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Article(id=1200383183184327439, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200383177295515678, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2024-0201, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1709654400000, receivedDateStr=2024-03-06, revisedDate=1715529600000, revisedDateStr=2024-05-13, acceptedDate=null, acceptedDateStr=null, onlineDate=1764123252912, onlineDateStr=2025-11-26, pubDate=1723392000000, pubDateStr=2024-08-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764123252912, onlineIssueDateStr=2025-11-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764123252912, creator=13701087609, updateTime=1764123252912, updator=13701087609, issue=Issue{id=1200383177295515678, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='8', pageStart='2183', pageEnd='2416', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764123251508, creator=13701087609, updateTime=1764225076631, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200810262870741499, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200383177295515678, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200810262870741500, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1200383177295515678, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2365, endPage=2371, ext={EN=ArticleExt(id=1200383183607952175, articleId=1200383183184327439, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Study on quality evaluation of Gegen Qinlian decoction based on UHPLC fingerprint and multi-component quantification, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Gegen Qinlian decoction has a wide range of clinical applications. However, there is a lack of systematic quality evaluation methods to ensure the safety and effectiveness of Gegen Qinlian decoction in clinical use. The UHPLC fingerprint and multi-component determination method of Gegen Qinlian decoction were established to provide scientific basis for the quality control and evaluation of Gegen Qinlian decoction. The chromatography was performed on a ZORBAX Eclipse Plus-C18 column (150 mm × 4.6 mm, 3.5 μm) with mobile phase consisted of acetonitrile (A) - 20 mmol·L-1 ammonium acetate (containing 0.8% acetic acid and 0.5% triethylamine) (B) and gradient elution at a flow rate of 1.0 mL·min-1. The column temperature was 25 ℃, the detection wavelength was 260 nm, the fingerprint of 10 batches of Gegen Qinlian decoction was determined, and the similarity evaluation system of TCM chromatographic fingerprint was used for comprehensive analysis, and 9 components were quantitatively analyzed. In the fingerprint study of Gegen Qinlian decoction, a total of 18 peaks were obtained, 12 of which were identified by reference substances. Moreover, the similarity of 10 batches of Gegen Qinlian decoction was good, and all of them were greater than 0.99. In the multi-component quantitative analysis, the linear relationship between the nine components and the peak area was good (r ≥ 0.999) in the corresponding mass concentration range. The average recovery rate was 94.4%-100.3%, and the RSD was 0.1%-1.4%. The fingerprint of Gegen Qinlian decoction was studied under the same wavelength, and the content of 9 main components were determined by our established method. The method has high sensitivity and strong specificity, which provided a comprehensive scientific basis for the comprehensive evaluation of the quality of Gegen Qinlian decoction.

, correspAuthors=Qing LI, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 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=Juan XIE, Qi TANG, Pan ZHANG, Xin LI, Kai-shun BI, Qing LI), CN=ArticleExt(id=1200383184639751044, articleId=1200383183184327439, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=基于UHPLC指纹图谱和多成分定量的葛根芩连汤质量评价研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

葛根芩连汤临床应用广泛, 但目前缺乏系统的质量评价方法以确保葛根芩连汤在临床使用的安全性和有效性。因此本文建立了葛根芩连汤的UHPLC指纹图谱及多成分含量测定方法, 为葛根芩连汤的质量控制与评价提供科学依据。采用UHPLC-DAD法检测, 色谱柱为ZORBAX Eclipse Plus-C18 (150 mm × 4.6 mm, 3.5 μm), 流动相为乙腈(A)-20 mmol·L-1乙酸铵(含0.8%乙酸及0.5%三乙胺, B), 梯度洗脱, 柱温25 ℃, 流速为1.0 mL·min-1, 检测波长为260 nm, 建立10批葛根芩连汤的指纹图谱, 结合中药色谱指纹图谱相似度评价系统进行全面分析, 并对其中9个成分进行定量分析。在葛根芩连汤指纹图谱研究中, 得到共有峰18个, 通过对照品指认了其中12个峰对应的成分, 10批葛根芩连汤相似度良好, 均大于0.99。多成分定量分析中9种成分在相应的质量浓度范围内与峰面积的线性关系良好(r ≥ 0.999), 平均加样回收率在94.4%~100.3%内, RSD在0.1%~1.4%内。本方法在同一波长下研究葛根芩连汤指纹图谱的同时能对其9个主要成分的含量进行测定, 方法灵敏度高、专属性强, 为全面评价葛根芩连汤的质量提供依据。

