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Article(id=1222469813075693817, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469808688451726, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2019-0449, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1567612800000, receivedDateStr=2019-09-05, revisedDate=1573315200000, revisedDateStr=2019-11-10, acceptedDate=null, acceptedDateStr=null, onlineDate=1769389115828, onlineDateStr=2026-01-26, pubDate=1576080000000, pubDateStr=2019-12-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1769389115828, onlineIssueDateStr=2026-01-26, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1769389115828, creator=13701087609, updateTime=1769389115828, updator=13701087609, issue=Issue{id=1222469808688451726, tenantId=1146029695717560320, journalId=1189982191388893191, year='2019', volume='54', issue='12', pageStart='2141', pageEnd='2352', issueExtLink='null', onlineDate='null', pubDate='1576080000000', pubDateStr='2019-12-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1769389114782, creator='13701087609', updateTime=1769389604971, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1222471864748859926, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469808688451726, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1222471864748859927, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1222469808688451726, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2149, endPage=2154, ext={EN=ArticleExt(id=1222469813679673629, articleId=1222469813075693817, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Quality control of Shuanghuanglian preparations using an effect-constituent index, columnId=1222469813071503566, journalTitle=Acta Pharmaceutica Sinica, columnName=Special Reports: Biological Evaluation and Precise Control for the Quality of Traditional Chinese Medicines, runingTitle=null, highlight=null, articleAbstract=

We established a quality evaluation method for Shuanghuanglian preparations based on an effect-constituent index (ECI), which is guided by the clinical efficacy of Shuanghuanglian and a dose-efficacy correlation. An HPLC method was used to establish the quantitative fingerprint of Shuanghuanglian from different manufacturers and to determine the content of 10 fingerprint components, including baicalin, chlorogenic acid, forsythin, galuteolin, wogonin, forsythoside A, luteolin, caffeic acid, baicalein, and scutellarin. Using Staphylococcus aureus as biological model, the potency of Shuanghuanglian preparations was determined by antibiotic microbial assay. Using the method of PLC-DA, the efficacious antibacterial components were screened by "dose-efficacy" correlation analysis. According to the antibacterial potency and content of the antibacterial ingredients, combined with the method of the custom weight coefficient, ECI was calculated and verified. The results show that the antibacterial ECI can facilitate evaluation of the efficacy of Shuanghuanglian based on the composition of its contents, providing a new method for the quality control of traditional Chinese medicine.

, authors=null, authorsList=Hui ZHANG, Si-yu LI, Hong-ling FENG, Mu-rui LI, Zhi-dong PEI, Ting-guo KANG, authorCompany=null, correspAuthors=Zhi-dong PEI, Ting-guo KANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2019 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1222469814195573054, articleId=1222469813075693817, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=基于效应成分指数的双黄连制剂质量控制研究, columnId=1222469813352521949, journalTitle=药学学报, columnName=专题报道：中药质量生物评价与精准控制, runingTitle=null, highlight=null, articleAbstract=

建立一种以临床功效为导向的，将功效作用与成分含量关联的，基于效应成分指数的双黄连制剂质量控制方法。采用HPLC法建立双黄连制剂的定量指纹图谱，分别测定不同来源批次双黄连制剂中黄芩苷、绿原酸、咖啡酸、木犀草苷、木犀草素、野黄芩苷、汉黄芩素、黄芩素、连翘苷、连翘酯苷A共10个指纹成分的含量；借鉴抗生素微生物检定法，以金葡菌为生物模型，检测不同来源双黄连制剂的抗菌效价；通过偏最小二乘-判别分析法（PLC-DA）进行"量-效"相关性分析，筛选出双黄连制剂的抗菌功效成分；根据各抗菌功效成分的抗菌效价和含量，结合自定义权重系数分配方法，计算抗菌效应成分指数并加以验证。结果，采用效应成分指数可以实现通过成分的含量评价双黄连制剂的抗菌功效作用，能够反映制剂的质量，为中药质量控制提供新的研究思路和方法。

, authors=null, authorsList=张慧, 李思雨, 冯宏玲, 李穆睿, 裴志东, 康廷国, authorCompany=null, correspAuthors=裴志东, 康廷国, authorNote=null, correspAuthorsNote=
*裴志东, E-mail:;
康廷国, E-mail:
