雪菊混伪品问题; 同时, 结合HPLC技术通过优化色谱条件获取特征性指纹图谱, 利用化学模式识别法(主成分分析、聚类分析、判别分析)挖掘共有峰与特征峰信息, 解析不同产地雪菊药材的化学物质基础差异。在多指标含量测定环节, 选取具有明确药理活性的标志性成分构建综合质量评价模型。将分子生物学技术的准确性与化学分析方法的直观性有机融合, 为雪菊药材质量标准提升提供科学依据, 也为雪菊药材的规范化种植、标准化栽培及规范化销售等全产业链环节提供量化质量控制标准, 为推动雪菊产业高质量发展提供可靠的科学依据与技术支撑。
材料与方法
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仪器   PCR扩增仪(C1000, 美国BIO-RAD公司); 电泳仪(DYY-6C, 北京市六一仪器厂); 凝胶成像系统(Gel Doc XR+, 美国BIO-RAD公司); 立式压力蒸汽灭菌器(LDZX-30L, 上海申安医疗器械厂); 电子天平(JW-100, 余姚纪铭称重校验设备有限公司); 万分之一天平(7D20002A型, 上海衡际科学仪器有限公司); 高速冷冻离心机(5424R, 德国Eppendorf公司); 实验室超纯水器(UPC-I-10T型, 四川优普超纯科技有限公司), HPLC液相色谱仪(1260型, 美国安捷伦公司), 冷却水循环装置(CA-1115A型, 上海爱朗仪器有限公司)。
试药   Ezup柱式植物基因组DNA抽提试剂盒[生工生物工程(上海)股份有限公司批号: K620KA4775]; RnaseA溶液(批号: 20231118)、10×Easy Tap Buffer (批号: R21128)、dNTPs (批号: S10219)、Taq DNA Polymerase (批号: R21128)、Gelstain 1000×核酸染料(批号: 20230925)、Trans 2K DNA Marker (20240228)均购自北京全式金生物技术股份有限公司; 琼脂糖(批号162135, 上海贝晶生物技术有限公司); 6×DNA Loading Buffer (批号: R11216)、绿原酸(质量分数: 99.48%, 批号: MUST-24013002)、马里苷(质量分数: 99.61%, 批号: MUST-23082908)、黄诺玛苷(质量分数: 95.33%, 批号: MUST-24063015)均购自成都曼思特生物科技有限公司; 3,5-O-二咖啡酰奎宁酸(异绿原酸A) (批号: 111782-202309)购自中国食品药品检定研究院; 乙腈(色谱纯)购自赛默飞世尔科技(中国)有限公司; 磷酸(批号: 20230501516)、甲醇(批号20230801)均购自天津市致远化学试剂有限公司; β-巯基乙醇(批号: 2603080, 美国赛默飞世尔); 无水乙醇(批号: 2024102901, 成都市科隆化学品有限公司); 氯仿(批号: 202201114, 福晨天津化学试剂); 异丙醇(批号: 20110330, 天津永晨精细化工有限公司); 引物由上海生物工程科技有限公司合成。
药材   本课题组于2022年8月至2023年11月间, 在新疆伊犁、喀什、和田等地区中药材市场、药店、栽培大田等地共收集了35批次雪菊药材样品, 每批次样品质量不少于250 g, 药材收集完毕后, 置于阴凉通风处自然晾干。药材经新疆维吾尔自治区药物研究院何江研究员鉴定为菊科植物雪菊C. tinctoria, 植株标本收藏于新疆维吾尔自治区药物研究院标本室, 样品产地信息见表1。
基因组DNA提取   用75%的乙醇彻底清洗雪菊药材样品, 待乙醇自然蒸发干燥后, 取药材样品组织约150 mg, 在液氮中迅速研磨成粉末状, 采用Ezup柱式植物基因组DNA抽提试剂盒, 按照说明书提供的操作步骤进行, 基因组DNA于-20 ℃冰箱中, 储存备用。
PCR扩增与测序   采用通用引物扩增ITS2基因间隔区序列[16, 17]S2F: 5′-ATGCGATACTTGGTGTGAAT-3′; S3R: 5′-GACGCTTCTCCAGACTACAAT-3′, 引物由上海生工生物科技有限公司合成。在PCR仪上进行扩增, 反应总体系为25 μL, 体系含10×Taq Buffer 2.5 μL, dNTPs 2 μL, 正反引物(10 μmol·L-1)各0.5 μL、Taq酶0.25 μL, 模板DNA 2 μL, 补足ddH2O至25 μL。扩增程序为: 94 ℃预变性5 min; 94 ℃变性30 s, 56 ℃退火30 s, 72 ℃延伸45 s, 共40个循环; 72℃最终延伸10 min。反应结束后, 取5 μL PCR反应产物在1%的琼脂糖凝胶上电泳检测, 于凝胶成像系统中成像。将条带明显且单一的PCR产物, 送至上海生工生物科技有限公司双向测序。
