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Article(id=1210517369538081775, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210517366081975259, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2022-0820, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1656950400000, receivedDateStr=2022-07-05, revisedDate=1660752000000, revisedDateStr=2022-08-18, acceptedDate=null, acceptedDateStr=null, onlineDate=1766539431223, onlineDateStr=2025-12-24, pubDate=1668182400000, pubDateStr=2022-11-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766539431223, onlineIssueDateStr=2025-12-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766539431223, creator=13701087609, updateTime=1766539431223, updator=13701087609, issue=Issue{id=1210517366081975259, tenantId=1146029695717560320, journalId=1189982191388893191, year='2022', volume='57', issue='11', pageStart='3259', pageEnd='3450', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1766539430399, creator=13701087609, updateTime=1766539608198, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1210518111875363690, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210517366081975259, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1210518111875363691, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1210517366081975259, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=3411, endPage=3418, ext={EN=ArticleExt(id=1210517370926395452, articleId=1210517369538081775, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Establishment and application of a rapid quality inspection method for Indigo Naturalis based on quantitative portrayal of water testing process, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

A new rapid, quality control method based on quantitative water tests has been established for the quality evaluation of Indigo Naturalis. The Turbiscan stability index (TSI) of 26 batches of Indigo Naturalis was measured by a stability analyzer. The parameters, including the method by which the ingredients are added, their particle size, amount, and the testing temperature, were systematically optimized and the methodological indexes such as repeatability and stability were determined. The content of indigo and indirubin in 26 batches of Indigo Naturalis was determined by high performance liquid chromatography and the total ash was measured. The correlation analysis between the active ingredients, total ash content and TSI value of Indigo Naturalis was determined by SPSS 26.0 and Origin 2021. This research shows that the best way to prepare samples for testing is to add 0.2 g of Indigo Naturalis powder which has passed through a No. 7 sieve but failed to pass through a No. 9 sieve to a glass bottle containing 20 mL pure water by a funnel and scan at 25 ℃ with a stability analyzer. Consistency analysis showed that the content ranking of indigo and indirubin is opposite to the TSI value, and the content ranking of total ash is generally consistent with the TSI value. Correlation analysis showed that the correlation coefficients of indigo and indirubin content and TSI value were -0.850 and -0.801, respectively, and R2 was 0.72 and 0.64. The correlation coefficient between total ash content and TSI value was 0.724, R2 was 0.52. Using the change in TSI value of Indigo Naturalis powder in water, this study establishes the range of classification of Indigo Naturalis decoction pieces and the content of relevant components, which can be used to authenticate Indigo Naturalis and evaluate its quality.

, correspAuthors=Run-chun XU, Ding-kun ZHANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2022 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=Xue-mei LIU, Ya-nan HE, Fang WANG, Ming YANG, Hai ZHANG, Xiang-bo YANG, Li HAN, Run-chun XU, Ding-kun ZHANG), CN=ArticleExt(id=1210517372016914573, articleId=1210517369538081775, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=基于水试过程定量刻画的青黛质量快检方法的建立与应用, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

建立一种基于定量水试的青黛质控快检新方法, 用于青黛的质量优劣评价。采用稳定性分析仪测定26批青黛粉末的Turbiscan稳定性参数(Turbiscan stability index, TSI), 系统优化加样方式、加样粒径、加样量、检测温度等参数, 并对重复性、稳定性等方法学指标进行考察。采用高效液相法测定26批青黛中靛蓝、靛玉红的含量, 并测定总灰分含量。运用SPSS 26.0、Origin 2021软件对青黛有效成分、总灰分含量与TSI值进行相关性分析。研究表明, 青黛供试品的最佳加样方式为: 取过七号筛不过九号筛的青黛粉末0.2g, 以漏斗加样方式装入含20mL纯水的玻璃瓶, 用稳定性分析仪在25℃下扫描。一致性分析表明, 靛蓝、靛玉红的含量排序与TSI值总体上呈相反趋势, 总灰分含量排序与TSI值总体上一致。相关性分析表明, 靛蓝、靛玉红含量与TSI值呈负相关, 相关系数分别为-0.850、-0.801, R2分别为0.72、0.64。总灰分值与TSI值存在正相关, 相关系数为0.724, R2为0.52。根据青黛粉末入水后的TSI值变化, 该研究构建了青黛饮片等级划分的范围及其对应成分含量, 可以判断青黛真伪以及对青黛进行分级评价, 为青黛快速鉴别方法提供参考依据。