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China J Chin Mater Med (中国中药杂志), 2012, 37: 2913-2916., articleTitle=Study on quality evaluation of Sparganii Rhizoma by biopotency determination method, refAbstract=null), Reference(id=1222469820336034578, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469813075693817, doi=null, pmid=null, pmcid=null, year=2016, volume=47, issue=null, pageStart=1443, pageEnd=1457, url=http://en.cnki.com.cn/Article_en/CJFDTOTAL-ZCYO201609005.htm, language=null, rfNumber=[12], rfOrder=11, authorNames=Liu CX, Chen SL, Xiao XH, journalName=Chin Tradit Herb Drugs (中草药), refType=null, unstructuredReference= Liu CX , Chen SL , Xiao XH . A new concept on quality marker of Chinese materia medica:Quality control for Chinese medicinal products[J]. Chin Tradit Herb Drugs (中草药), 2016, 47: 1443-1457., articleTitle=A new concept on quality marker of Chinese materia medica:Quality control for Chinese medicinal products, refAbstract=null), Reference(id=1222469820436697880, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469813075693817, doi=null, pmid=null, pmcid=null, year=2015, volume=40, issue=null, pageStart=1027, pageEnd=1031, url=http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgzyzz201506008, language=null, rfNumber=[13], rfOrder=12, authorNames=Jiang H, Gao Y, Yang JM, journalName=China J Chin Mater Med (中国中药杂志), refType=null, unstructuredReference= Jiang H , Gao Y , Yang JM et al . Overview of traditional Chinese medicine quality evaluation method based on overall research[J]. 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A: Reference standard (324 nm); B: Reference standard (280 nm); C: Sample (324 nm); D: Sample (280 nm). 1: Chlorogenic acid; 2: Caffeic acid; 3: Forsythoside A; 4: Galuteolin; 5: Luteolin; 6: Scutellarin; 7: Forsythin; 8: Baicalin; 9: Baicalein; 10: Wogonin , figureFileSmall=kWAci/b9xO/wou5D2FTocQ==, figureFileBig=QC4EaACcpKVkz3sqMzaKyw==, tableContent=null), ArticleFig(id=1222469817928503899, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469813075693817, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
No. Batch Inhibitory zone D/mm PT (U·g-1 medicinal materials) FL/%
TL TM TH
S-1 130802 18.54 19.53 20.24 4.79 4.731
S-2 150409 16.83 17.94 18.74 3.94 5.462
S-3 1411003 18.11 18.98 19.87 4.28 8.843
S-4 150719 15.29 16.06 16.97 3.91 7.361
S-5 160426 16.05 16.97 18.01 4.23 6.482
S-6 150724 15.50 16.35 17.27 4.16 6.292
S-7 160305 15.58 16.39 17.31 4.04 9.613
S-8 151102 14.97 15.82 16.99 3.83 10.461
S-9 160624 15.48 16.20 17.16 3.86 7.427
S-10 160718 15.21 16.13 16.92 3.84 6.840
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Biological potency assay results of Shuanghuanglian from different manufacturers. TL, TM and TH represent low, medium, and high doses, respectively. FL: Fiducial limit

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No. Batch Inhibitory zone D/mm PT (U·g-1 medicinal materials) FL/%
TL TM TH
S-1 130802 18.54 19.53 20.24 4.79 4.731
S-2 150409 16.83 17.94 18.74 3.94 5.462
S-3 1411003 18.11 18.98 19.87 4.28 8.843
S-4 150719 15.29 16.06 16.97 3.91 7.361
S-5 160426 16.05 16.97 18.01 4.23 6.482
S-6 150724 15.50 16.35 17.27 4.16 6.292
S-7 160305 15.58 16.39 17.31 4.04 9.613
S-8 151102 14.97 15.82 16.99 3.83 10.461
S-9 160624 15.48 16.20 17.16 3.86 7.427
S-10 160718 15.21 16.13 16.92 3.84 6.840
), ArticleFig(id=1222469818167579251, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469813075693817, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Sample Chemical constituent assay/mg·g-1
S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10
Baicalin 8.145 8.098 8.082 8.178 8.254 8.321 8.287 8.047 8.138 8.259
Chlorogenic acid 2.747 2.282 2.535 1.827 1.594 2.293 1.955 1.793 1.847 1.737
Forsythin 0.253 0.273 0.242 0.228 0.241 0.274 0.213 0.274 0.251 0.279
Galuteolin 0.243 0.229 0.209 0.470 0.349 0.371 0.430 0.462 0.438 0.369
Wogonin 0.316 0.344 0.273 0.432 0.380 0.354 0.564 0.297 0.412 0.378
Forsythoside A 0.337 0.306 0.365 0.364 0.453 0.298 0.348 0.438 0.312 0.436
Luteolin 0.677 0.677 0.648 0.792 0.648 0.731 0.829 0.634 0.712 0.835
Caffeic acid 0.055 0.057 0.046 0.051 0.075 0.057 0.045 0.061 0.079 0.070
Baicalein 0.264 0.226 0.209 0.247 0.325 0.418 0.253 0.372 0.237 0.342
Scutellarin 0.355 0.179 0.198 0.197 0.267 0.161 0.187 0.212 0.181 0.205
), ArticleFig(id=1222469818301796988, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469813075693817, language=CN, label=Table 2, caption=

Chemical constituent assay results of Shuanghuanglian form different manufacturers

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Sample Chemical constituent assay/mg·g-1
S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10
Baicalin 8.145 8.098 8.082 8.178 8.254 8.321 8.287 8.047 8.138 8.259
Chlorogenic acid 2.747 2.282 2.535 1.827 1.594 2.293 1.955 1.793 1.847 1.737
Forsythin 0.253 0.273 0.242 0.228 0.241 0.274 0.213 0.274 0.251 0.279