序列分析与处理   采用CodonCode Aligner软件对测序峰图进行序列拼接, 去除引物区以及低质量序列, 同时基于隐马尔可夫模型HMMer注释去除5.8S rDNA和28S rDNA, 从而获得完整的ITS2基因内转录间隔区序[18]。用MEGA 6.0软件对拼接后的序列进行多重序列比对分析, 并基于(Kimura2-Parameter, K2P)双参数模型采用邻接法(neighbor joining, NJ), 构建系统进化树(1 000次重复bootstrap检验各分支的支持率)。
供试品溶液的制备   取雪菊药材适量研磨成粉, 精密称取约200 mg置于100 mL锥形瓶中, 加入70%乙醇40 mL, 加热回流提取30 min, 滤过, 滤液置50 mL量瓶中, 加70%乙醇定容至刻度, 摇匀, 0.45 μm滤膜过滤, 即得。
对照品溶液的制备   分别精密称取绿原酸、黄诺玛苷、马里苷和异绿原酸A对照品适量, 精密加入甲醇溶液配制成质量浓度分别为18.8、20.6、15.7、5.1 µg·mL-1的单个对照品储备液, 摇匀, 储存于4 ℃条件下备用。
色谱条件   ZORBAX SB-C18色谱柱(250 mm × 4.6 mm, 5 μm); 流动相为0.05%磷酸水(A)-乙腈(B), 梯度洗脱(0~5 min, 90%~88% A; 5~10 min, 88%~84% A; 10~18 min, 84%~83.2% A; 18~28 min, 83.2% A; 28~33 min, 83.2%~76% A; 33~38 min, 76% A; 38~48 min, 76%~74% A; 48~50 min, 74%~72% A; 50~55 min, 72%~60% A; 55~57 min, 60%~90% A; 57~60 min, 90% A); 体积流量1.0 mL·min-1; 柱温30 ℃; 进样量10 µL; 检测波长280 nm。
指纹图谱方法学考察
精密度  精密称取雪菊粉末1份(S8), 按“供试品溶液的制备”项下制备供试品溶液。在“色谱条件”项下连续进样6次, 计算共有峰相对保留时间RSD和相对峰面积RSD。
重复性  精密称取雪菊粉末6份(S8), 按“供试品溶液的制备”项下制备供试品溶液, 在“色谱条件”项下, 计算共有峰相对保留时间RSD和相对峰面积RSD。
稳定性  精密称取雪菊粉末1份(S8), 按“供试品溶液的制备”项下制备供试品溶液, 在“色谱条件”项下于0、2、4、6、8、10、12、18 h进样分析, 计算共有峰相对保留时间RSD和相对峰面积RSD。
共有峰的指认及相似度分析
共有峰的指认  35批次雪菊样品按“供试品溶液的制备”项下制备供试品溶液, 在“色谱条件”项下测定, 记录色谱图。将AIA数据导入《中药色谱指纹图谱相似度评价系统(2012版)》软件中, 以S8样品色谱图为参照指纹图谱, 时间窗宽度设置为0.5 min, 采用多点校正后Mark峰自动匹配建立指纹图谱。
相似度分析  设定对照指纹图谱(R)为参照图谱, 计算35批次雪菊样品与对照指纹图谱的相似度。
化学模式识别
聚类分析(cluster analysis, CA)  以35批次雪菊样品中的16个共有峰峰面积为变量, 导入Origin 2021软件, 采用组间连接法, 平方欧式距离为样品的测度, 进行CA。
主成分分析(principal component analysis, PCA)  主成分因子特征值和方差贡献率分析以35批次雪菊样品中的16个共有峰峰面积为变量, 导入SPSS 25.0软件, 进行Z-Score标准化处理, 进行PCA, 以主成分特征值大于1为标准, 进行因子分析, 计算相关系数矩阵、特征值和方差贡献率。再以35批次雪菊样品中的16个共有峰峰面积为变量, 导入SIMCA-14.1软件, 进行PCA。
