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Article(id=1190335355799830776, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1190335347767743264, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2024-1011, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1729094400000, receivedDateStr=2024-10-17, revisedDate=1739376000000, revisedDateStr=2025-02-13, acceptedDate=null, acceptedDateStr=null, onlineDate=1761727664184, onlineDateStr=2025-10-29, pubDate=1744387200000, pubDateStr=2025-04-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1761727664184, onlineIssueDateStr=2025-10-29, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1761727664184, creator=13701087609, updateTime=1761727664184, updator=13701087609, issue=Issue{id=1190335347767743264, tenantId=1146029695717560320, journalId=1189982191388893191, year='2025', volume='60', issue='4', pageStart='843', pageEnd='1182', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1761727662269, creator=13701087609, updateTime=1761729313427, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1190342273276678997, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1190335347767743264, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1190342273276678998, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1190335347767743264, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1115, endPage=1123, ext={EN=ArticleExt(id=1190335356085043452, articleId=1190335355799830776, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Study on quality control of Guanxin Qiwei tablets based on fingerprint and chemometrics, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

The study aims to establish HPLC fingerprint and multi-index content determination method of Guanxin Qiwei tablets and provide scientific basis for its quality control. The fingerprints of 18 batches of Guanxin Qiwei tablets were established by Shim-pack GGIST HP C18 chromatographic column, and analyzed comprehensively in combination with the Similarity Evaluation System of TCM Chromatographic Fingerprint (2012 edition). Cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for stoichiometric study. HPLC-MS/MS method was established for simultaneous determination of 9 components. A total of 22 common peaks were identified in 18 batches of Guanxin Qiwei tablets fingerprint, and the similarity was 0.952-0.998, 9 common peaks were identified. They were No. 1 gallic acid, No. 3 protocatechuic acid, No. 8 ellagic acid, No. 9 salvianolic acid B, No. 12 luteolin, No. 13 apigenin, No. 19 dehydrodiisoeugenol, No. 20 cryptotanshinone and No. 21 tanshinone ⅡA. CA and PCA analysis grouped 18 batches of Guanxin Qiwei tablets into 3 categories: S1-S4 (manufacturer A) was grouped into one category, S5-S8 (manufacturer B) was grouped into one category, and S9-S18 (manufacturer C and D) was grouped into one category. Under OPLS-DA analysis mode, 14 quality differentiators were selected with the variable important projection (VIP) greater than 1 as the standard, and all of them had significant differences. The linear relationship of the 9 components was good in their respective ranges, and the linear correlation coefficient r was greater than or equal to 0.099 9. Precision RSDS were all lower than 3.00%. The stability and repeatability were good, RSD lower than 5.00%; The average recoveries were 96.58%-106.28%, RSD was 2.68%-6.45%. This method is efficient and stable, and can be used for quality control of Guanxin Qiwei tablets.

, correspAuthors=Jun LI, Yue-wu WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2025 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, authorCompany=null, fund=null, authors=null, authorsList=Cheng-dong LIU, Li YANG, Jia-hui CHEN, Peng-peng ZHANG, Jing LIU, Qian ZHANG, Jun LI, Yue-wu WANG), CN=ArticleExt(id=1190335863813931372, articleId=1190335355799830776, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=基于指纹图谱和化学计量学的冠心七味片质量控制研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

本研究建立了冠心七味片的HPLC指纹图谱与化学计量学结合的多指标含量测定方法, 为其质量控制提供科学依据。采用Shim-pack GIST-HP C18色谱柱建立18批冠心七味片的指纹图谱, 结合《中药色谱指纹图谱相似度评价系统(2012版)》进行全面分析; 采用聚类分析(CA)、主成分分析(PCA) 和正交偏最小二乘判别分析(OPLS-DA) 进行化学计量学研究, 并建立HPLC-MS/MS法同时测定9个成分含量。18批冠心七味片指纹图谱共标定了22个共有峰, 相似度为0.952~0.998, 9个共有峰被指认, 分别为1号峰没食子酸、3号峰原儿茶酸、8号峰鞣花酸、9号峰丹酚酸B、12号峰木犀草素、13号峰芹菜素、19号峰去氢二异丁香酚、20号峰隐丹参酮、21号峰丹参酮ⅡA; CA和PCA分析将18批冠心七味片聚为3大类: S1~S4 (A厂家) 聚为一类, S5~S8 (B厂家) 聚为一类, S9~S18 (C、D厂家) 聚为一类, 在OPLS-DA分析模式下, 以变量权重值(VIP) > 1为标准, 筛选出了14个质量差异物, 其均有不同的显著性差异; 9个成分在各自范围内线性关系良好, 线性相关系数r均≥ 0.999 0; 精密度RSD均 < 3.00%; 稳定性和重复性良好, RSD均 < 5.00%; 平均加样回收率为96.58%~106.28%, RSD为2.68%~6.45%。此方法高效稳定, 可用于冠心七味片质量控制。