Galuteolin 0.243 0.229 0.209 0.470 0.349 0.371 0.430 0.462 0.438 0.369
Wogonin 0.316 0.344 0.273 0.432 0.380 0.354 0.564 0.297 0.412 0.378
Forsythoside A 0.337 0.306 0.365 0.364 0.453 0.298 0.348 0.438 0.312 0.436
Luteolin 0.677 0.677 0.648 0.792 0.648 0.731 0.829 0.634 0.712 0.835
Caffeic acid 0.055 0.057 0.046 0.051 0.075 0.057 0.045 0.061 0.079 0.070
Baicalein 0.264 0.226 0.209 0.247 0.325 0.418 0.253 0.372 0.237 0.342
Scutellarin 0.355 0.179 0.198 0.197 0.267 0.161 0.187 0.212 0.181 0.205
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Var ID (Primary) VIP
Scutellarin 1.680 46
Baicalin 1.228 42
Forsythoside A 1.190 33
Chlorogenic acid 1.064 05
Luteolin 0.949 583
Wogonin 0.814 821
Caffeic acid 0.752 117
Galuteolin 0.651 074
Baicalein 0.534 419
Forsythin 0.526 433
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Correlation analysis of chemical constituent and biological potency assay

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Var ID (Primary) VIP
Scutellarin 1.680 46
Baicalin 1.228 42
Forsythoside A 1.190 33
Chlorogenic acid 1.064 05
Luteolin 0.949 583
Wogonin 0.814 821
Caffeic acid 0.752 117
Galuteolin 0.651 074
Baicalein 0.534 419
Forsythin 0.526 433
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Sample Effect-constituent index Real antibacterial potency (U·g-1 medicinal materials)
S1 1.126 4.79
S2 0.969 3.94
S3 1.035 4.28
S4 0.936 3.91
S5 0.970 4.23
S6 0.972 4.16
S7 0.951 4.04
S8 0.958 3.83
S9 0.911 3.86
S10 0.958 3.84
), ArticleFig(id=1222469818767364773, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1222469813075693817, language=CN, label=Table 4, caption=

Correlation analysis between ECI and biological potency assay of Shuanghuanglian from different manufacturers

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Sample Effect-constituent index Real antibacterial potency (U·g-1 medicinal materials)
S1 1.126 4.79
S2 0.969 3.94
S3 1.035 4.28
S4 0.936 3.91
S5 0.970 4.23
S6 0.972 4.16
S7 0.951 4.04
S8 0.958 3.83
S9 0.911 3.86
S10 0.958 3.84
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基于效应成分指数的双黄连制剂质量控制研究
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张慧 , 李思雨 , 冯宏玲 , 李穆睿 , 裴志东 * , 康廷国 *
药学学报 | 专题报道：中药质量生物评价与精准控制 2019,54(12): 2149-2154
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药学学报 |专题报道：中药质量生物评价与精准控制 2019 , 54 (12) : 2149 -2154
基于效应成分指数的双黄连制剂质量控制研究
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张慧, 李思雨, 冯宏玲, 李穆睿, 裴志东* , 康廷国*
作者信息
  • 辽宁中医药大学药学院, 辽宁 大连 116600
通讯作者:
*裴志东, E-mail:;
康廷国, E-mail:
Quality control of Shuanghuanglian preparations using an effect-constituent index
Hui ZHANG, Si-yu LI, Hong-ling FENG, Mu-rui LI, Zhi-dong PEI* , Ting-guo KANG*
Affiliations
  • College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China
出版时间: 2019-12-12 doi: 10.16438/j.0513-4870.2019-0449
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摘要
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建立一种以临床功效为导向的，将功效作用与成分含量关联的，基于效应成分指数的双黄连制剂质量控制方法。采用HPLC法建立双黄连制剂的定量指纹图谱，分别测定不同来源批次双黄连制剂中黄芩苷、绿原酸、咖啡酸、木犀草苷、木犀草素、野黄芩苷、汉黄芩素、黄芩素、连翘苷、连翘酯苷A共10个指纹成分的含量；借鉴抗生素微生物检定法，以金葡菌为生物模型，检测不同来源双黄连制剂的抗菌效价；通过偏最小二乘-判别分析法（PLC-DA）进行"量-效"相关性分析，筛选出双黄连制剂的抗菌功效成分；根据各抗菌功效成分的抗菌效价和含量，结合自定义权重系数分配方法，计算抗菌效应成分指数并加以验证。结果，采用效应成分指数可以实现通过成分的含量评价双黄连制剂的抗菌功效作用，能够反映制剂的质量，为中药质量控制提供新的研究思路和方法。

双黄连制剂  /  效应成分指数  /  抗菌效价  /  定量指纹图谱  /  质量控制

We established a quality evaluation method for Shuanghuanglian preparations based on an effect-constituent index (ECI), which is guided by the clinical efficacy of Shuanghuanglian and a dose-efficacy correlation. An HPLC method was used to establish the quantitative fingerprint of Shuanghuanglian from different manufacturers and to determine the content of 10 fingerprint components, including baicalin, chlorogenic acid, forsythin, galuteolin, wogonin, forsythoside A, luteolin, caffeic acid, baicalein, and scutellarin. Using Staphylococcus aureus as biological model, the potency of Shuanghuanglian preparations was determined by antibiotic microbial assay. Using the method of PLC-DA, the efficacious antibacterial components were screened by "dose-efficacy" correlation analysis. According to the antibacterial potency and content of the antibacterial ingredients, combined with the method of the custom weight coefficient, ECI was calculated and verified. The results show that the antibacterial ECI can facilitate evaluation of the efficacy of Shuanghuanglian based on the composition of its contents, providing a new method for the quality control of traditional Chinese medicine.