偏最小二乘法判别分析(partial least squares-discriminant analysis, PLS-DA)  为进一步筛选鉴别和区分不同产地雪菊药材标志性成分, 以35批次雪菊样品中的16个共有峰峰面积为变量, 导入SIMCA-14.1软件, 进行PLS-DA建模分析。变量重要性投影(variable importance in projection, VIP)值是筛选差异性化合物的重要指标, VIP值越大, 差异越显著。通过提取PLS-DA模型中16个VIP值, 以VIP > 1为筛选条件, 得到差异性标志物的VIP得分图。
HPLC定量分析方法学考察
线性关系考察  精密量取4个对照品溶液各1、2、3、4、5、6 mL, 分别置于不同25 mL量瓶中, 加甲醇稀释至刻度, 摇匀, 制备系列混合对照品溶液。精密吸取该系列混合对照品溶液适量, 在“色谱条件”项下进样。以各成分质量浓度(mg·mL-1)为横坐标(X)、峰面积为纵坐标(Y), 进行线性回归, 得到4个成分的回归方程与线性范围。
精密度  精密量取2 mL混合对照品溶液, 在“色谱条件”项下分别测定6次, 计算绿原酸、黄诺玛苷、马里苷、异绿原酸A的峰面积RSD。
稳定性  精密称取雪菊粉末1份(S8), 按“供试品溶液的制备”项下方法制备供试品溶液, 在“色谱条件”项下于0、2、4、6、8、10、12、18 h进样测定, 计算绿原酸、黄诺玛苷、马里苷、异绿原酸A的峰面积RSD。
重复性  精密称取雪菊粉末6份(S8), 按“供试品溶液的制备”项下方法制备供试品溶液, 在“色谱条件”项下分别进样测定, 计算绿原酸、黄诺玛苷、马里苷、异绿原酸A的平均含量和RSD。
加样回收率  精密称取雪菊粉末0.1g (S8), 置具塞锥形瓶中, 平行6份, 分别加入与供试品溶液中相应指标成分含量约1∶1的对照品溶液, 按“供试品溶液的制备”项下制备供试品溶液, 在“色谱条件”项下分别进样测定, 计算各成分平均加样回收率和RSD。
结果
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1　ITS2序列分析
本研究成功扩增了35份雪菊药材样本的核苷酸序列, PCR扩增效率和测序成功率均为100%。成功测序后, 所得药材样品ITS2序列大小为494~499 bp, 经过去除5.8S rDNA和28S rDNA区核苷酸序列后, 完整的ITS2序列大小为225 bp。共计有3个单倍型(H1、H2和H3), 主体单倍型为H1和H2, 长度均为225 bp; GC含量分别为54.67%、55.11%、55.56%; 存在2个变异位点, 分别发生在106位点的T/C转换, 195位点的C/T转换。进一步说明, 雪菊ITS2序列较为保守, 种内发生变异较小。
2　BLAST法
BLAST法对雪菊样品进行鉴定, 依据国际DNA条形码协会(CBOL)的通用标准。采用BLAST工具对NCBI数据库进行BLAST搜索, 如果查询序列的最佳匹配均为目标物种且排名靠前则认为该查询序列最接近的物种是目标物种。通过对雪菊35批次药材ITS2基因序列进行在线比对, 序列的相似度均在99.55%以上, 结果再次佐证了35批次药材样品的基原均为雪菊C. tinctoria。
3　构建NJ进化树法
金鸡菊属植物在全世界约有100种, 生长于亚洲、美洲、非洲南部及夏威夷等地, 其中我国常见的该属近缘种植物有大花金鸡菊(Coreopsis grandiflora Hogg.)、剑叶金鸡菊(Coreopsis lanceolata L.)、大叶金鸡菊(Coreopsis major Watt.)、三叶金鸡菊(Coreopsis tripteris L.)和轮叶金鸡菊(Coreopsis verticillata L.)等。选择雪菊单倍型Coreopsis tinctoria-1 (H1)、Coreopsis tinctoria-2 (H2)、Coreopsis tinctoria-3 (H3)作为参考序列, 采用邻接法对不同来源的ITS2序列进行聚类分析并构建系统发育树, 结果见图1。由图1可知, 雪菊ITS2序列聚在一起, 而与其他相关近缘物种很清晰地分开, 说明雪菊ITS2序列可以作为鉴别雪菊与其他近缘种植物的条形码序列。
4　指纹图谱方法学考察
4.1　精密度
各共有峰相对保留时间和相对峰面积RSD值均小于5%, 表明本研究所用的仪器的精密度良好。