, correspAuthors=李清, authorNote=null, correspAuthorsNote=
*李清, Tel: 86-24-43510589, E-mail:
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No.Puerariae Lobatae RadixScutellariae RadixCoptidis RhizomaGlycyrrhizae Radix et Rhizoma Praeparata Cum Melle
OriginBatch No.OriginBatch No.OriginBatch No.OriginBatch No.
S1Anhui AnqingA220329Shanxi220701ChongqingA220222Xinjiang220601
S2Anhui AnqingA220107Shanxi Yuncheng211215ChongqingA220503Xinjiang220801
S3Anhui Suzhou22042101Shanxi Yuncheng220303Chongqing ShizhuZ2103001Gansu ZhangyeA220727
S4Anhui Suzhou22072101Shanxi Yuncheng220401Sichuan Pengzhou210421Gansu Longnan200902
S5Anhui Anqing21082001Shanxi Yuncheng220202Sichuan22012602Gansu Zhangye220202
S6Anhui Yuexi220600119Hebei Chengde220500181Sichuan Pengzhou220801Gansu20220801
S7Anhui Anqing21120801Hebei Chengde220601Sichuan Meishan220401Inner Mongolia Chifeng210702
S8Anhui AnqingA220812Hebei220901Sichuan Hongya220801Inner Mongolia22021002
S9Anhui220502Shaanxi Shangzhou220401Sichuan220501CP0544Inner Mongolia Baotou220501741
S10Sichuan Hongya220401Gansu Longxi22072601Hubei LichuanHL180322Inner Mongolia Alxa Left Banner220801
), ArticleFig(id=1201107067558912617, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200383183184327439, language=CN, label=Table 1, caption=

Information of 10 batches of Gegen Qinlian decoction with decoction pieces

, figureFileSmall=null, figureFileBig=null, tableContent=
No.Puerariae Lobatae RadixScutellariae RadixCoptidis RhizomaGlycyrrhizae Radix et Rhizoma Praeparata Cum Melle
OriginBatch No.OriginBatch No.OriginBatch No.OriginBatch No.
S1Anhui AnqingA220329Shanxi220701ChongqingA220222Xinjiang220601
S2Anhui AnqingA220107Shanxi Yuncheng211215ChongqingA220503Xinjiang220801
S3Anhui Suzhou22042101Shanxi Yuncheng220303Chongqing ShizhuZ2103001Gansu ZhangyeA220727
S4Anhui Suzhou22072101Shanxi Yuncheng220401Sichuan Pengzhou210421Gansu Longnan200902
S5Anhui Anqing21082001Shanxi Yuncheng220202Sichuan22012602Gansu Zhangye220202
S6Anhui Yuexi220600119Hebei Chengde220500181Sichuan Pengzhou220801Gansu20220801
S7Anhui Anqing21120801Hebei Chengde220601Sichuan Meishan220401Inner Mongolia Chifeng210702
S8Anhui AnqingA220812Hebei220901Sichuan Hongya220801Inner Mongolia22021002
S9Anhui220502Shaanxi Shangzhou220401Sichuan220501CP0544Inner Mongolia Baotou220501741
S10Sichuan Hongya220401Gansu Longxi22072601Hubei LichuanHL180322Inner Mongolia Alxa Left Banner220801
), ArticleFig(id=1201107067659575918, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200383183184327439, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
AnalyteLinearityLOQ/μg·mL-1LOD/μg·mL-1
Range/mg·mL-1EquationR2
Puerarin3.048-0.121 9y = 15 675x + 672.770.999 10.327 10.109 0
Daidzin0.717 0-0.028 68y = 17 943x - 68.1770.999 80.399 10.133 0
Baicalin1.948-0.077 92y = 9 940.1x - 238.260.999 93.6181.206
Wogonoside0.739 0-0.029 56y = 14 428x - 103.80.999 81.8580.619 2
Barmatine0.318 0-0.012 72y = 18 000x - 44.2510.999 72.9040.823
Berberine0.338 0-0.013 52y = 20 818x - 58.7810.999 73.6911.119
Lycyrrhizic acid0.273 0-0.010 92y = 3 435.4x - 11.6050.999 91.9020.543 5
Baicalein0.099 0-0.003 960y = 14 440x - 52.0630.999 11.0420.347 5
Wogonin0.043 00-0.001 720y = 17 510x - 4.421 30.999 70.666 00.222 0
), ArticleFig(id=1201107067772822128, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200383183184327439, language=CN, label=Table 2, caption=

Results of linearity, limit of quantitation (LOQ) and limit of detection (LOD)

, figureFileSmall=null, figureFileBig=null, tableContent=
AnalyteLinearityLOQ/μg·mL-1LOD/μg·mL-1
Range/mg·mL-1EquationR2
Puerarin3.048-0.121 9y = 15 675x + 672.770.999 10.327 10.109 0
Daidzin0.717 0-0.028 68y = 17 943x - 68.1770.999 80.399 10.133 0
Baicalin1.948-0.077 92y = 9 940.1x - 238.260.999 93.6181.206
Wogonoside0.739 0-0.029 56y = 14 428x - 103.80.999 81.8580.619 2
Barmatine0.318 0-0.012 72y = 18 000x - 44.2510.999 72.9040.823
Berberine0.338 0-0.013 52y = 20 818x - 58.7810.999 73.6911.119
Lycyrrhizic acid0.273 0-0.010 92y = 3 435.4x - 11.6050.999 91.9020.543 5
Baicalein0.099 0-0.003 960y = 14 440x - 52.0630.999 11.0420.347 5
Wogonin0.043 00-0.001 720y = 17 510x - 4.421 30.999 70.666 00.222 0
), ArticleFig(id=1201107067898651252, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200383183184327439, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
AnalyteContent/mg·g-1
S1S2S3S4S5S6S7S8S9S10
Puerarin16.9015.9019.3619.3618.7414.0316.1019.6517.1514.29
Daidzin3.2812.8423.7643.7643.8402.4783.2383.6792.9803.348
Baicalin10.8310.8410.1410.1410.009.3610.1912.8610.489.14
Wogonoside3.6023.6463.2703.2703.0504.1023.5173.7173.5933.453
Barmatine1.4901.5631.3701.3701.2701.5311.5890.8701.5971.492
Berberine2.5362.1691.7801.7801.5391.0812.2931.3771.8101.556
Lycyrrhizic acid1.2401.3581.1381.1381.5800.9851.2941.4631.1241.272
Baicalein0.282 20.342 20.234 00.234 00.334 80.177 30.553 00.423 40.326 70.461 3
Wogonin0.131 60.140 60.120 60.120 60.162 90.088 80.245 10.199 90.243 50.194 6
), ArticleFig(id=1201107067978343030, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1200383183184327439, language=CN, label=Table 3, caption=