Shuanghuanglian preparation  /  effect-constituent index  /  antibacterial potency  /  quantitative fingerprint chromatogram  /  quality control
张慧, 李思雨, 冯宏玲, 李穆睿, 裴志东, 康廷国. 基于效应成分指数的双黄连制剂质量控制研究. 药学学报, 2019 , 54 (12) : 2149 -2154 . DOI: 10.16438/j.0513-4870.2019-0449
Hui ZHANG, Si-yu LI, Hong-ling FENG, Mu-rui LI, Zhi-dong PEI, Ting-guo KANG. Quality control of Shuanghuanglian preparations using an effect-constituent index[J]. Acta Pharmaceutica Sinica, 2019 , 54 (12) : 2149 -2154 . DOI: 10.16438/j.0513-4870.2019-0449
正文
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双黄连制剂是由金银花、黄芩、连翘3种中药组成, 具有疏风解表, 清热解毒的作用, 目前在临床应用广泛, 主要用于外感风热所致的感冒, 症见发热, 咳嗽, 咽痛等[1]。现代药理研究证实, 其具有抗菌[2]、抗病毒[3]、解热抗炎[4]等作用, 对金葡菌等多种革兰氏阳性[2, 5]及阴性菌[2, 5]均具有较强的抑制作用, 是临床抗菌抗病毒的代表性药物, 其药效明确, 化学成分清楚。2015版《中国药典》收载的质量标准仅通过测定黄芩中的黄芩苷、连翘中的连翘苷、金银花中的绿原酸3个成分的含量来控制制剂的质量[2], 由于中药含有多种化学成分, 具有整体性特点, 是通过多成分多靶点协调起效而发挥药效作用, 为此测定单一或几个成分含量不能真正反映制剂的临床疗效, 不能准确控制中药的质量。
中药“效应成分指数(effect-constituent index, ECI)”是基于化学成分分析和效应检测共同加权的质量评价指标, 其可通过中药中活性成分的“量-效”关系方程来建立。这一指标的建立, 不仅能使中药质量标准在目前可控的基础上, 实现与药效密切关联, 为临床应用提供参考, 同时能使不同成分所关联的药效大小得以表征, 使中药质量评价更加综合全面[6, 7]。
基于上述, 本论文建立一种以临床功效为导向的, 将功效作用与成分含量关联的, 基于效应成分指数的双黄连制剂质量控制方法。其是借鉴化药抗生素测定的生物效价经典方法, 以金葡菌为生物模型, 建立反映双黄连制剂整体药效的抗菌效价测定法[8-11]; 采用HPLC法建立双黄连制剂中共有指纹峰的含量测定方法; 通过偏最小二乘-判别分析法(PLC-DA)进行“量-效”相关性分析, 以筛选出功效成分, 并通过功效成分与抗菌效价测定结果, 结合自定义权重系数分配方法, 计算效应成分指数, 并以该指数为指标评价该制剂的质量。本论文旨在为中药质量控制研究提供新思路和新方法。
材料与方法
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仪器与试药  高效液相色谱仪(Agilent 1260, 四元泵, DAD检测器, 安捷伦科技有限公司); 十万分之一电子天平(METTLER AB135-S, 瑞士); 数控超声波清洗器(KQ-250D, 昆山市超声仪器有限公司); 二氧化碳培养箱(SANYO MCO175, 日本三洋公司); 生物洁净工作台(BCN-1360B, 北京东联哈尔仪器制造有限公司); 立式压力蒸汽灭菌器(BXM-30R, 上海博迅实业有限公司医疗设备厂)。
绿原酸(批号:110753-201314, 含量96.6%)、黄芩苷(批号: 110715-201117, 含量91.7%)、庆大霉素(批号: 130326-201015, 含量630 U·mg-1)、金葡菌[CMCC (B) 26003]、大肠埃希菌[CMCC (B) 44103]、变形杆菌[CMCC (B) 49027], 均购自中国食品药品检定研究院; 咖啡酸(批号: Y17D6C7672)、连翘苷(批号: P20J7F9279)、连翘酯苷A (批号: HN1116XA13)、木犀草苷(批号: P06F6R1)、黄芩素(批号: C02A6Y1)、野黄芩苷(批号: KS0902CA14)、汉黄芩素(批号: P1906F4649)、木犀草素(批号Y01D6S6815), 均购自上海源叶生物科技有限公司, 含量≥98%;铜绿色假单胞菌[CMCC (B) 10104]、肺炎双球菌[CMCC (B) 29010], 均购自中国医学细菌保藏管理中心; 营养肉汤(批号: 20170215, 北京索莱宝科技有限公司), 营养琼脂培养基(批号: 20170103, 北京奥博星生物技术有限责任公司)。