, correspAuthors=许润春, 张定堃, authorNote=null, correspAuthorsNote=
*许润春, E-mail: ;
张定堃, E-mail:
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A: No. S9; B: No. S23; C: No. S25; D: No. S4 , figureFileSmall=2XbDR8YQ53zJjn9gp9xYUw==, figureFileBig=jS+8zR15GOvp/Nj6iZK0Ag==, tableContent=null), ArticleFig(id=1210517380191613470, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517369538081775, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
No.Batch No.Place of originSample
S120150402Xianyou, FujianIndigo Naturalis
S220170916Xianyou, FujianIndigo Naturalis
S320190414Jiangyou, SichuanIndigo Naturalis
S420180305Xianyou, FujianIndigo Naturalis
S520160425Xianyou, FujianIndigo Naturalis
S620200414Yaan, SichuanIndigo Naturalis
S720200523Yaan, SichuanIndigo Naturalis
S820191114Jiangyou, SichuanIndigo Naturalis
S920210501Xianyou, FujianPurified Indigo Naturalis
S1020160912Xianyou, FujianIndigo Naturalis
S1120210515Xianyou, FujianPurified Indigo Naturalis
S1220210418Xianyou, FujianPurified Indigo Naturalis
S1320211106Xianyou, FujianPurified Indigo Naturalis
S1420210412Xianyou, FujianPurified Indigo Naturalis
S1520210311Xianyou, FujianPurified Indigo Naturalis
S1620211007Xianyou, FujianPurified Indigo Naturalis
S1720211213Xianyou, FujianPurified Indigo Naturalis
S1820210407Xianyou, FujianPurified Indigo Naturalis
S1920210327Xianyou, FujianPurified Indigo Naturalis
S2020191113Yaan, SichuanIndigo Naturalis
S2120180301Xianyou, FujianIndigo Naturalis
S2220210114Yaan, SichuanIndigo Naturalis
S2320180521Jiangyou, SichuanIndigo Naturalis
S2420211223Jiangyou, SichuanIndigo Naturalis
S2520161012Xianyou, FujianIndigo Naturalis
S2620191128Xianyou, FujianIndigo Naturalis
), ArticleFig(id=1210517380313248291, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517369538081775, language=CN, label=Table 1, caption=