4.2　重复性
各共有峰相对保留时间和相对峰面积RSD值均小于5%, 表明该方法重复性良好。
4.3　稳定性
各共有峰相对保留时间和相对峰面积RSD值均小于5%, 表明供试品溶液在室温放置18 h内稳定性良好。
4.4　指纹图谱的建立及分析
35批次药材的HPLC叠加指纹图谱共有模式及其对照指纹图谱, 见图2A。通过与对照品比对, 指纹图谱共指认出4个色谱峰, 5号为绿原酸、8号为黄诺玛苷、11号为马里苷、12号为异绿原酸A, 混合对照品溶液HPLC色谱图, 见图2B。35批次药材中16个共有指纹峰的峰面积RSD为22.91%~67.50%, 表明每批次药材质量存在一定差距。
4.5　相似度分析
HPLC指纹图谱相似度分析结果可看出, S1~S35批次样品和对照指纹图谱的相似度范围为0.936~0.998, 整体相似度较高; S21与对照指纹图谱的相似度最低, 为0.936, S20与对照指纹图谱的相似度最高, 为0.998。
5　化学模式识别
5.1　CA
不同产地的35批次雪菊药材HPLC指纹图谱的CA结果见图3A, 树状图分支较少, 表明采集的35批次药材之间的质量差异较小。
从树状谱系图结果可以看到, 不同产地的35批次药材可聚为3类, S1、S5、S9、S10、S16、S17、S19~S22、S24、S26~S29和S32~S35聚为第1类; S2~S4、S6~S8、S13、S15、S23、S30、和S31聚为第Ⅱ类; S11、S12、S14、S18、S25聚为第Ⅲ类; 聚类1中包含19批次药材, 表明多批次药材质量上较为相似, 其产地主要是乌鲁木齐、喀什、伊犁等; 聚类Ⅱ中包含11批次药材, 其产地主要是阿勒泰、吐鲁番等; 聚类Ⅲ包含5批次药材, 其产地主要是皮山。CA结果表明, 不同产地药材在质量上有一定的差异, 但差异较小。聚类分析结果较好吻合了不同产地的药材指纹图谱与系统相似度的直观差异, 正确体现出指纹图谱之间的相似程度。
5.2　PCA
结果显示, KMO检验系数为0.620, Bartlett球形度检验显著性P < 0.000 1, 适合进行因子分析, 得到5个主成分, 碎石图和主成分特征值和贡献率的结果见图3B和表2。由图3B可知, 前5个成分变化曲线倾斜程度较大, 从第6个成分开始曲线逐渐趋于平缓。由表2可知, 特征值分别为5.915、3.163、1.968、1.505、1.042, 方差贡献率分别为36.970、19.767、12.297、9.404、6.515, 累积方差贡献率达到84.953%, 表明这5个主成分可以比较全面概括不同批次雪菊药材的大部分信息。基于PCA建立无监督的模式识别模型, 模型拟合参数R2X为1, 预测参数Q2为1, 以上参数均大于0.5, 表明该模型具有较高的解释和预测性[19-21], PCA得分图见图3C。从图中看出, 35批次药材聚为3类, 不同聚类内部聚集得较好, 35批次药材整体呈现按产地不同分布趋势, 特别是聚类Ⅰ中多个批次如S1、S10、S26、S27; S5、S24、S29、S32分布较聚集; PCA中有34批次样品点均落在95%的置信区间内, S21批次样品的点落在95%的置信区间外, 且与聚类Ⅰ中的其他批次样品分布较为离散, 说明该批次药材与同一聚类的其他批次样品在质量上相差较大。上述结果表明主成分与雪菊指纹图谱的差异性有一定关联性, 通过观察样品的组内聚集和组间分离趋势, 表明主成分分析与聚类分析结果基本一致。
利用SPSS 25.0软件对主成分分析结果进行因子提取, 采用凯撒正态化最大方差法正交旋转, 迭代25次后得到主成分载荷矩阵, 其中第1主成分主要反映了色谱峰4、8、9、11的信息, 第2主成分主要反映了色谱峰2、15、16的信息, 第3主成分主要反映了色谱峰5、6、7、13、14的信息, 第4主成分主要反映了色谱峰1、3、10、12的信息。
5.3　PLS-DA
PLS-DA建模分析结果见图4A, PLS-DA显示在该模型下R2X (cum) = 0.603, R2Y (cum) = 0.606, Q2 (cum) = 0.359, 表明拟合的预测模型具有较好的解释性能, 但其预测性能欠佳[22], 为验证该模型没有过拟合现象, 随机排列200次做置换检验, 得置换检验结果, 见图4B, 结果表明R2和Q2的回归线斜率均大于0, 且R2和Q2的回归线截距为0.198和-0.288, 表明模型不存在过拟合现象[23]。PLS-DA得分图结果表明35批药材样品被分为3类, PLS-DA结果与CA、PCA结果一致, 进一步验证了CA和PCA的结果。