双黄连片(S-1, 批号150802, 药都制药集团股份有限公司; S-2, 批号1511003, 哈尔滨圣泰生物制药有限公司; S-3, 批号150409, 陕西白鹿制药股份有限公司); 双黄连颗粒(S-4, 批号150719;S-5, 批号160426; S-6, 批号150724; S-7, 批号160305; S-8, 批号151102; S-9, 批号160624; S-10, 批号160718), 均由哈尔滨儿童制药厂有限公司提供。
抗菌效价
效价测定用溶液的制备  精密称取庆大霉素标准品, 用pH 7.8的磷酸盐缓冲液稀释制成12.53 μg·mL-1标准品溶液作为贮备液, 分别按1:0.8剂距用pH 7.8的灭菌磷酸盐缓冲溶液稀释制成8.02、6.42、5.14 μg·mL-1的对照品溶液。分别精密称取双黄连片和颗粒, 用pH 7.8的灭菌磷酸盐缓冲溶液溶解, 超声20 min, 放冷, 制成200.11 mg·mL-1片剂和500.29 mg·mL-1颗粒剂贮备液, 分别按1:0.8剂距稀释制成为片剂65.57、81.97和102.46 mg·mL-1和颗粒剂204.92、256.15和320.18 mg·mL-1的供试品溶液。
菌悬液的制备  取金葡菌的营养琼脂培养物, 接种在营养琼脂, 并在37 ℃下培养22 h, 根据菌群生长状态, 取3代细菌的培养物, 用无菌水洗下菌苔, 制成浊度为0.5麦氏比浊度的菌悬液, 备用。
双碟的制备  取平底双碟, 分别注入经加热灭菌后融化的培养基20 mL, 使其在碟底内均匀摊布并凝固, 作为底层。另取灭菌培养基加热融化后, 放冷至48~50 ℃, 加入1%上述制备的菌悬液, 摇匀, 每一碟中分别加入培养基5 mL, 均匀摊布, 作为菌层, 冷却后, 在每一双碟中均匀等距放置6个牛津杯, 备用。
抗菌效价检测  采用三剂量法进行实验。分别取对照品溶液和供试品溶液按照剂距1:0.8随机制成高、中、低3个不同剂量组, 取上述制备的双碟, 在每一双碟中的6个牛津杯中, 其中3个分别滴加高、中、低3个浓度的庆大霉素磷酸盐溶液, 另外3个牛津杯分别滴加高、中、低3个浓度的双黄连供试品溶液, 加样量均为0.1 mL, 置于37 ℃恒温培养箱中培养18 h后, 取出, 精密测量各个抑菌圈直径, 运用生物检定统计法, 采用生物检定统计软件BS2000版, 对样品进行可靠性测验及效价计算。
定量指纹图谱
色谱条件  色谱柱为Inertsil ODS-2色谱柱(150 mm×4.6 mm, 5 μm); 连翘苷检测波长280 nm, 其他成分检测波长324 nm; 柱温30 ℃; 流速1.0 mL·min-1; 进样量10 μL; 流动相为甲醇(A)-0.1%甲酸水溶液(B), 梯度洗脱条件为0~10min, 20%~25% A; 10~35 min, 25%~40% A; 35~50 min, 40%~50% A; 50~55 min, 50%~20% A。
定量测定用溶液的制备  取对照品适量加50%甲醇定容, 制成绿原酸0.0842 mg·mL-1、连翘苷0.025 4 mg·mL-1、连翘酯苷A 0.012 9 mg·mL-1、黄芩苷0.050 2 mg·mL-1、黄芩素0.047 0 mg·mL-1、汉黄芩素0.051 4 mg·mL-1、野黄芩苷0.001 0 mg·mL-1、芦丁0.005 8 mg·mL-1、木犀草苷0.051 1 mg·mL-1、咖啡酸0.005 0 mg·mL-1对照品溶液; 另取双黄连制剂, 研细, 精密称定适量, 置50 mL量瓶中, 加50%甲醇适量, 超声处理(功率250 W, 频率33 kHz) 20 min, 放冷, 加50%甲醇稀释至刻度, 摇匀, 制成相当于生药2.83 g·mL-1的供试品溶液。
定量指纹图谱的建立和测定  取10批双黄连制剂供试品溶液10 μL注入液相色谱仪进行测定, 按外标法测定各标定成分含量, 并将测定图谱导入中国药典委员会相似度评价软件(2012版), 以S-1为参照图谱, 进行多点校正, 自动匹配, 按中位数法生成对照图谱R, 对10批制剂指纹图谱进行相似度评价。
结果
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1 抗菌效价的检测