Content of nine components in ten batches of Gegen Qinlian decoction

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AnalyteContent/mg·g-1
S1S2S3S4S5S6S7S8S9S10
Puerarin16.9015.9019.3619.3618.7414.0316.1019.6517.1514.29
Daidzin3.2812.8423.7643.7643.8402.4783.2383.6792.9803.348
Baicalin10.8310.8410.1410.1410.009.3610.1912.8610.489.14
Wogonoside3.6023.6463.2703.2703.0504.1023.5173.7173.5933.453
Barmatine1.4901.5631.3701.3701.2701.5311.5890.8701.5971.492
Berberine2.5362.1691.7801.7801.5391.0812.2931.3771.8101.556
Lycyrrhizic acid1.2401.3581.1381.1381.5800.9851.2941.4631.1241.272
Baicalein0.282 20.342 20.234 00.234 00.334 80.177 30.553 00.423 40.326 70.461 3
Wogonin0.131 60.140 60.120 60.120 60.162 90.088 80.245 10.199 90.243 50.194 6
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基于UHPLC指纹图谱和多成分定量的葛根芩连汤质量评价研究
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谢娟 2 , 唐琪 1 , 张盼 1 , 李欣 2 , 毕开顺 1, 2 , 李清 1, *
药学学报 | 研究论文 2024,59(8): 2365-2371
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药学学报 | 研究论文 2024, 59(8): 2365-2371
基于UHPLC指纹图谱和多成分定量的葛根芩连汤质量评价研究
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谢娟2, 唐琪1, 张盼1, 李欣2, 毕开顺1, 2, 李清1, *
作者信息
  • 1.沈阳药科大学药学院, 辽宁 沈阳 110016
  • 2.南方医科大学药学院, 广东 广州 510515

通讯作者:

*李清, Tel: 86-24-43510589, E-mail:
Study on quality evaluation of Gegen Qinlian decoction based on UHPLC fingerprint and multi-component quantification
Juan XIE2, Qi TANG1, Pan ZHANG1, Xin LI2, Kai-shun BI1, 2, Qing LI1, *
Affiliations
  • 1. School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
  • 2. School of Pharmacy, Southern Medical University, Guangzhou 510515, China
出版时间: 2024-08-12 doi: 10.16438/j.0513-4870.2024-0201
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葛根芩连汤临床应用广泛, 但目前缺乏系统的质量评价方法以确保葛根芩连汤在临床使用的安全性和有效性。因此本文建立了葛根芩连汤的UHPLC指纹图谱及多成分含量测定方法, 为葛根芩连汤的质量控制与评价提供科学依据。采用UHPLC-DAD法检测, 色谱柱为ZORBAX Eclipse Plus-C18 (150 mm × 4.6 mm, 3.5 μm), 流动相为乙腈(A)-20 mmol·L-1乙酸铵(含0.8%乙酸及0.5%三乙胺, B), 梯度洗脱, 柱温25 ℃, 流速为1.0 mL·min-1, 检测波长为260 nm, 建立10批葛根芩连汤的指纹图谱, 结合中药色谱指纹图谱相似度评价系统进行全面分析, 并对其中9个成分进行定量分析。在葛根芩连汤指纹图谱研究中, 得到共有峰18个, 通过对照品指认了其中12个峰对应的成分, 10批葛根芩连汤相似度良好, 均大于0.99。多成分定量分析中9种成分在相应的质量浓度范围内与峰面积的线性关系良好(r ≥ 0.999), 平均加样回收率在94.4%~100.3%内, RSD在0.1%~1.4%内。本方法在同一波长下研究葛根芩连汤指纹图谱的同时能对其9个主要成分的含量进行测定, 方法灵敏度高、专属性强, 为全面评价葛根芩连汤的质量提供依据。