, correspAuthors=李君, 王跃武, authorNote=null, correspAuthorsNote=
李君, E-mail:
王跃武, E-mail:
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China J Tradit Chin Med Pharm (中华中医药杂志), 2024, 39: 2444-2450., articleTitle=null, refAbstract=null), Reference(id=1190349893802758187, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=27, rfOrder=26, authorNames=null, journalName=null, refType=null, unstructuredReference=Bu R, Zhang PP, Wang YW, et al. Simultaneous determination of fifteen constituents in Zhuriheng Dropping Pills by UPLC-MS/MS [J]. Chin Tradit Pat Med (中成药), 2023, 45: 3890-3894., articleTitle=null, refAbstract=null), Reference(id=1190349893882449964, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=28, rfOrder=27, authorNames=null, journalName=null, refType=null, unstructuredReference=Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (中华人民共和国药典) [S]. 2020 Ed. 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Fingerprints of 18 batches of Guanxin Qiwei tablets (A) and comparison of reference mother liquor with sample S1 chromatogram (B). 1: Gallic acid; 3: Protocatechuic acid; 8: Ellagic acid; 9: Salvianolic acid B; 12: Luteolin; 13: Apigenin; 19: Dehydrodiisoeugenol; 20: Cryptotanshinone; 21: TanshinoneⅡA; 2, 4, 5, 6, 7, 10, 11, 14, 15, 16, 17, 18, 19, 22: Other common peaks

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Cluster heat map analysis of 18 batches of Guanxin Qiwei tablets. Group A: Manufacturer A; Group B: Manufacturer B; Group C: Manufacturer C; Group D: Manufacturer D

, figureFileSmall=qah4Rcezb4oilQ9exzRNGQ==, figureFileBig=w1zi2g11oMb+DEFUb/1R4w==, tableContent=null), ArticleFig(id=1190349890564755456, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, language=EN, label=null, caption=null, figureFileSmall=zSJxjOwiIKBmWM9sAVTfvg==, figureFileBig=pB1U0Xy5Im8GEYXIeMRnNQ==, tableContent=null), ArticleFig(id=1190349890615087105, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, language=CN, label=Figure 3, caption=

PCA score of 18 batches of Guanxin Qiwei tablets. A: Manufacturer A; B: Manufacturer B; C: Manufacturer C; D: Manufacturer D

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OPLS-DA score plot for 18 batches of Guanxin Qiwei tablets (A), OPLS-DA permutation test diagram (B) and VIP plot for 22 total peaks from18 batches of Guanxin Qiwei tablet (C)

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Analysis of variance of quality differentials. A: Salvianolic acid B; B: Tanshinone ⅡA; C: Peak 16; D: Gallic acid; E: Peak 15; F: Peak 4; G: Cryptotanshinone; H: Peak 22; I: Peak 2; J: Peak 5; K: Peak 7; L: Peak 10; M: Peak 18; N: Ellagic acid. n = 18, $ \stackrel{-}{x} $ ± s. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1

, figureFileSmall=q/QsCXSmrE+SDvQBJH/yWw==, figureFileBig=+yb3nDrEEtC7DhU1dpDdNA==, tableContent=null), ArticleFig(id=1190349890963214342, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, language=EN, label=null, caption=null, figureFileSmall=wfGdyhII2PpeVkOkqs+YXw==, figureFileBig=2k+gU8ea+8NC/ELo9KaMGg==, tableContent=null), ArticleFig(id=1190349891021934599, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, language=CN, label=Figure 6, caption=

Extraction ion flow chromatogram of each component in MRM mode. A: Reference solution; B: Sample solution; C: Blank solution; 1: Gallic acid; 2: Protocatechuic acid; 3: Ellagic acid; 4: Salvianolic acid B; 5: Luteolin; 6: Apigenin; 7: Dehydrodiisoeugenol; 8: Cryptotanshinone; 9: Tanshinone ⅡA

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Compound Equation r Range/ng·mL-1 LOQ/ng·mL-1 LOD/ng·mL-1
Gallic acid Y = 4.961×104X + 4.036×107 0.999 4 8.025-80.25 0.095 0.031
Protocatechuic acid Y = 9.467×103X + 1.332×106 0.999 2 97.6-976 0.275 0.091
Ellagic acid Y = 6.520×103X + 1.640×103 0.999 0 13.992-139.92 0.275 0.091
Salvianolic acid B Y = 2.407×103X + 7.346×106 0.999 1 688.5-6 885 0.870 0.287
Luteolin Y = 3.989×103X + 4.212×104 0.999 3 187.32-1 873.2 0.193 0.064
Apigenin Y = 6.575×102X + 2.101×103 0.999 6 25.912-259.12 0.452 0.149
Dehydrodiisoeugenol Y = 5.615×103X + 1.801×106 0.999 4 39.0-390 0.029 0.010
Cryptotanshinone Y = 3.737×104X + 1.336×107 0.999 7 187.32-1 873.2 0.082 0.027
Tanshinone ⅡA Y = 3.011×104X + 1.426×107 0.999 4 146.4-1 464 0.394 0.130
), ArticleFig(id=1190349891156152329, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, language=CN, label=Table 1, caption=