1.1 菌种的筛选
以双黄连片0.1 g·mL-1, 双黄连颗粒0.25 g·mL-1作为原液, 采用试管2倍稀释法, 取无菌试管10支, 每管加入MH肉汤1 mL, 在第1管加入药液1.0 mL混匀, 然后吸取1 mL至第2管, 混匀后再吸取1 mL至第3管, 如此按倍数稀释后, 每管再分别加入金葡菌、大肠埃希菌、铜绿色假单胞菌、肺炎双球菌、变形杆菌的菌液各1 mL, 使每管菌液终浓度约5×105 cfu·mL-1, 37 ℃培养18~24 h, 检测最低抑菌浓度(MIC), 并采用平板活菌计数法, 取MIC终点以上未长菌的各管培养液0.1 mL转种到营养琼脂平皿上, 继续培养, 检测最低杀菌浓度(MBC)。结果, 双黄连制剂对5种致病菌均有一定抑菌作用, 其中双黄连片对上述顺序排列的5种菌种MIC/MBC依次为1.563/3.125、6.25/6.25、3.125/6.25、12.50/12.50、6.25/12.50 mg·mL-1, 双黄连颗粒MIC/MBC依次为7.81/15.63、31.25/31.25、15.63/31.25、62.50/62.50、31.25/62.50 mg·mL-1, 提示双黄连制剂对金葡菌抑菌效果明显, 为此选择金葡菌为药效抑菌菌种。
1.2 剂量关系考察
分别取对照品和供试品贮备液按1:0.8剂距, 用磷酸盐缓冲液分别稀释至不同浓度溶液, 测定抑菌圈直径, 并以抑菌圈直径均值为纵坐标, 以对照品或供试品浓度对数值为横坐标, 进行线性回归。结果对照品回归方程为y = 1.267 7 x + 13.423, 双黄连片剂和颗粒回归方程分别为y = 19.035 x - 16.243、y = 10.77 x - 6.251 7, 其中庆大霉素在2.10~12.53 μg·mL-1内、双黄连片在41.97~200.11mg·mL-1和颗粒在83.93~500.29mg·mL-1内浓度对数值与抑菌圈直径呈良好线性关系, 三者R均大于0.99。
根据《中国药典》四部附录“抗生素微生物检定法”规定, 抗菌效价检测时, 对照品溶液的中心浓度所致的抑菌圈直径应在15~18 mm之间, 依据这个抑菌圈直径范围, 结合上述剂量关系考察的抑菌圈直径所对应的样品浓度的线性结果, 确定了庆大霉素对照品和双黄连制剂的测试浓度。
1.3 碟内精密度实验
取制备的3个浓度的双黄连片供试品溶液, 每种浓度分别放置于同一碟内, 按三剂量法重复测定6次, 精密测量各抑菌圈直径D值, 计算RSD值, 结果3种浓度的抗菌效价的RSD值分别为4.16%、4.16%和3.73%, 精密度良好。
1.4 碟间精密度实验
取双黄连片供试品溶液, 分别稀释至上述3种浓度, 每种浓度分别加于不同双碟内, 按上述条件重复测定6次, 精密测量各抑菌圈直径D值, 计算RSD值, 结果3种浓度碟间精密度的RSD值分别3.22%、3.49%和4.13%, 低于5%, 具有较好的精密度。
1.5 中间精密度
3个不同实验人员, 在不同时间点, 取双黄连片稀释至上述3种浓度, 按抗菌效价测定法, 重复测定6次, 精密测量各抑菌圈直径D值, 计算效价值。结果中间精密度的RSD值为4.79%, 低于5%, 中间精密度符合要求。
1.6 稳定性实验
取供试品溶液, 按中间精密度浓度分别于制备后0、2、4、6、8和12 h按上述条件测定各抑菌圈直径D, 计算抗菌效价。结果表明, RSD为3.26%, 供试品溶液放置12 h内稳定性良好。
1.7 重复性实验
取同一批次双黄连片, 按供试品溶液制备方法制备6份, 按抗菌效价测定法检测各抑菌圈直径D值, 计算效价值和6个样品效价的RSD, 结果显示RSD值为4.19%, 3个剂量组的重复性良好。
1.8 加样回收率实验
分别按1:0.8剂距的1/2量制备双黄连片供试品溶液的3个剂量组, 并分别加入1/2量的8.02、6.42和5.14 μg·mL-1对照品溶液, 按抗菌效价测定法测定样品效价值, 计算回收率。结果显示, 本品回收率在94.69%~103.59%之间, 3个剂量组的平均回收率及RSD分别为98.65% ± 3.52%、100.42% ± 3.11%和99.13% ± 2.5%, 符合规定。
1.9 抗菌效价测定
取不同厂家10批双黄连制剂, 按照“效价测定用溶液的制备”和“抗菌效价检测”项下方法制备供试品溶液和对照品溶液, 采用生物检定统计程序BS2000版中的(3·3)法进行可靠性测验、效价计算和实验误差估计。结果显示, 10批不同厂家双黄连制剂的效价值在3.83~4.28 (U·g-1生药)之间, 说明不同厂家及批次的双黄连制剂存在质量差异。结果见表 1。
2 定量指纹图谱的测定