Sample information of Indigo Naturalis

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No.Batch No.Place of originSample
S120150402Xianyou, FujianIndigo Naturalis
S220170916Xianyou, FujianIndigo Naturalis
S320190414Jiangyou, SichuanIndigo Naturalis
S420180305Xianyou, FujianIndigo Naturalis
S520160425Xianyou, FujianIndigo Naturalis
S620200414Yaan, SichuanIndigo Naturalis
S720200523Yaan, SichuanIndigo Naturalis
S820191114Jiangyou, SichuanIndigo Naturalis
S920210501Xianyou, FujianPurified Indigo Naturalis
S1020160912Xianyou, FujianIndigo Naturalis
S1120210515Xianyou, FujianPurified Indigo Naturalis
S1220210418Xianyou, FujianPurified Indigo Naturalis
S1320211106Xianyou, FujianPurified Indigo Naturalis
S1420210412Xianyou, FujianPurified Indigo Naturalis
S1520210311Xianyou, FujianPurified Indigo Naturalis
S1620211007Xianyou, FujianPurified Indigo Naturalis
S1720211213Xianyou, FujianPurified Indigo Naturalis
S1820210407Xianyou, FujianPurified Indigo Naturalis
S1920210327Xianyou, FujianPurified Indigo Naturalis
S2020191113Yaan, SichuanIndigo Naturalis
S2120180301Xianyou, FujianIndigo Naturalis
S2220210114Yaan, SichuanIndigo Naturalis
S2320180521Jiangyou, SichuanIndigo Naturalis
S2420211223Jiangyou, SichuanIndigo Naturalis
S2520161012Xianyou, FujianIndigo Naturalis
S2620191128Xianyou, FujianIndigo Naturalis
), ArticleFig(id=1210517380493603370, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1210517369538081775, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
No.Indigo contentIndirubin contentTotal ash contentTSI value
S13.32% ± 0.03%0.28% ± 0.00%68.62% ± 0.70%1.11 ± 0.08
S20.00% ± 0.00%0.00% ± 0.00%82.61% ± 0.49%36.27 ± 0.42
S32.46% ± 0.05%0.16% ± 0.00%75.03% ± 2.36%1.73 ± 0.06
S40.00% ± 0.00%0.00% ± 0.00%58.36% ± 0.88%72.65 ± 1.06
S51.90% ± 0.03%0.00% ± 0.00%82.18% ± 1.44%26.51 ± 1.42
S63.23% ± 0.03%0.27% ± 0.00%60.67% ± 1.10%1.64 ± 0.11
S73.54% ± 0.03%0.18% ± 0.00%63.35% ± 1.32%1.04 ± 0.10
S82.07% ± 0.05%0.17% ± 0.01%84.15% ± 1.33%1.53 ± 0.06
S99.00% ± 0.38%0.60% ± 0.00%61.74% ± 1.26%0.61 ± 0.03
S101.17% ± 0.01%0.00% ± 0.00%55.23% ± 0.61%73.28 ± 1.56
S115.35% ± 0.06%0.46% ± 0.00%56.73% ± 1.18%0.92 ± 0.01
S124.51% ± 0.02%0.29% ± 0.00%52.34% ± 1.35%0.91 ± 0.09
S136.90% ± 0.13%0.50% ± 0.00%57.21% ± 0.99%0.82 ± 0.06
S146.21% ± 0.18%0.44% ± 0.00%53.28% ± 2.26%0.86 ± 0.06
S155.51% ± 0.53%0.46% ± 0.00%54.50% ± 1.12%0.97 ± 0.08
S166.86% ± 0.12%0.48% ± 0.00%54.88% ± 1.60%0.87 ± 0.09
S176.47% ± 0.07%0.49% ± 0.00%56.49% ± 2.14%0.93 ± 0.03
S186.34% ± 0.27%0.41% ± 0.00%64.07% ± 1.75%0.94 ± 0.05
S196.44% ± 0.16%0.43% ± 0.00%59.79% ± 0.15%0.82 ± 0.04
S202.98% ± 0.01%0.21% ± 0.00%70.50% ± 1.39%1.20 ± 0.08
S213.02% ± 0.10%0.14% ± 0.00%83.75% ± 1.56%1.18 ± 0.07
S222.82% ± 0.02%0.21% ± 0.00%65.90% ± 1.38%1.26 ± 0.02
S233.63% ± 0.31%0.16% ± 0.00%73.64% ± 1.36%1.36 ± 0.04
S241.67% ± 0.73%0.14% ± 0.00%86.48% ± 0.43%33.24 ± 1.36
S251.40% ± 0.02%0.00% ± 0.00%88.65% ± 0.28%20.33 ± 1.73
S262.17% ± 0.21%0.14% ± 0.00%85.04% ± 1.80%1.88 ± 0.19
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Determination results of indigo content, indirubin content, total ash content, and TSI value in Indigo Naturalis (n = 3, $ \overline{x} $ ± s)

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No.Indigo contentIndirubin contentTotal ash contentTSI value
S13.32% ± 0.03%0.28% ± 0.00%68.62% ± 0.70%1.11 ± 0.08
S20.00% ± 0.00%0.00% ± 0.00%82.61% ± 0.49%36.27 ± 0.42
S32.46% ± 0.05%0.16% ± 0.00%75.03% ± 2.36%1.73 ± 0.06
S40.00% ± 0.00%0.00% ± 0.00%58.36% ± 0.88%72.65 ± 1.06
S51.90% ± 0.03%0.00% ± 0.00%82.18% ± 1.44%26.51 ± 1.42
S63.23% ± 0.03%0.27% ± 0.00%60.67% ± 1.10%1.64 ± 0.11
S73.54% ± 0.03%0.18% ± 0.00%63.35% ± 1.32%1.04 ± 0.10
S82.07% ± 0.05%0.17% ± 0.01%84.15% ± 1.33%1.53 ± 0.06
S99.00% ± 0.38%0.60% ± 0.00%61.74% ± 1.26%0.61 ± 0.03
S101.17% ± 0.01%0.00% ± 0.00%55.23% ± 0.61%73.28 ± 1.56
S115.35% ± 0.06%0.46% ± 0.00%56.73% ± 1.18%0.92 ± 0.01
S124.51% ± 0.02%0.29% ± 0.00%52.34% ± 1.35%0.91 ± 0.09
S136.90% ± 0.13%0.50% ± 0.00%57.21% ± 0.99%0.82 ± 0.06
S146.21% ± 0.18%0.44% ± 0.00%53.28% ± 2.26%0.86 ± 0.06
S155.51% ± 0.53%0.46% ± 0.00%54.50% ± 1.12%0.97 ± 0.08
S166.86% ± 0.12%0.48% ± 0.00%54.88% ± 1.60%0.87 ± 0.09
S176.47% ± 0.07%0.49% ± 0.00%56.49% ± 2.14%0.93 ± 0.03
S186.34% ± 0.27%0.41% ± 0.00%64.07% ± 1.75%0.94 ± 0.05
S196.44% ± 0.16%0.43% ± 0.00%59.79% ± 0.15%0.82 ± 0.04
S202.98% ± 0.01%0.21% ± 0.00%70.50% ± 1.39%1.20 ± 0.08
S213.02% ± 0.10%0.14% ± 0.00%83.75% ± 1.56%1.18 ± 0.07
S222.82% ± 0.02%0.21% ± 0.00%65.90% ± 1.38%1.26 ± 0.02
S233.63% ± 0.31%0.16% ± 0.00%73.64% ± 1.36%1.36 ± 0.04
S241.67% ± 0.73%0.14% ± 0.00%86.48% ± 0.43%33.24 ± 1.36
S251.40% ± 0.02%0.00% ± 0.00%88.65% ± 0.28%20.33 ± 1.73
S262.17% ± 0.21%0.14% ± 0.00%85.04% ± 1.80%1.88 ± 0.19
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基于水试过程定量刻画的青黛质量快检方法的建立与应用
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刘雪梅 1 , 贺亚男 1 , 王芳 2 , 杨明 2 , 张海 3 , 阳向波 3 , 韩丽 1 , 许润春 1, * , 张定堃 1, *
药学学报 | 研究论文 2022,57(11): 3411-3418
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药学学报 | 研究论文 2022, 57(11): 3411-3418
基于水试过程定量刻画的青黛质量快检方法的建立与应用
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刘雪梅1, 贺亚男1, 王芳2, 杨明2, 张海3, 阳向波3, 韩丽1, 许润春1, * , 张定堃1, *
作者信息
  • 1.成都中医药大学药学院, 西南特色中药资源国家重点实验室, 四川 成都 611137
  • 2.江西中医药大学, 现代中药制剂教育部重点实验室, 江西 南昌 330004
  • 3.雅安迅康药业有限公司, 四川 雅安 625600