Results of linear relationship study on 9 components of Guanxin Qiwei tablets

, figureFileSmall=null, figureFileBig=null, tableContent=
Compound Equation r Range/ng·mL-1 LOQ/ng·mL-1 LOD/ng·mL-1
Gallic acid Y = 4.961×104X + 4.036×107 0.999 4 8.025-80.25 0.095 0.031
Protocatechuic acid Y = 9.467×103X + 1.332×106 0.999 2 97.6-976 0.275 0.091
Ellagic acid Y = 6.520×103X + 1.640×103 0.999 0 13.992-139.92 0.275 0.091
Salvianolic acid B Y = 2.407×103X + 7.346×106 0.999 1 688.5-6 885 0.870 0.287
Luteolin Y = 3.989×103X + 4.212×104 0.999 3 187.32-1 873.2 0.193 0.064
Apigenin Y = 6.575×102X + 2.101×103 0.999 6 25.912-259.12 0.452 0.149
Dehydrodiisoeugenol Y = 5.615×103X + 1.801×106 0.999 4 39.0-390 0.029 0.010
Cryptotanshinone Y = 3.737×104X + 1.336×107 0.999 7 187.32-1 873.2 0.082 0.027
Tanshinone ⅡA Y = 3.011×104X + 1.426×107 0.999 4 146.4-1 464 0.394 0.130
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Sample No. Gallic acid Protocatechuic acid Ellagic acid Salvianolic acid B Luteolin Apigenin Dehydrodiisoeugenol Cryptotanshinone Tanshinone ⅡA
S1 2.941 28.534 4.007 412.402 6.175 3.217 15.701 166.060 139.349
S2 2.967 34.382 3.955 417.903 5.743 3.064 11.265 169.940 142.438
S3 2.939 34.139 4.014 404.292 6.234 3.276 12.927 135.251 109.998
S4 3.230 15.405 3.970 412.994 6.458 3.135 11.367 162.645 117.271
S5 1.402 26.225 1.572 375.195 2.555 2.812 33.035 130.917 97.961
S6 1.459 17.371 1.494 374.257 2.863 2.942 36.943 140.704 99.027
S7 2.348 15.980 1.598 434.682 2.255 3.194 36.885 140.442 99.065
S8 1.362 30.352 1.565 359.978 2.418 2.799 26.680 145.028 93.083
S9 3.050 30.606 2.331 634.100 12.962 15.721 16.951 177.191 142.933
S10 2.799 13.116 2.332 579.439 13.231 13.932 13.789 159.237 131.985
S11 2.687 18.185 1.943 447.025 4.985 16.332 9.694 124.166 120.006
S12 2.655 29.319 2.569 559.121 9.448 14.513 12.962 171.727 140.115
S13 2.401 27.135 3.084 615.211 7.737 2.927 8.834 163.741 130.090
S14 2.361 26.027 4.038 549.642 7.162 2.813 9.775 165.463 128.718
S15 2.721 26.732 3.064 597.875 5.147 2.728 8.577 163.615 129.986
S16 1.800 14.875 2.949 612.730 6.981 9.492 5.909 111.250 112.538
S17 1.708 29.802 2.343 574.082 7.081 8.451 7.070 116.509 110.449
S18 2.003 32.172 3.043 603.768 6.991 8.426 6.315 113.972 101.227
), ArticleFig(id=1190349891319730187, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190335355799830776, language=CN, label=Table 2, caption=

Determination of 9 components in 18 batches of Guanxin Qiwei tablets (μg·g-1)

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample No. Gallic acid Protocatechuic acid Ellagic acid Salvianolic acid B Luteolin Apigenin Dehydrodiisoeugenol Cryptotanshinone Tanshinone ⅡA
S1 2.941 28.534 4.007 412.402 6.175 3.217 15.701 166.060 139.349
S2 2.967 34.382 3.955 417.903 5.743 3.064 11.265 169.940 142.438
S3 2.939 34.139 4.014 404.292 6.234 3.276 12.927 135.251 109.998
S4 3.230 15.405 3.970 412.994 6.458 3.135 11.367 162.645 117.271
S5 1.402 26.225 1.572 375.195 2.555 2.812 33.035 130.917 97.961
S6 1.459 17.371 1.494 374.257 2.863 2.942 36.943 140.704 99.027
S7 2.348 15.980 1.598 434.682 2.255 3.194 36.885 140.442 99.065
S8 1.362 30.352 1.565 359.978 2.418 2.799 26.680 145.028 93.083
S9 3.050 30.606 2.331 634.100 12.962 15.721 16.951 177.191 142.933
S10 2.799 13.116 2.332 579.439 13.231 13.932 13.789 159.237 131.985
S11 2.687 18.185 1.943 447.025 4.985 16.332 9.694 124.166 120.006
S12 2.655 29.319 2.569 559.121 9.448 14.513 12.962 171.727 140.115
S13 2.401 27.135 3.084 615.211 7.737 2.927 8.834 163.741 130.090
S14 2.361 26.027 4.038 549.642 7.162 2.813 9.775 165.463 128.718
S15 2.721 26.732 3.064 597.875 5.147 2.728 8.577 163.615 129.986
S16 1.800 14.875 2.949 612.730 6.981 9.492 5.909 111.250 112.538
S17 1.708 29.802 2.343 574.082 7.081 8.451 7.070 116.509 110.449
S18 2.003 32.172 3.043 603.768 6.991 8.426 6.315 113.972 101.227
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基于指纹图谱和化学计量学的冠心七味片质量控制研究
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刘成东 1, 2 , 杨利 2 , 陈佳惠 1 , 张彭鹏 2 , 刘静 1 , 张谦 1 , 李君 1, * , 王跃武 1, *
药学学报 | 研究论文 2025,60(4): 1115-1123
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药学学报 | 研究论文 2025, 60(4): 1115-1123
基于指纹图谱和化学计量学的冠心七味片质量控制研究
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刘成东1, 2, 杨利2, 陈佳惠1, 张彭鹏2, 刘静1, 张谦1, 李君1, * , 王跃武1, *
作者信息
  • 1 内蒙古医科大学药学院, 内蒙古 呼和浩特 010110
  • 2 包头中药有限公司, 内蒙古 包头 014040