葛根芩连汤  /  指纹图谱  /  多成分定量  /  质量控制

Gegen Qinlian decoction has a wide range of clinical applications. However, there is a lack of systematic quality evaluation methods to ensure the safety and effectiveness of Gegen Qinlian decoction in clinical use. The UHPLC fingerprint and multi-component determination method of Gegen Qinlian decoction were established to provide scientific basis for the quality control and evaluation of Gegen Qinlian decoction. The chromatography was performed on a ZORBAX Eclipse Plus-C18 column (150 mm × 4.6 mm, 3.5 μm) with mobile phase consisted of acetonitrile (A) - 20 mmol·L-1 ammonium acetate (containing 0.8% acetic acid and 0.5% triethylamine) (B) and gradient elution at a flow rate of 1.0 mL·min-1. The column temperature was 25 ℃, the detection wavelength was 260 nm, the fingerprint of 10 batches of Gegen Qinlian decoction was determined, and the similarity evaluation system of TCM chromatographic fingerprint was used for comprehensive analysis, and 9 components were quantitatively analyzed. In the fingerprint study of Gegen Qinlian decoction, a total of 18 peaks were obtained, 12 of which were identified by reference substances. Moreover, the similarity of 10 batches of Gegen Qinlian decoction was good, and all of them were greater than 0.99. In the multi-component quantitative analysis, the linear relationship between the nine components and the peak area was good (r ≥ 0.999) in the corresponding mass concentration range. The average recovery rate was 94.4%-100.3%, and the RSD was 0.1%-1.4%. The fingerprint of Gegen Qinlian decoction was studied under the same wavelength, and the content of 9 main components were determined by our established method. The method has high sensitivity and strong specificity, which provided a comprehensive scientific basis for the comprehensive evaluation of the quality of Gegen Qinlian decoction.