为进一步探究35批次雪菊药材质量呈现差异性的本质原因, PLS-DA模型中16个变量重要性投影值的VIP得分图, 见图4C, 得分见表3。得到VIP > 1[24, 25]的共有峰, 分别为16、15、4、5 (绿原酸)、10、8 (黄诺玛苷)、11 (马里苷)和12 (异绿原酸A)号峰, 说明这8个成分对不同产地雪菊药材质量有较大影响, 可作为不同产地雪菊药材质量差异的标志性成分。
6　HPLC定量分析方法学考察
6.1　线性关系考察
绿原酸、黄诺玛苷、马里苷、异绿原酸A各成分在线性范围内线性关系良好, 4个成分的回归方程与线性范围结果见表4。
6.2　精密度
绿原酸、黄诺玛苷、马里苷、异绿原酸A峰面积RSD分别为0.41%、0.32%、1.87%、0.25%, 表明仪器精密度良好。
6.3　稳定性
绿原酸、黄诺玛苷、马里苷、异绿原酸A峰面积RSD分别为1.89%、0.82%、0.21%、0.34%, 表明样品溶液18 h内具有良好稳定性。
6.4　重复性
绿原酸、黄诺玛苷、马里苷、异绿原酸A平均含量分别为4.83、8.36、42.84、3.60 mg·g-1, RSD分别为0.98%、0.83%、0.47%、2.05%, 表明该实验重复性良好。
6.5　加样回收率
绿原酸、黄诺玛苷、马里苷、异绿原酸A的平均加样回收率分别为98.27、97.95%、103.45%、104.00%, RSD分别为2.39%、1.76%、4.81%、2.46%。
6.6　样品含量测定
35批雪菊样品中各成分的质量分数, 结果见表5绿原酸、黄诺玛苷、马里苷、异绿原酸A的质量分数分别为0.860~7.504、3.368~19.182、19.719~83.492、2.039~12.568 mg·g-1, 提示不同批次产品中各成分的含量差异明显。
讨论
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在中药材DNA条形码鉴定中, 样品采集与贮藏过程中的环境因素易导致DNA降解, 使得片段长度不足, 制约了鉴定准确性。为确保可靠性, 需从两方面进行优化: 一是规范样品采集与贮藏流程, 优先采用新鲜材料以获取完整DNA; 二是筛选更短、扩增效率更高的条形码标记以适配降解样本。值得注意的是, DNA条形码技术无法用于矿物药鉴定, 因此必须结合形态学等传统方法, 多种技术联用, 才能实现全面、准确的中药材鉴定。
本研究基于色谱峰数量、峰形及分离度等评价指标, 先比较了超声与回流两种提取方法, 再对提取溶剂(甲醇、乙醇)和溶剂倍量(1∶100、1∶150、1∶200)进行优化。最终确定以70%乙醇为溶剂、1∶200的溶剂倍量加热回流30 min, 作为供试品溶液的前处理方法。
此外, 对流动相系统(甲醇-0.05%磷酸、乙腈-冰醋酸、乙腈-0.05%磷酸、乙腈-0.1%磷酸)与检测波长(285、378、280 nm)进行了筛选。最终条件确定为: 以乙腈-0.05%磷酸水溶液为流动相, 梯度洗脱, 检测波长280 nm, 流速1.0 mL·min-1。该条件下, 绿原酸、黄诺玛苷、马里苷与异绿原酸A均吸收良好, 峰形与分离度良好。
本研究建立了35批次雪菊药材的指纹图谱, 标定16个共有峰。相似度评价显示, 所有批次与对照图谱的相似度均高于0.93, 表明样品间一致性较好, 质量较稳定。同时, 指认了4个成分: 绿原酸(5号峰)、黄诺玛苷(8号峰)、马里苷(11号峰)和异绿原酸A (12号峰)。化学模式识别分析结果一致, 将样品分为3类, 呈现较明显的产地聚集趋势。除S21批次位于PCA 95%置信区间外且分布离散外, 其余34批均落于区间内, 说明S21批次质量与同类其余样品差异较大。基于PLS-DA模型的VIP分析进一步识别出8个标志性成分(峰16、15、4、5、10、8、11、12), 其对不同产地雪菊质量差异贡献较大, 可作为产地区分的潜在标志物。