2.2.1 指纹图谱的建立
对10个不同厂家的双黄连制剂进行指纹图谱的测定, 通过中药色谱指纹图谱相似度评价系统2012版进行了共有峰评价, 确定了14个共有峰, 10批双黄连制剂指纹图谱的相似度在0.93~0.98之间。通过对照品核对, 鉴定了其中10个指纹峰, 分别是黄芩苷、绿原酸、咖啡酸、木犀草苷、木犀草素、野黄芩苷、汉黄芩素、黄芩素、连翘苷、连翘酯苷A。
2.2.2 指纹图谱的含量测定
取10个不同厂家双黄连制剂, 按“定量指纹图谱测定方法”分别在324和280 nm项下测定样品的峰面积, 对指认的10个共有指纹峰进行了含量测定。结果显示, 不同厂家黄芩苷含量基本稳定, 均约8 mg·g-1药材, 其他9个成分有一定差异, 10个成分的总量差异较小, 均约12 mg·g-1药材, 在双黄连制剂中以黄芩苷的含量最高。结果见图 1, 表 2。
3 PLS-DA分析筛选功效成分
应用SIMCA-P软件, 以测得10个不同厂家双黄连制剂的抗菌效价作为因变量, 10个成分的含量为自变量, 采用偏最小二乘-判别分析(PLS-DA)法对成分的含量与抗菌效价进行相关性分析, 将因变量根据效价的高低, 分成较高(S-1)、高(S-4、S-5、S-10)、中(S-2、S-6、S-7)、低(S-3、S-8、S-9)共4组, 再以10个成分的含量作为自变量, 进行统计分析。结果所建立的PLS-DA模型中变量的解释度R2Y为0.911, 模型的预测度Q2为0.862, 均接近1, 表明所建模型可靠。通过模型给出的权重(VIP)值筛选功效成分, 成分VIP值大于1, 表明与抗菌效价相关, 且其越大则对抑菌作用贡献越大。结果野黄芩苷、绿原酸、黄芩苷、连翘酯苷A与制剂的抗菌效价有明显相关性, VIP值均大于1, 其相关顺序为野黄芩苷 > 绿原酸 > 黄芩苷 > 连翘酯苷A, 说明这4个成分的含量与制剂的质量和临床疗效相关, 为效应成分。结果见表 3。
4 效应成分指数的构建
4.1 效应成分抗菌效价测定
按双黄连制剂的抗菌效价检测方法, 以庆大霉素为对照, 取野黄芩苷、绿原酸、黄芩苷、连翘酯苷A共4个功效成分, 分别用pH 7.8磷酸盐缓冲溶液稀释, 采用三剂量管碟法分别测定功效成分抗菌效价。结果显示, 野黄芩苷、绿原酸、黄芩苷、连翘酯苷A的抗菌效价分别为144.90、49.56、18.59、126.62 U·g-1。
4.2 效应成分指数的建立
采用自定义权重系数的方法, 根据4个功效活性成分的抗菌效价, 对各功效成分的药效权重进行分配。公式如下:
${W_i} = \frac{{{P_T}}}{{\mathop \sum \nolimits_{i = 1}^n {P_T}}}$
其中, Wi为各功效成分的权重系数, PT为以金葡菌为生物模型, 以庆大霉素效价为参比获得的4个功效成分的抗菌效价值, n为成分个数, 通过公式可得到野黄芩苷、绿原酸、黄芩苷、连翘酯苷A的药效权重系数分别为0.427、0.146、0.055和0.373。
效应成分指数通过综合指数评价法来进行反映, 公式如下:
${Z_{\rm{m}}} = 100 \times \mathop \sum \limits_{i = 1}^n {W_i} \times {X_i}$
其中Zm为不同厂家双黄连制剂的“效应成分指数”, Xi为不同来源双黄连制剂中功效成分的含量。将公式(1)和公式(2)合并, “效应成分指数”公式如下:
$\begin{array}{*{20}{c}}{{Z_{\rm{m}}} = 100 \times (0.427 \times {X_{{\rm{SCU}}}} + 0.373 \times {X_{{\rm{FOR}}}} + }\\{{\rm{}}0.146 \times {X_{{\rm{CHL}}}} + 0.055 \times {X_{{\rm{BAI}}}})}\end{array}$
由公式可知, 野黄芩苷权重系数最大, 说明其对药效贡献较大; 而黄芩苷权重系数最小, 其对药效贡献最小。现有的双黄连制剂质量标准中主要检测绿原酸、黄芩苷、连翘苷3个成分, 对制剂药效贡献最大的野黄芩苷未收入标准, 而与制剂疗效无关的连翘苷却列入标准中, 提示现有质量标准不能真正反映临床功效。
4.3 效应成分指数的验证
以庆大霉素为对照, 分别检测不同厂家各批次双黄连制剂的抗金葡菌的抗菌效价, 同时采用上述定量指纹图谱色谱条件分别测定不同厂家样品的4个功效成分的含量, 并将测得的含量值代入上述效应成分指数公式中, 计算相应的“效应成分指数”。采用SPSS 24.0统计分析软件, 对实测抗菌效价值与效应成分指数进行相关性分析, 结果见表 4。结果显示, 不同厂家的双黄连制剂抗菌效价实测值与效应成分指数相关性为0.919, 具有显著正相关性(P < 0.001), 即效应成分指数越大, 制剂的抗菌效价就越大, 由此证实效应成分指数可以评价制剂的质量。