Gegen Qinlian decoction  /  fingerprint  /  multicomponent quantification  /  quality control
谢娟, 唐琪, 张盼, 李欣, 毕开顺, 李清. 基于UHPLC指纹图谱和多成分定量的葛根芩连汤质量评价研究. 药学学报, 2024 , 59 (8) : 2365 -2371 . DOI: 10.16438/j.0513-4870.2024-0201
Juan XIE, Qi TANG, Pan ZHANG, Xin LI, Kai-shun BI, Qing LI. Study on quality evaluation of Gegen Qinlian decoction based on UHPLC fingerprint and multi-component quantification[J]. Acta Pharmaceutica Sinica, 2024 , 59 (8) : 2365 -2371 . DOI: 10.16438/j.0513-4870.2024-0201
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葛根芩连汤(Gegen Qinlian decoction, GQD) 为传统中医药常用的经典古方, 来源于张仲景所著《伤寒论》, 表里同治, 具有解表证兼里热之功效。葛根芩连汤由四味中药组成, 分别为葛根、黄芩、黄连及炙甘草, 其中葛根为君药, 在中药归经理论中归脾胃经, 可疏散表邪, 解热发汗生津, 治疗阴虚之证; 臣药之一黄芩, 在中药归经理论中归肺、胆、脾、大肠经, 可清肠热治泄泻; 臣药黄连在中药归经理论中归心、脾、胃、胆、大肠经, 可解毒清热, 治疗湿热痞满之证; 使药炙甘草起调和诸药之效[1]。现代研究表明葛根芩连汤含有葛根素、大豆苷、黄芩苷、小檗碱、甘草酸等多种有效成分[2], 具有解热、抗菌、抗炎、止泻、降糖等多重药理作用[3-6]。目前中国药典(2020年版) 对葛根芩连丸、葛根芩连片这两种剂型均有收载, 片剂的剂型最为常见, 故研究者较多, 质量控制较为完善, 而葛根芩连丸的研究较少, 整体质量控制有待提升。汤剂是我国应用时间最早且范围最广的一类剂型, 汤剂以水作为溶剂, 能够快速在体内发挥疗效, 治疗效果好的同时不良反应小, 同时, 汤剂的剂量还能随证加减, 具有针对性强和灵活度高的特点, 并且对于吞咽困难者, 在丸剂与片剂无法服用的时候, 可以选择汤剂进行治疗, 故对葛根芩连汤这一剂型进行质量控制存在一定的必要性。但由于制备工艺和剂型相差较大, 无法利用片剂或丸剂的质量标准对葛根芩连汤进行评价。目前对葛根芩连汤的研究主要集中在化学成分[7]及药理药效[8,9]方面, 在对葛根芩连汤的鉴别研究上, 有学者[10]利用高效液相色谱对葛根芩连汤的指纹图谱进行了分析, 在三个波长(250、280、346 nm) 下对其共有峰进行了识别, 在含量测定的研究中, 大部分的学者[11-13]也均采用多波长对葛根芩连汤中的有效成分进行测定, 分析成分选择不完全且分析时间较长, 有些学者开发的液相条件中流动相制备还需调整pH值, 分析方法较为繁琐。目前葛根芩连汤的质量控制方面缺乏整体性的评价, 未见葛根芩连汤指纹图谱与含量测定相结合的整体质量分析方法, 并且存在分析时间长、方法较复杂以及指标成分分析不全面等问题, 鉴别及含量测定的方法有待进一步提升。因此, 有必要建立一套较为全面的质量分析方法对葛根芩连汤的质量进行控制和评价。本文运用超高效液相色谱法, 在260 nm波长下对葛根芩连汤指纹图谱方法进行研究, 并能同时测定葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素9个指标成分的含量, 建立了葛根芩连汤指纹图谱与含量测定相结合的质量控制方法, 本研究开发的液相条件具有洗脱方式简单、分析时间短和检测波长简便等优点, 为葛根芩连汤质量控制体系的完善建立提供了参考依据。
材料与方法
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仪器  1290 Infinity Ⅱ型UHPLC (美国安捷伦公司); TDZ24 WS低速离心机(湖南湘仪实验室仪器开发有限公司); MT-XS105分析天平(瑞士Mettler Toledo公司); BP210S电子分析天平(德国Sartorius公司); MTAB135-S分析天平(日本岛津公司); KQ-500DB超声波清洗器(昆山市超声仪器有限公司); 45U2紫砂煎药壶(潮州市潮安区康雅顺电器有限公司)。
材料与试剂  葛根素对照品(批号CHB201203, 纯度≥ 98%)、3′-甲氧基葛根素对照品(批号CHB210107, 纯度≥ 98%)、葛根素芹菜糖苷对照品(批号CHB201204, 纯度≥ 98%)、大豆苷对照品(批号CHB201119, 纯度≥ 98%)、黄芩苷对照品(批号CHB201101, 纯度≥ 98%)、汉黄芩苷对照品(批号CHB190115, 纯度≥ 98%)、大豆苷元对照品(批号CHB201120, 纯度≥ 98%)、盐酸巴马汀对照品(批号CHB201223, 纯度≥ 98%)、盐酸小檗碱对照品(批号CHB210108, 纯度≥ 98%)、甘草酸对照品(批号CHB1810610, 纯度≥ 98%)、黄芩素对照品(批号CHB201102, 纯度≥ 98%)、汉黄芩素对照品(批号CHB201221, 纯度≥ 98%), 均购于成都克洛玛生物科技有限公司, 购买的对照品经标化质量均满足要求; 纯净水(杭州娃哈哈集团有限公司); 乙腈(色谱纯, 美国Sigma-Aldrich公司); 乙酸(色谱纯, 天津市富宇精细化工有限公司); 三乙胺和乙酸铵(HPLC级, 天津市大茂化学试剂厂); 甲醇(色谱纯, 山东禹王和天下新材料有限公司); 葛根、黄连、黄芩和炙甘草均购于其道地产区或主产区, 经沈阳药科大学药学院王东教授鉴定, 确认葛根饮片为豆科植物葛属藤本植物野葛Pueraria lobata (Willd.) Ohwi的根经干燥后得到的, 黄连饮片为毛茛科黄连属草本植物Coptis chinensis Franch的根经干燥后得到的, 黄芩饮片为唇形科植物黄芩属草本植物Scutellaria baicalensis Georgi的根经干燥后得到的, 炙甘草饮片为豆科植物甘草属双子叶植物Glycyrrhiza uralensis Fisch的根经蜜炙后得到的。10批葛根芩连汤配伍后将其命名编号为S1~S10, 配伍饮片来源信息见表 1。