通讯作者:

李君, E-mail:
王跃武, E-mail:
Study on quality control of Guanxin Qiwei tablets based on fingerprint and chemometrics
Cheng-dong LIU1, 2, Li YANG2, Jia-hui CHEN1, Peng-peng ZHANG2, Jing LIU1, Qian ZHANG1, Jun LI1, * , Yue-wu WANG1, *
Affiliations
  • 1School of Pharmacy, Inner Mongolia Medical University, Hohhot 010110, China
  • 2Baotou Traditional Chinese Medicine Co., Ltd., Baotou 014040, China
出版时间: 2025-04-12 doi: 10.16438/j.0513-4870.2024-1011
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摘要
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本研究建立了冠心七味片的HPLC指纹图谱与化学计量学结合的多指标含量测定方法, 为其质量控制提供科学依据。采用Shim-pack GIST-HP C18色谱柱建立18批冠心七味片的指纹图谱, 结合《中药色谱指纹图谱相似度评价系统(2012版)》进行全面分析; 采用聚类分析(CA)、主成分分析(PCA) 和正交偏最小二乘判别分析(OPLS-DA) 进行化学计量学研究, 并建立HPLC-MS/MS法同时测定9个成分含量。18批冠心七味片指纹图谱共标定了22个共有峰, 相似度为0.952~0.998, 9个共有峰被指认, 分别为1号峰没食子酸、3号峰原儿茶酸、8号峰鞣花酸、9号峰丹酚酸B、12号峰木犀草素、13号峰芹菜素、19号峰去氢二异丁香酚、20号峰隐丹参酮、21号峰丹参酮ⅡA; CA和PCA分析将18批冠心七味片聚为3大类: S1~S4 (A厂家) 聚为一类, S5~S8 (B厂家) 聚为一类, S9~S18 (C、D厂家) 聚为一类, 在OPLS-DA分析模式下, 以变量权重值(VIP) > 1为标准, 筛选出了14个质量差异物, 其均有不同的显著性差异; 9个成分在各自范围内线性关系良好, 线性相关系数r均≥ 0.999 0; 精密度RSD均 < 3.00%; 稳定性和重复性良好, RSD均 < 5.00%; 平均加样回收率为96.58%~106.28%, RSD为2.68%~6.45%。此方法高效稳定, 可用于冠心七味片质量控制。

冠心七味片  /  指纹图谱  /  化学计量学  /  方差分析  /  定量分析  /  质量控制

The study aims to establish HPLC fingerprint and multi-index content determination method of Guanxin Qiwei tablets and provide scientific basis for its quality control. The fingerprints of 18 batches of Guanxin Qiwei tablets were established by Shim-pack GGIST HP C18 chromatographic column, and analyzed comprehensively in combination with the Similarity Evaluation System of TCM Chromatographic Fingerprint (2012 edition). Cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used for stoichiometric study. HPLC-MS/MS method was established for simultaneous determination of 9 components. A total of 22 common peaks were identified in 18 batches of Guanxin Qiwei tablets fingerprint, and the similarity was 0.952-0.998, 9 common peaks were identified. They were No. 1 gallic acid, No. 3 protocatechuic acid, No. 8 ellagic acid, No. 9 salvianolic acid B, No. 12 luteolin, No. 13 apigenin, No. 19 dehydrodiisoeugenol, No. 20 cryptotanshinone and No. 21 tanshinone ⅡA. CA and PCA analysis grouped 18 batches of Guanxin Qiwei tablets into 3 categories: S1-S4 (manufacturer A) was grouped into one category, S5-S8 (manufacturer B) was grouped into one category, and S9-S18 (manufacturer C and D) was grouped into one category. Under OPLS-DA analysis mode, 14 quality differentiators were selected with the variable important projection (VIP) greater than 1 as the standard, and all of them had significant differences. The linear relationship of the 9 components was good in their respective ranges, and the linear correlation coefficient r was greater than or equal to 0.099 9. Precision RSDS were all lower than 3.00%. The stability and repeatability were good, RSD lower than 5.00%; The average recoveries were 96.58%-106.28%, RSD was 2.68%-6.45%. This method is efficient and stable, and can be used for quality control of Guanxin Qiwei tablets.