通讯作者:

*许润春, E-mail: ;
张定堃, E-mail:
Establishment and application of a rapid quality inspection method for Indigo Naturalis based on quantitative portrayal of water testing process
Xue-mei LIU1, Ya-nan HE1, Fang WANG2, Ming YANG2, Hai ZHANG3, Xiang-bo YANG3, Li HAN1, Run-chun XU1, * , Ding-kun ZHANG1, *
Affiliations
  • 1. Pharmacy School, State Key Laboratory of Southwestern Chinese Medicine Resources, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China
  • 2. State Key Laboratory of Innovation Drug and Efficient Energy-saving Pharmaceutical Equipment, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
  • 3. Ya'an Xunkang Pharmaceutical Co., Ltd., Ya'an 625600, China
出版时间: 2022-11-12 doi: 10.16438/j.0513-4870.2022-0820
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摘要
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建立一种基于定量水试的青黛质控快检新方法, 用于青黛的质量优劣评价。采用稳定性分析仪测定26批青黛粉末的Turbiscan稳定性参数(Turbiscan stability index, TSI), 系统优化加样方式、加样粒径、加样量、检测温度等参数, 并对重复性、稳定性等方法学指标进行考察。采用高效液相法测定26批青黛中靛蓝、靛玉红的含量, 并测定总灰分含量。运用SPSS 26.0、Origin 2021软件对青黛有效成分、总灰分含量与TSI值进行相关性分析。研究表明, 青黛供试品的最佳加样方式为: 取过七号筛不过九号筛的青黛粉末0.2g, 以漏斗加样方式装入含20mL纯水的玻璃瓶, 用稳定性分析仪在25℃下扫描。一致性分析表明, 靛蓝、靛玉红的含量排序与TSI值总体上呈相反趋势, 总灰分含量排序与TSI值总体上一致。相关性分析表明, 靛蓝、靛玉红含量与TSI值呈负相关, 相关系数分别为-0.850、-0.801, R2分别为0.72、0.64。总灰分值与TSI值存在正相关, 相关系数为0.724, R2为0.52。根据青黛粉末入水后的TSI值变化, 该研究构建了青黛饮片等级划分的范围及其对应成分含量, 可以判断青黛真伪以及对青黛进行分级评价, 为青黛快速鉴别方法提供参考依据。