对照品溶液的制备  精密称取葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素适量, 加75%甲醇制成浓度分别为6.096、1.434、3.896、1.478、0.636 4、0.676 2、0.546 0、0.198 3、0.086 2 mg·mL-1的混合对照品溶液, 精密称取3′-甲氧基葛根素、葛根素芹菜糖苷和大豆苷元适量, 加75%甲醇制成浓度分别为0.688 4、0.944、0.068 24 mg·mL-1的混合对照品溶液。
供试品溶液的制备  按照《伤寒论》记载及《医疗机构中药煎药室管理规范》相关规定制备葛根芩连汤。取葛根16 g、黄芩6 g、黄连6 g、炙甘草4 g, 加入14倍量水浸泡30 min, 葛根先煎15 min, 再加入黄芩、黄连及炙甘草煎煮45 min, 待到一煎完毕利用筛网(200目) 趁热过滤, 向药渣中加入12倍量水, 煎煮40 min, 200目筛趁热过滤, 合并两煎液后60 ℃减压浓缩至75 mL, 即得葛根芩连汤。精密量取葛根芩连汤1 mL至5 mL量瓶中, 加75%甲醇稀释, 摇匀并超声30 min后定容, 使用有机系滤膜(0.22 μm微孔) 过滤, 取续滤液即得。
阴性对照溶液的制备  按“供试品溶液制备”项下方法分别制备不含葛根、黄连、黄芩、炙甘草的阴性对照溶液。
色谱条件  色谱柱: ZORBAX Eclipse Plus-C18 (150 mm × 4.6 mm, 3.5 μm); 流动相: 乙腈(A)-20 mmol·L-1乙酸铵(含0.8%乙酸及0.5%三乙胺, B); 梯度洗脱程序如下: 0~5 min, 10% A~11% A; 5~14 min, 11% A~15% A; 14~17 min, 15% A~17% A; 17~18 min, 17% A~19% A; 18~35 min, 19% A~20% A; 35~44 min, 20% A~22% A; 44~46 min, 22% A~34% A; 46~53 min, 34% A~36% A; 53~54 min, 36% A~45% A; 54~58 min, 45% A~58% A; 柱温: 25 ℃; 流速: 1.0 mL·min-1; 检测波长: 260 nm; 进样量: 5 μL。
指纹图谱方法学考察  
精密度  按“供试品溶液的制备”项下方法, 取同一批葛根芩连汤(S3) 制得供试品溶液, 在上述色谱条件下连续进样6次, 记录色谱图中各峰的保留时间及峰面积, 计算各共有峰与内参比峰之间的相对保留时间(RRT) 和相对峰面积(RPA)。
重复性  按“供试品溶液的制备”项下方法, 取同一批葛根芩连汤(S3) 平行制备6份供试品溶液, 在上述色谱条件下对6份重复性样品进行进样分析, 记录色谱图中各峰的保留时间及峰面积, 计算各共有峰与内参比峰之间的RRT和RPA。
稳定性  按“供试品溶液的制备”项下方法, 取葛根芩连汤(S3) 制备供试品溶液, 在上述色谱条件下分别放置0、2、4、6、8、10、12和24 h后, 再对样品进行进样分析, 记录色谱图中各峰的保留时间及峰面积, 计算各共有峰与内参比峰之间的RRT和RPA。
指纹图谱建立及相似度评价  收集10批样品, 按“供试品溶液的制备”项下方法制备10批葛根芩连汤的供试品溶液, 在上述色谱条件下进样分析, 记录色谱图。将图谱导入国家药典委员会开发的《中药色谱指纹图谱相似度评价系统》2012年版进行分析, 计算各样品指纹图谱与对照图谱的相似度。
系统适用性  分别取混合对照品溶液和供试品溶液进样分析, 计算葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素和汉黄芩素的分离度、理论塔板数、各色谱峰的拖尾及色谱峰信噪比。取混合对照品溶液, 连续进样6针, 计算待测成分的RSD。
多指标含量测定方法学考察  
专属性取对照品溶液、供试品溶液、葛根阴性对照溶液、黄连阴性对照溶液、黄芩阴性对照溶液和炙甘草阴性对照溶液各5 μL, 按上述色谱条件进行检测分析, 比较色谱图。
准确度  精密量取已知待测成分含量的葛根芩连汤9份, 每份精密移吸取0.5 mL, 分别加入葛根芩连汤中相当于各待测成分的50%、100%和150%水平的对照品, 制备低、中、高浓度的供试品溶液, 每一种浓度平行制备3份, 按照上述色谱条件进样分析, 计算各成分的加样回收率和RSD。
精密度  取同一批葛根芩连汤, 分别精密量取0.5、1.0、1.5 mL置于5 mL量瓶中, 制备低、中、高3个浓度的供试品溶液, 每一种浓度平行制备3份, 按照上述色谱条件进样分析, 测定各待测成分的含量并计算RSD, 作为重复性实验。取同一批葛根芩连汤, 制备相同浓度的供试品溶液3份, 每天平行制备3份, 连续三天进样分析, 计算各待测成分含量并计算RSD, 作为中间精密度实验。
检测限和定量限  取混合对照品溶液, 用75%甲醇进行逐步稀释, 分别以信噪比3∶1和10∶1测定各成分的检测限和定量限, 并测定精密度和准确度。
线性与范围  分别依次精密量取葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素的混合对照品溶液0.10、0.50、0.75、1.00、1.50、2.00 mL置于5 mL量瓶中, 用75%甲醇定容至刻度, 摇匀, 制得标准系列溶液。按上述色谱条件分析, 记录峰面积, 以对照品浓度C (µg·mL-1) 为横坐标, 峰面积A为纵坐标, 绘制标准曲线, 计算回归方程和相关系数。
耐用性  分别考察色谱条件在不同进样体积(4、5和6 μL)、不同柱温(24、25和26 ℃)、不同检测波长(255、260和265 nm)、不同配比的水相流动相[18 mmol·L-1乙酸铵(含0.78%乙酸及0.48%三乙胺)、20 mmol·L-1乙酸铵(含0.8%乙酸及0.5%三乙胺)、22 mmol·L-1乙酸铵(含0.82%乙酸及0.52%三乙胺)] 条件下, 测定待测成分的含量, 并计算不同条件的RSD。