Guanxin Qiwei tablet  /  fingerprint  /  chemometrics  /  analysis of variance  /  quantitative analysis  /  quality control
刘成东, 杨利, 陈佳惠, 张彭鹏, 刘静, 张谦, 李君, 王跃武. 基于指纹图谱和化学计量学的冠心七味片质量控制研究. 药学学报, 2025 , 60 (4) : 1115 -1123 . DOI: 10.16438/j.0513-4870.2024-1011
Cheng-dong LIU, Li YANG, Jia-hui CHEN, Peng-peng ZHANG, Jing LIU, Qian ZHANG, Jun LI, Yue-wu WANG. Study on quality control of Guanxin Qiwei tablets based on fingerprint and chemometrics[J]. Acta Pharmaceutica Sinica, 2025 , 60 (4) : 1115 -1123 . DOI: 10.16438/j.0513-4870.2024-1011
正文
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冠状动脉粥样硬化性心脏病(简称冠心病, CHD) 是由动脉粥样硬化导致冠状动脉狭窄, 进而引起心肌缺血和坏死的疾病[1]。近年来, CHD在全球范围内的发病率和死亡率持续上升, 已对人类健康构成了严重威胁[2]。CHD的治疗方法有心肌血运重建手术[3], 及时的手术治疗可以减轻CHD对心脏的损害, 但无法完全恢复心肌结构[4], 因此, 寻找一种药物治疗CHD是非常重要和迫切的。随着我国传统医药的发展, 其在医疗领域已经展现出独特的优势。蒙医将冠心病归为“心刺痛”范畴[5], 蒙医治疗“心刺痛”具有悠久的历史, 经典方剂丰富, 亟待进一步挖掘。
冠心七味片是治疗“心刺痛”的经典验方之一, 该方由丹参、檀香、降香、山柰、肉豆蔻、广枣和沙棘七味药组成, 具有活血化瘀、强心止痛功效, 主要用于治疗冠心病、心绞痛、心烦心悸等疾病[6]。方中丹参为主药, 具有活血祛瘀、痛经止痛、清心除烦等功效[7, 8], 现代研究表明丹参中丹酚酸B具有抗动脉粥样硬化和抗氧化等药理活性[9], 隐丹参酮则有抗缺血性心肌损伤和抑制炎症反应等药理活性[10]。此外, 方中檀香可行气止痛、降香化瘀止血、山柰活血化瘀、肉豆蔻温中行气、广枣活血化瘀、沙棘活血散瘀[11-16], 诸药合用强化活血化瘀、强心止痛之功。此方为治疗CHD的良方。
目前关于冠心七味片的质量研究文献较为陈乏[17, 18], 质量研究较为薄弱。中药指纹图谱在中药整体质量分析中已得到普遍认可和使用[19]。化学计量学能够实现中药质量信息的标准化, 并对中药质量信息进行全面把控[20]。高效液相色谱-三重四极杆质谱(HPLC-MS) 可采用多反应监测模式(MRM) 分析, 具有高灵敏度、高选择性和高效性等优势[21]。基于此, 本研究基于高效液相色谱(HPLC) 技术建立18批冠心七味片指纹图谱与化学计量学分析结合HPLC-MS/MS法同时测定冠心七味片中没食子酸、原儿茶酸、鞣花酸、丹酚酸B、木犀草素、芹菜素、去氢二异丁香酚、隐丹参酮、丹参酮ⅡA等9个成分含量, 旨在为该制剂质量控制提供参考。
材料与方法
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仪器    LC-2030C Puls型高效液相色谱、LC-MS 8045高效液相色谱-三重四级杆质谱联用仪, 配备DGU-20A型脱气机, LC-20AD型输液泵、AUW120D十万分之一电子天平(日本岛津公司); BSA224S型万分之一天平(赛多利斯科学仪器有限公司); KQ5200E型超声波清洗器(广州博勒泰科有限公司)。
试药    收集4个厂家18个批次冠心七味片, 分别为A厂家(S1~S4, 批号分别为06230701042、06230601002、06230701003、06230704040)、B厂家(S5~S8, 批号分别为220640022、211002116、211002211、210121108)、C厂家(S9~S12, 批号分别为22302016、22309003、22203011、22302019)、D厂家(S13~S18, 批号分别为20210907、20210908、20210909、C24016、C24017、C24018), A、D厂家规格均为0.31 g/片、B厂家规格为0.3 g/片、C厂家规格为0.32 g/片; 对照品没食子酸(批号: 110831-202407, 纯度≥ 98.6%)、原儿茶酸(批号: 110809-202207, 纯度≥ 98.2%)、鞣花酸(批号: 111959-201903, 纯度≥ 98.4%)、丹酚酸B (批号: 111562-202318, 纯度≥ 98.2%)、木犀草素(批号: 111520-202107, 纯度≥ 98.6%)、芹菜素(批号: 111901-202205, 纯度≥ 98.6%)、去氢二异丁香酚(批号: 111838-201804, 纯度≥ 98.4%)、隐丹参酮(批号: 110852-201807, 纯度≥ 99.2%)、丹参酮ⅡA (批号: 110776-202323, 纯度≥ 99.6%) 均来自中国食品药品检定研究院; 甲醇、乙腈(色谱纯, 美国Thermo Fisher公司); 磷酸(分析纯, 天津市富宇精细化工有限公司); 甲酸(质谱纯, 上海起发生物科技有限公司); 蒸馏水(屈臣氏中国有限公司)。