此外, 本研究建立的PLS-DA模型Q2值为0.359 (< 0.5), 表明模型预测能力较弱, 泛化性能有待提高。为增强模型的科学性与可靠性, 后续拟从以下方面进行优化: 优化数据预处理方法, 加强标准化与归一化, 剔除异常值与冗余变量以降低噪声; 扩大样本量, 尤其补充分类边界样本, 提升模型稳定性与预测力; 调整模型参数, 如主成分数, 或采用正态化处理; 并尝试引入稀疏偏最小二乘判别分析(sparse partial least squares-discriminant analysis, sPLS-DA)、随机森林等其他多元分析方法, 以构建预测性能更优的模型。
雪菊含黄酮类、酚类、多糖及皂苷等多种活性成分, 具有降血压、调血脂、控血糖、抗肿瘤及抗氧化等药理作用[8, 26-33]。其中黄酮类成分是其主要活性组分。刘萍等[34]研究发现, 昆仑雪菊中的原花青素B2与绿原酸可能通过提高抗氧化酶活性保护胰岛β细胞, 从而改善2型糖尿病小鼠血糖水平。高飞[35]基于油酸诱导的HepG2细胞脂肪变性模型, 进一步证实雪菊总黄酮及其中成分马里苷可显著降低细胞内甘油三酯、游离脂肪酸等脂质含量, 提示马里苷可能是雪菊降血脂作用的关键成分之一。
本研究采用HPLC法测定了35批次雪菊中4种指标成分的含量。结果显示, 马里苷的整体含量显著高于绿原酸、黄诺玛苷和异绿原酸A, 其中S4批次含量最高(83.492 mg·g-1), S20批次最低(19.719 mg·g-1)。绿原酸含量普遍较低, 最高为S31批次(7.504 mg·g-1), 最低为S22批次(0.860 mg·g-1)。黄诺玛苷和异绿原酸A的最高含量分别出现在S4 (19.182 mg·g-1)和S8批次(12.568 mg·g-1), 最低含量分别见于S26 (3.368 mg·g-1)与S27批次(2.039 mg·g-1)。上述成分含量差异可能与产地、气候、采收时间、加工及储存等因素密切相关。中药材历来强调“性随地异”, 产地是影响药材品质的关键因素。唐代《新修本草》中“离其本土, 则质同而效异”的记载, 亦明确指出原生环境对药材质量与疗效具有重要影响, 进一步印证了本研究结果的合理性。
综上所述, 本研究基于ITS2基因序列鉴定方法、HPLC指纹图谱、多指标成分含量测定结合化学模式识别的方法, 对不同产地的35批次雪菊药材进行质量控制评价。结果表明, 35批次雪菊药材样本的PCR扩增效率和测序成功率均为100%, 表明雪菊ITS2序列较为保守, 种内发生变异较小。雪菊ITS2序列鉴定结果表明雪菊ITS2序列可以作为鉴别雪菊与其他近缘种植物的条形码序列; HPLC指纹图谱匹配了16个共有峰, CA、PCA及PLS-DA均将35批次雪菊药材区分为3类, PCA分析中5个主成分累积方差贡献率为84.758%, PLS-DA筛选出16、15、4、5 (绿原酸)、10、8 (黄诺玛苷)、11 (马里苷)和12 (异绿原酸A)号峰, 说明这8个成分对不同产地雪菊药材质量有较大影响, 可作为不同生长期雪菊药材质量差异的标志性成分; 采用HPLC法同时测定药材中绿原酸、黄诺玛苷、马里苷和异绿原酸A等4个指标成分的含量。本研究建立的DNA条形码鉴定技术及HPLC指纹图谱-多指标含量测定方法简单可行、结果可靠, 为雪菊药材质量评价提供参考, 同时也为进一步深入研究和推动雪菊产业高质量发展提供了科学依据

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2026年第61卷第4期
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doi: 10.16438/j.0513-4870.2025-0659
  • 接收时间：2025-06-12
  • 首发时间：2026-09-21
  • 出版时间：2026-04-12
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  • 收稿日期：2025-06-12
  • 修回日期：2026-01-12
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    新疆维吾尔自治区药物研究院, 维吾尔药重点实验室, 新疆 乌鲁木齐 830010

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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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