稳定性  取同一份葛根芩连汤供试品溶液, 室温放置, 分别于0、2、4、6、8、12和24 h后进样分析, 计算各待测成分含量RSD。
样品含量测定  将10批不同批次的葛根芩连汤按照上述“供试品的制备”项下进行制备, 进样分析, 计算各批次葛根芩连汤中9种待测成分的含量。
结果
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1 指纹图谱方法学考察
在上述色谱条件下进行指纹图谱分析, 共检测出18个特征峰, 各个特征峰之间分离度良好, 混合对照品溶液与供试品溶液的UHPLC色谱图见图 1。
1.1 精密度
18个共有峰RRT的RSD均小于1.6%, RRA的RSD均小于1.5%, 表明仪器精密度良好。
1.2 重复性
18个共有峰RRT的RSD均小于0.6%, RRA的RSD均小于1.9%, 表明方法重复性良好。
1.3 稳定性
18个共有峰RRT的RSD均小于1.5%, RRA的RSD均小于2.3%, 表明供试品溶液在24 h内稳定性良好。
2 葛根芩连汤指纹图谱的建立及分析
葛根芩连汤成分复杂, 10批葛根芩连汤指纹图谱见图 2, 共有18个特征峰, 通过对照品可指认12个色谱峰, 其中黄芩苷为葛根芩连汤的主要活性成分和指标成分之一, 其响应与保留时间适中, 保留时间处于整个图谱中较中间的位置, 分离度良好, 且对照品易获得, 符合内参比峰的要求, 因此选择黄芩苷作为葛根芩连汤指纹图谱的内参比峰。利用《中药色谱指纹图谱相似度评价系统》2012年版建立10批葛根芩连汤UHPLC指纹图谱共有模式, S1~S10的相似度分别为0.998、0.998、0.995、0.998、0.992、0.995、0.998、0.997、0.997、0.998, 相似度均 > 0.99, 表明10批葛根芩连汤相似性良好, 质量稳定。
3 系统适用性考察
取混合对照品溶液和供试品溶液进样分析, 分析结果显示9个待测成分的分离度(R) 均大于1.5, 且理论塔板数(n) 均不低于8 000, 各色谱峰对称性良好, 色谱峰信噪比均大于10。将混合对照品溶液连续进样6针, 葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素9个待测成分的RSD分别为0.1%、0.3%、0.5%、0.5%、0.4%、0.2%、0.5%、1.4%、1.2%, 表明仪器精密度良好。
4 多指标含量测定方法学考察
4.1 专属性
在对照品溶液、供试品溶液及各阴性对照溶液的色谱图中发现, 葛根素、大豆苷来自葛根, 黄芩苷、汉黄芩苷、黄芩素、汉黄芩素来自黄芩, 小檗碱、巴马汀来自黄连, 甘草酸来自甘草, 阴性溶液均无干扰, 结果表明方法专属性良好。
4.2 准确度
葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素的平均加样回收率为100.3%、94.5%、99.2%、98.2%、97.9%、98.1%、98.7%、94.3%、96.0%, RSD分别为0.2%、0.6%、0.2%、0.1%、0.1%、0.2%、0.3%、1.4%、0.9%, 表明方法的准确度良好。
4.3 精密度
记录低、中、高共9份样品的峰面积, 测定各待测成分的含量并计算RSD, 葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素的RSD分别为1.6%、0.9%、1.0%、0.9%、0.3%、0.6%、1.0%、3.0%、3.1%, 结果表明样品重复性良好。葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素的RSD分别为0.6%、0.6%、2.5%、0.9%、0.4%、2.5%、0.7%、3.2%、1.4%, 表明方法精密度良好。
4.4 检测限与定量限
稀释各混合对照品溶液的浓度, 进样分析, 使定量限S/N ≥ 10, 检测限S/N ≥ 3, 结果如表 2所示, 各对照品的检测限在0.109 0~1.206 µg·mL-1之间, 定量限在0.327 1~3.691 µg·mL-1之间。取定量限溶液分别进行精密度和准确度试验, 结果表明, 各对照品的峰面积RSD (%) 在0.1%~2.9%之间, 平均加样回收率和回收率RSD (%) 分别在95.0%~100.8%之间和0.9%~3.1%之间, 方法在定量限浓度下精密度和准确度良好。
4.5 线性与范围
葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素在考察范围内呈现良好的线性关系, 相关系数均大于0.999, 结果见表 2。
4.6 耐用性
分别考察色谱条件在不同进样体积、不同柱温、不同检测波长以及不同配比的水相流动相的条件下, 葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素的RSD在0.2%~3.4%内, 结果表明, 方法耐用性良好。
4.7 稳定性
葛根素、大豆苷、黄芩苷、汉黄芩苷、巴马汀、小檗碱、甘草酸、黄芩素、汉黄芩素的RSD分别为0.8%、0.7%、2.0%、0.6%、1.0%、1.8%、0.6%、1.1%、0.2%, 结果表明, 供试品溶液在24 h内稳定。
5 样品含量测定
取混合对照品溶液和10批葛根芩连汤供试品溶液, 按上述色谱条件进样分析, 按外标法以峰面积计算含量, 结果见表 3。
讨论
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葛根芩连汤为传统中医药的经典古方, 出自《伤寒论》, 君药为葛根, 臣药为黄芩与黄连, 使药为炙甘草, 主治下利身热、心烦躁扰、面赤口渴等。中药材质量的优劣与药材的产地、品种、栽培等密切相关[14], 故收集了葛根、黄芩、黄连和炙甘草各十批不同批次的药材饮片, 对指纹图谱的相似度进行了计算, 结果显示十批葛根芩连汤的相似度均大于0.99, 表明不同批次样品的相似度较高, 整体质量较为稳定。