对照品溶液的制备    精密称取没食子酸3.21 mg、原儿茶酸2.44 mg、鞣花酸2.12 mg、丹酚酸B2.55 mg、木犀草素2.33 mg、芹菜素3.16 mg、去氢二异丁香酚3.00 mg、隐丹参酮4.46 mg、丹参酮ⅡA 3.66 mg, 置于不同10 mL量瓶中, 甲醇溶解并定容, 即得9个对照品母液, 精密量取各母液适量置于同一10 mL量瓶中, 甲醇定容, 得质量浓度分别为80.25、976.00、139.92、6885.00、209.70、259.12、390.00、1 873.20、1 464.00 ng·mL-1的混合对照品溶液。全部储存于4 ℃条件下备用。
供试品溶液的制备    取冠心七味片适量研磨成粉, 称取约0.500 g置于50 mL锥形瓶中, 加70%甲醇5 mL, 称量, 密封, 超声45 min (频率90 kHz), 待冷却室温, 70%甲醇补重, 摇匀, 0.22 μm有机滤膜过滤, 即得。
色谱条件    色谱柱为Shim-pack GIST-HP C18 (2.1 mm × 100 mm, 3 μm), 流动相为甲醇(A)-0.2%磷酸水溶液(B), 流速为0.80 mL·min-1, 柱温为35 ℃, 进样量为10 μL, 检测波长为254 nm; 梯度洗脱: 0~8 min, 15%→25% A; 8~23 min, 25%→38% A; 23~45 min, 38%→46% A; 45~65 min, 46%→48% A; 65~90 min, 48%→65% A; 90~110 min, 65%→80% A; 110~120 min, 80%→95% A; 120~130 min, 95%→15% A。
色谱-质谱条件    色谱柱为Agilent Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm); 流动相: 甲醇(A)-水(含0.1%甲酸, B), 流速为0.25 mL·min-1, 柱温为35 ℃, 进样量为3 μL; 梯度洗脱: 0~0.8 min, 5%~15% A; 0.8~1.5 min, 15%~25% A; 1.5~5.0 min, 25%~65% A; 5.0~6.0 min, 65%~75% A; 6.0~6.5 min, 75%~85% A; 6.5~7.0 min, 95% A; 7.0~8.0 min, 85%~10% A; 8.0~9.5 min, 5% A。质谱喷雾电压3.80 kV (+)、3.50 kV (-), 雾化气体积流量3 L·min-1; 加热气体积流量10 L·min-1; 接口温度300 ℃; 脱溶剂温度526 ℃; 加热块温度400 ℃; 采用电喷雾离子源(ESI); 正负离子扫描; 多重反应监测(MRM) 模式; 正离子模式下的质谱参数: 去氢二异丁香酚m/z 327.2→188.0, 碰撞能量-21.0 eV、隐丹参酮m/z 297.1→251.1, 碰撞能量-24.0 eV、丹参酮ⅡA m/z 295.1→249.1, 碰撞能量-22.0 eV。负离子模式下的质谱参数: 没食子酸m/z 168.9→125.0, 碰撞能量15.0 eV、原儿茶酸m/z 152.9→109.1, 碰撞能量15.0 eV、鞣花酸m/z 300.8→283.8, 碰撞能量32.0 eV、丹酚酸B m/z 716.9→519.3, 碰撞能量21.0 eV、木犀草素m/z 285.0→133.0, 碰撞能量35.0 eV、芹菜素m/z 260.1→117.0, 碰撞能量34.0 eV。
指纹图谱方法学考察
精密度    取S1号样品(批号: 06230701042), 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。
稳定性    取S1号样品, 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, 于0、3、6、12、18、24 h进样分析, 计算共有峰相对保留时间RSD和相对峰面积RSD。
重复性    取S1号样品6份, 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。
化学计量学分析及统计学处理    化学计量学分析采用微生信平台和SIMCA 14.1软件。方差分析采用GraphPad Prism 9.0软件, P < 0.05定义为具有统计学差异, P < 0.01表示为具有显著性差异, P < 0.001表示为具有极显著性差异。
HPLC-MS定量分析方法学考察
线性关系考察    分别精密量取混合对照品溶液100、200、400、600、800、1 000 μL, 分别置于不同1 mL量瓶中, 甲醇定容, 在“色谱-质谱条件”项下进样。将对照品进样浓度设为横坐标(X, ng·mL-1), 峰面积设为纵坐标(Y) 进行回归分析计算。
精密度    精密量取400 μL混合对照品溶液, 置于1 mL量瓶, 甲醇定容, 在“色谱-质谱条件”项下分别测定6次分析, 计算上述各成分峰面积RSD。
稳定性    取S1号样品, 按“供试品溶液的制备”项下制备供试品溶液, 在“色谱-质谱条件”项下, 于0、3、6、12、18、24 h进样分析, 计算各成分峰面积RSD。
重复性    取S1号样品6份, 按“供试品溶液的制备”项下制备供试品溶液, 在“色谱-质谱条件”项下分别进样分析, 计算上述各成分平均含量和RSD。
加样回收    称取已知质量浓度S1号样品6份, 每份约0.250 0 g, 以样品质量浓度约100%的比例加入对照品溶液。按“供试品溶液的制备”项下制备样品溶液。在“色谱-质谱条件”项下进样测定, 计算各成分平均加样回收率和RSD。
结果
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1 指纹图谱方法学考察