讨论
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目前中药质量控制方法多以测定一种或几种成分的含量来评价药材的质量, 定量成分指标的确定主要以通过成分的药效高通量筛选来确定, 脱离中药多成分协同起效的整体性特点, 不能真正的反映临床的疗效, 评价药材的质量, 为此如何寻找到可代表中药疗效, 且反映中药整体性特点的质量评价方法, 是现今中药质量研究的难点和热点[12, 13]。为此, 本论文尝试建立一种以临床功效为导向的, 将功效作用与成分含量关联的, 基于效应成分指数的双黄连制剂的质量控制方法。
为评价双黄连制剂的功效作用, 本研究借鉴化学药品质量检测中抗生素微生物检定法-管碟法的经典方法, 以金葡菌为生物模型, 以庆大霉素为参比效价, 建立测定双黄连制剂抗菌效价的方法, 其抗菌效价的高低能够客观评价制剂的临床功效。实验结果显示, 建立的方法精密度、重复性、回收率等满足要求, 方法稳定, 10批不同来源的双黄连制剂的抗菌效价有一定差异, 每个单位剂量的效价以庆大霉素效价计, 在3.83~4.28 (U·g-1生药)之间。
为测定双黄连制剂中共有指纹成分的含量, 本论文建立了定量指纹图谱的测定方法。采用HPLC法建立双黄连制剂的化学成分指纹图谱, 并测定与制剂抗菌抗病毒临床功效相关的10个指纹峰的含量, 实验结果显示, 10个不同厂家的指纹峰含量差异明显, 但黄芩苷含量基本一致, 其他成分有一定差异性。
为确定与临床功效相关的药效成分, 采用偏最小二乘-判别分析(PLS-DA)法, 分别对双黄连制剂抗菌效价和定量指纹峰含量进行相关性分析, 结果显示10个化学成分中仅有野黄芩苷、绿原酸、黄芩苷、连翘酯苷A与制剂的抗菌效价有显著相关性, 为此这4个成分可作为评价制剂质量的效应成分。
采用自定义权重系数的方法, 结合4种与功效相关成分的抗菌效价, 计算各功效成分的药效权重系数, 并测定各功效成分的含量, 以二者为参数建立效应成分指数的计算公式, 通过该指数可以评价制剂的质量。功效成分指数将功效和含量关联, 通过验证, 证实双黄连制剂的实测抗菌效价与功效成分指数呈现正相关, 相关系数大于0.9 (P < 0.001), 说明效应成分指数可反映药材的临床功效, 可以真正的评价药材质量。
本研究建立的基于功效成分指数的双黄连制剂质量评价的方法, 是以临床功效为导向, 将功效作用与成分含量关联的一种质量评价方法, 通过功效成分指数可以评价制剂的质量, 反映制剂的临床功效, 本研究为中药质量控制提供了新的研究思路。
基金
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  • 国家自然科学基金资助项目(81874338)
  • 辽宁省教育厅优秀人才（第一层次）基金资助项目(LR2015042)
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2019年第54卷第12期
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doi: 10.16438/j.0513-4870.2019-0449
  • 接收时间：2019-09-05
  • 首发时间：2026-01-26
  • 出版时间：2019-12-12
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  • 收稿日期：2019-09-05
  • 修回日期：2019-11-10
基金
国家自然科学基金资助项目(81874338)
辽宁省教育厅优秀人才（第一层次）基金资助项目(LR2015042)
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    辽宁中医药大学药学院, 辽宁 大连 116600

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*裴志东, E-mail:;
康廷国, E-mail:
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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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