中药煎煮的方法和方式直接影响了中药汤剂临床药效, 因此为了达到更高的提取效率, 对提取工艺进行了考察。根据文献的报道以及方解的研究, 葛根芩连汤的配比存在5∶3∶3∶2以及8∶3∶3∶2两种情况, 但由于《伤寒论》出自东汉时期, 古书中描写葛根用量为半斤、黄芩用量为三两、黄连用量为三两、炙甘草用量为二两, 而东汉时期半斤为八两, 故确定配比为8∶3∶3∶2, 确定配比后对葛根芩连汤的制备工艺进行了考察, 发现在浸泡时间为30 min、一煎和二煎时间分别为45和40 min、一煎加水量和二煎加水量分别为14和12倍量水时, 待测成分的含量能得到最大程度的提升, 精密量取1 mL复方汤剂后以75%甲醇定容至5 mL量瓶时, 色谱图基线平稳, 峰面积相对较高且干扰较少, 最终确定了本研究的提取工艺及前处理方法。本研究通过对提取工艺及前处理方法的考察, 提示在复方汤剂的煎煮过程中, 需进行标准化的提取方法, 从而保证产品的质量稳定均一。
中药的质量是中医临床疗效的基本前提和根本保证, 为了辨别中药质量的好坏, 首先要解决复杂样品的组分分析问题, 开发效果好、专属性强以及操作简便的分析方法, 从而建立起相应的质量标准[15]。中药质量标准应具备科学性、合理性、简便性和实用性[16]。在选择指标成分时, 遵循组方配伍及君臣佐使原则, 在君药中选择了葛根中含量较高的葛根素与大豆苷两种成分; 在选择臣药黄芩指标成分时, 发现黄芩中的黄芩苷与黄芩素、汉黄芩苷与汉黄芩素之间均为苷与苷元的关系, 苷是糖或糖的衍生物通过端基碳的半缩醛羟基或半缩酮羟基与非糖部分脱水缩合而成, 苷元就是苷中的非糖部分, 苷与苷元之间存在互相转化, 是整体与部分的关系, 所以在选择指标成分时, 将这四种成分均纳入含量测定的范围; 在臣药黄连中选择了活性效果好且含量高的小檗碱和巴马汀两种成分; 炙甘草作为使药起调和诸药的作用, 在全方中药量使用最小, 故选择甘草酸这一含量较高的成分作为指标性成分进行测定, 君药葛根同中国药典指标成分相比, 增加了大豆苷的测定, 臣药黄芩增加了汉黄芩苷、黄芩素和汉黄芩素的测定, 黄连增加了巴马汀的测定, 从而更加全面地对葛根芩连汤进行质量控制。目前现有关于葛根芩连汤液相色谱条件研究及含量测定研究中采用多波长[17,18]对其进行检测, 本研究在260 nm的单波长下, 基线较为平整, 各峰之间分离度良好, 能够同时完成指纹图谱的建立与9种成分的含量测定。本研究开发的液相条件具有洗脱方式简单、分析时间短和检测波长简便等优点, 为葛根芩连汤进一步质量控制和方法开发的建立提供了参考依据。
结论
收起
本实验建立了在同一波长下同时完成指纹图谱的研究及多成分含量测定的方法, 收集10批饮片所制备的葛根芩连汤质量一致性较好, 在此基础上利用该色谱方法对葛根芩连汤中9种有效成分进行了含量测定, 并进行了方法学考察, 发现10批葛根芩连汤中各成分的含量存在一定差异, 这可能是中药饮片在生长环境、采收时间、炮制工艺和贮藏条件等不同所引起的。综上, 本研究利用UHPLC方法建立了一套完整的质量分析方法, 从定性与定量两个方面为对葛根芩连汤的质量进行评价, 该方法具有专属性强、灵敏度高、操作简便等优点, 为葛根芩连汤的质量控制体系的完善提供参考, 为提高其整体质量的一致性提供依据, 从而确保葛根芩连汤在临床使用上的安全性与有效性。
作者贡献  : 谢娟主要进行了本文的实验设计、样品前处理条件研究、色谱方法建立、实验数据处理、撰写文章等; 唐琪对实验思路及文章修改提供了宝贵意见; 张盼对本文分析方法的建立、样品的制备及数据处理提供了帮助; 李欣对文献调研及实验数据分析处理提供了帮助; 毕开顺对本文的选题与设计、实验思路及方法建立提供了专业的指导; 李清对本文的选题与设计、实验思路、方法建立及文章修改提供了专业的指导。
利益冲突  : 无任何利益冲突。
基金
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  • 国家自然科学基金(81973464/H3203)
  • 国家自然科学基金(8210142956/H3203)
  • 国家自然科学基金(8210132176/H3410)
  • 沈阳市中青年人才科技创新项目(RC190505)
  • 辽宁省教育厅高校基础研究项目(LJKZ0929)
  • 辽宁省教育厅高校基础研究项目(LJKQZ2021033)
文献
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参考文献 引证文献
排序方式：
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2024年第59卷第8期
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文章信息
doi: 10.16438/j.0513-4870.2024-0201
  • 接收时间：2024-03-06
  • 首发时间：2025-11-26
  • 出版时间：2024-08-12
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  • 收稿日期：2024-03-06
  • 修回日期：2024-05-13
基金
国家自然科学基金(81973464/H3203)
国家自然科学基金(8210142956/H3203)
国家自然科学基金(8210132176/H3410)
沈阳市中青年人才科技创新项目(RC190505)
辽宁省教育厅高校基础研究项目(LJKZ0929)
辽宁省教育厅高校基础研究项目(LJKQZ2021033)
作者信息
    1.沈阳药科大学药学院, 辽宁 沈阳 110016
    2.南方医科大学药学院, 广东 广州 510515

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2种不同金属材料的力学参数
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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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