1.1 精密度
所得色谱图中, 3号峰的响应度较高, 峰形优异。即以3号峰作为参照, 22个共有峰相对保留时间RSD均 < 0.09%, 相对峰面积RSD均 < 2.61%, 表明仪器精密度良好。
1.2 稳定性
以3号峰为参照峰, 22共有峰的相对保留时间RSD均 < 0.04%, 相对峰面积RSD均 < 2.42%, 表明样品溶液24 h内具有良好稳定性。
1.3 重复性
以3号峰为参照峰, 22共有峰的相对保留时间RSD均 < 0.35%, 相对峰面积RSD均 < 2.92%, 表明该方法具有较好的重复性。
2 冠心七味片指纹图谱的建立及相似度分析
取4个厂家18批次样品, 按“供试品溶液的制备”项下制备供试品溶液, 在“色谱条件”项下连续进样分析。将所得色谱数据转为CDF格式, 导入中药色谱指纹图谱相似度评价系统(2012版)。设S1号为参照图谱, 采用中位数法, 时间窗宽度设为0.1 min, 经过多点校正和Mark峰匹配, 生成对照谱图R, 得指纹图谱, 如图 1A所示, 共标定22个共有峰。通过对照品母液色谱与S1号样品色谱比对, 如图 1B所示, 9个色谱峰被指认, 分别为1号峰没食子酸、3号峰原儿茶酸、8号峰鞣花酸、9号峰丹酚酸B、12号峰木犀草素、13号峰芹菜素、19号峰去氢二异丁香酚、20号峰隐丹参酮、21号峰丹参酮ⅡA; 相似度评价结果, 18批冠心七味片样品相似度依次为0.986、0.965、0.982、0.952、0.985、0.980、0.981、0.983、0.997、0.994、0.990、0.996、0.995、0.995、0.998、0.993、0.996、0.996, 相似度在0.952~0.998之间, 表明样品具有良好的相似度, 整体性良好; 以原儿茶酸为参照峰, 计算得到18批次样品22个共有峰相对峰面积RSD为21.85%~77.74%, 表明18批样品在某些成分含量上存在差异。
3 化学计量学分析
3.1 聚类分析(CA)
利用微生信平台, 以18批样品22个共有峰峰面积为指标, 聚类方法采用complete法, 距离度量设置为euclidean[22], 结果如图 2, 根据峰面积表达量可将18批样品聚为3大类, A厂家(S1~S4) 聚为第1类, B厂家(S5~S8) 聚为第2类, C、D厂家(S9~S18) 聚为第3类, 表明18批冠心七味片样品有质量差异。
3.2 主成分分析(PCA)
以22个共有峰峰面积为自变量, 18个批次样品为因变量, 导入SIMCA 14.1软件进行无监督判别的主成分分析, 结果见图 3, 与CA分析结果一致, 其中A、B厂家不同批次样品间离散性较小, C、D厂家不同批次样品间离散性较大, 表明A、B厂家样品质量间较均一, C、D厂家样品间质量差异较大, 亦验证了18批冠心七味片具有质量差异。
3.3 正交偏最小二乘法-判别分析(OPLS-DA)
为排除无关变量对样品质量信息的影响, 采用SIMCA 14.1软件有监督模式的OPLS-DA分析, 模型参数R2X、R2Y和Q2分别为0.805、0.717和0.667, 均大于0.5, 说明该模型预测能力和稳定性较好[23], 结果见图 4A, 再经200次置换检验, Q2回归线与纵轴的相交点小于0, 表明该模型有效, 无过度拟合[24], 结果见图 4B, OPLS-DA分析结果与CA和PCA分析结果相同, 但OPLS-DA分类结果更为明显, 进一步表明冠心七味片样品存在质量差异。
为筛选出导致样品间质量差异的主要成分, 以变量权重值(VIP) > 1为标准[25]筛选出14个质量差异物, 如图 4C所示, 分别为峰9 (丹酚酸B)、峰12 (丹参酮ⅡA)、峰16、峰1 (没食子酸)、峰15、峰4、峰20 (隐丹参酮)、峰22、峰2、峰5、峰7、峰10、峰18、峰8 (鞣花酸), 即这14个成分是造成18批冠心七味片样品质量差异的主要来源。
3.4 质量差异物的方差分析
利用OPLS-DA分析中VIP值可筛选出样品间的质量差异物, 但无法判别样品间质量差异物是否具有统计学意义。因此, 根据以上CA、PCA、OPLS-DA分析结果, 将18批冠心七味片分为3类(A、B厂家分别为第1类和第2类, C、D厂家共为第3类)。采用GraphPad Prism 9.0软件, 以14个质量差异物峰面积为指标, 进行单因素方差分析[26], 结果如图 5所示, 1类中丹参酮ⅡA、峰16、没食子酸、峰15、峰4、隐丹参酮、峰22、峰2、峰5、峰7、峰18、鞣花酸峰面积显著高于2类; 2类中丹酚酸、丹参酮Ⅱ、峰16、没食子酸、隐丹参酮、峰2、峰5、峰7、鞣花酸峰面积显著低于3类, 而峰2、峰10峰面积显著高于3类; 3类中没食子酸、峰15、峰4、峰22、峰2、峰10、峰18、鞣花酸峰面积显著低于1类, 仅有丹酚酸B峰面积显著高于3类。上述各成分峰面积的相对显著性结果, 可能是造成4个厂家18批次冠心七味片分为3大类的主要原因。
4 HPLC-MS定量分析方法学考察
在上述色谱-质谱条件下, 各成分在MRM模式下的提取离子流色谱图见图 6。
4.1 线性关系考察
上述9个成分在各自范围内呈现线良好性关系, 线性相关系数r均≥ 0.999 0, 结果见表 1。
4.2 精密度
上述9个成分峰面积RSD分别为2.62%、0.90%、2.53%、1.95%、2.23%、2.55%、1.31%、0.79%、1.69%, 表明仪器精密度良好。
4.3 稳定性
上述9个成分峰面积RSD分别为2.76%、2.62%、4.29%、1.66%、1.67%、1.22%、2.81%、1.56%、3.05%, 表明样品溶液24 h内具有良好稳定性。
4.4 重复性
9个成分平均含量分别为2.991、28.294、4.078、412.490、6.076、3.248、15.714、167.948、139.360 μg·g-1, RSD分别为3.27%、3.30%、2.68%、1.62%、1.87%、1.57%、1.75%、1.32%、2.19%, 表明该方具有良好的重复性。
4.5 加样回收率
上述9个成分平均加样回收率分别为102.88%、96.98%、103.87%、101.07%、97.68%、106.28%、96.58%、98.08%、97.73%, RSD分别为3.07%、4.80%、4.38%、2.68%、3.12%、6.45%、3.14%、4.62%、4.59%。
4.6 样品含量测定
取18批冠心七味片样品, 按“供试品溶液的制备”项下制备供试品溶液, 稀释10倍, 在“色谱-质谱”项下进样测定3次, 计算上述9个成分平均含量, 结果见表 2。
讨论
为在指纹图谱实验中获得较高的色谱峰响应度、良好的分离度和峰形, 前期考察了不同流动相(甲醇-0.2%磷酸水、乙腈-0.2%磷酸水)、洗脱时间(90、110、130 min) 和流速(0.80、0.90、1.0 mL·min-1)。最终确定, 流动相为甲醇-0.2%磷酸水、洗脱时间为130 min、流速为0.80 mL·min-1时效果最佳; 在定量分析中, 考察了不同流动相(甲醇-0.1%甲酸水、乙腈-0.1%甲酸水) 对色谱峰的影响, 最终选择甲醇-0.1%甲酸水作为流动相。在质谱检测模式的考察中, 发现丹酚酸B在正负离子扫描模式下均能出峰, 但在负离子模式下的响应度更高。通过相关文献[27]发现去氢二异丁香酚具有三个定量离子, 当定量离子为188时, 峰形较优, 响应度最高。为兼顾各成分的测定, 本实验采用正负离子扫描和多反应监测模式进行定量分析。
本研究建立了HPLC指纹图谱, 结合化学计量学对4个厂家18批冠心七味片样品进行质量研究。结果共标定了22个共有峰, 其中9个共有峰被指认出, 分别为没食子酸、原儿茶酸、鞣花酸、丹酚酸B、木犀草素、芹菜素、去氢二异丁香酚、隐丹参酮、丹参酮ⅡA。在相似度分析中, 18个批样品相似度均在0.952以上, 表明整体性良好, 然而, 以原儿茶酸为参照时, 共有峰的相对峰面积RSD在21.85%~77.74%之间, 表明某些成分含量存在较大差异。为进一步研究其质量差异, 分别进行了CA、PCA和OPLS-DA分析, 结果将样品分为了3大类。为探究质量差异来源, 在OPLS-DA分析模式下, 以VIP > 1为标准, 筛选出了14个质量差异物, 其中丹酚酸B、丹参酮ⅡA、没食子酸、隐丹参酮、鞣花酸已被指认, 由于在该制剂在《中华人民共和国卫生部药品标准》 (蒙药分册) 中除了制法、检查、用法用量项, 尚无含量测定项, 即这5个成分可作为其含量测定指标, 且丹酚酸B、丹参酮ⅡA和隐丹参酮均为主药丹参含量测定项中主要成分[28], 说明此指标作为该制剂的质量控制依据是合理的。由于无法确定14个质量差异物是否具有统计学意义, 对其进行了单因素方差分析, 结果显示, 14个成分在3类样品之间均存在显著性差异, 这些差异可能是导致冠心七味片质量差异的主要原因。
本研究对所指认的9个成分进行了定量分析。结果显示, 18批样品中丹酚酸B、丹参酮ⅡA、没食子酸、隐丹参酮和鞣花酸的含量波动较大, 这可能是造成上述聚类现象的主要原因。根据相似度结果, A、B厂家样品的相似度分别在0.952~0.986和0.980~0.985之间, 而C、D厂家样品的相似度均在0.990以上, 与聚类结果相同, 也与不同厂家样品含量结果范围对应。此外, 丹酚酸B和丹参酮ⅡA的平均含量最高, 可为临床用药的有效性和安全性提供参考依据。
综上所述, 本研究建立了冠心七味片指纹图谱与化学计量学分析结合HPLC-MS/MS法同时测定多成分的方法高效且稳定, 可用于冠心七味片的质量控制。
基金
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  • 内蒙古自治区科技厅, 内蒙古自治区科技计划项目(2023YFHH0082)
  • 内蒙古自治区蒙医药协同创新中心, 国际合作项目(MYYXTGJ202306)
  • 内蒙古自治区蒙医药协同创新中心, 成果转化培育项目(MYYXTPY202306)
  • 内蒙古医科大学“致远”人才计划项目(ZY20242115)
  • 内蒙古医科大学面上项目(YKD2024MS015)
文献
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doi: 10.16438/j.0513-4870.2024-1011
  • 接收时间：2024-10-17
  • 首发时间：2025-10-29
  • 出版时间：2025-04-12
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  • 收稿日期：2024-10-17
  • 修回日期：2025-02-13
基金
内蒙古自治区科技厅, 内蒙古自治区科技计划项目(2023YFHH0082)
内蒙古自治区蒙医药协同创新中心, 国际合作项目(MYYXTGJ202306)
内蒙古自治区蒙医药协同创新中心, 成果转化培育项目(MYYXTPY202306)
内蒙古医科大学“致远”人才计划项目(ZY20242115)
内蒙古医科大学面上项目(YKD2024MS015)
作者信息
    1 内蒙古医科大学药学院, 内蒙古 呼和浩特 010110
    2 包头中药有限公司, 内蒙古 包头 014040

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李君, E-mail:
王跃武, 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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