青黛  /  定量水试  /  质量评价  /  多重光散射

A new rapid, quality control method based on quantitative water tests has been established for the quality evaluation of Indigo Naturalis. The Turbiscan stability index (TSI) of 26 batches of Indigo Naturalis was measured by a stability analyzer. The parameters, including the method by which the ingredients are added, their particle size, amount, and the testing temperature, were systematically optimized and the methodological indexes such as repeatability and stability were determined. The content of indigo and indirubin in 26 batches of Indigo Naturalis was determined by high performance liquid chromatography and the total ash was measured. The correlation analysis between the active ingredients, total ash content and TSI value of Indigo Naturalis was determined by SPSS 26.0 and Origin 2021. This research shows that the best way to prepare samples for testing is to add 0.2 g of Indigo Naturalis powder which has passed through a No. 7 sieve but failed to pass through a No. 9 sieve to a glass bottle containing 20 mL pure water by a funnel and scan at 25 ℃ with a stability analyzer. Consistency analysis showed that the content ranking of indigo and indirubin is opposite to the TSI value, and the content ranking of total ash is generally consistent with the TSI value. Correlation analysis showed that the correlation coefficients of indigo and indirubin content and TSI value were -0.850 and -0.801, respectively, and R2 was 0.72 and 0.64. The correlation coefficient between total ash content and TSI value was 0.724, R2 was 0.52. Using the change in TSI value of Indigo Naturalis powder in water, this study establishes the range of classification of Indigo Naturalis decoction pieces and the content of relevant components, which can be used to authenticate Indigo Naturalis and evaluate its quality.

Indigo Naturalis  /  quantitative water test  /  quality evaluation  /  multiple light scattering
刘雪梅, 贺亚男, 王芳, 杨明, 张海, 阳向波, 韩丽, 许润春, 张定堃. 基于水试过程定量刻画的青黛质量快检方法的建立与应用. 药学学报, 2022 , 57 (11) : 3411 -3418 . DOI: 10.16438/j.0513-4870.2022-0820
Xue-mei LIU, Ya-nan HE, Fang WANG, Ming YANG, Hai ZHANG, Xiang-bo YANG, Li HAN, Run-chun XU, Ding-kun ZHANG. Establishment and application of a rapid quality inspection method for Indigo Naturalis based on quantitative portrayal of water testing process[J]. Acta Pharmaceutica Sinica, 2022 , 57 (11) : 3411 -3418 . DOI: 10.16438/j.0513-4870.2022-0820
正文
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青黛为爵床科植物马蓝Baphicacanthus cusia (Nees) Bremek.、蓼科植物蓼蓝Polygonum tinctorium Ait.或十字花科植物菘蓝Isatis indigotica Fort.的叶或茎叶经加工制得的干燥粉末、团块或颗粒, 具有清热解毒、凉血消斑、泻火定惊的功效, 对于小儿高热、银屑病、白血病、溃疡性结肠炎等疾病有显著疗效[1]。现代研究表明, 靛蓝、靛玉红是青黛的主要药效成分。现行版《中华人民共和国药典》采用高效液相色谱法测定靛蓝、靛玉红的含量, 规定靛蓝含量不低于2.0%, 靛玉红含量不低于0.13%[2]。而靛蓝、靛玉红因其溶解度差, 现采用两种方法分别制备与检测, 供试品制备过程需要消耗大量的三氯甲烷、N, N-二甲基甲酰胺等有机溶剂, 仅50 mg青黛粉末需250 mL三氯甲烷提取, 费时、费力, 成本高, 且对环境不友好。因此, 亟待探索快速方便、绿色环保的青黛质量评价新方法。
青黛因其体轻质松、易漂浮的特点, 水试法成为其传统经验鉴别的主要方法之一。本课题组前期研究发现, 正品青黛的强疏水性与其特殊的微观结构密切相关[3]。靛蓝、靛玉红为平面对称结构分子, 溶解度差, 在炮制打靛过程中, 吸附于碳酸钙、二氧化硅等无机物表面, 形成了青黛的微观结构[4, 5]。靛蓝、靛玉红等有机物吸附于无机物表面越多, 青黛疏水性强, 越易漂浮于水面, 不易润湿浸入水中, 这也是优质青黛的物质内涵与水试表现。相反, 掺伪青黛或有机物较低的劣质青黛, 质地较重, 不易飞扬, 加入水中, 部分微粒快速下沉。
Turbiscan Lab稳定性分析仪主要用于分析乳状液和悬浮液的稳定性, 可同时检测光散射后透射光和背散射光的强度, 定量分析上述现象所发生的速率, 给出样品的分散稳定性参数, 快速准确地评价分散体系的稳定性, 在食品、医药、石油、涂料、墨水等领域应用广泛[6-8]。因此, 本研究提出了一种基于定量水试判定的质控新方法, 通过引入稳定性分析仪, 准确捕捉青黛粉末入水后的整体轨迹与沉浮行为, 建立以样品的Turbiscan稳定性参数(Turbiscan stability index, TSI) 为指标的青黛质量快速评价方法(图 1)。该研究有望为青黛饮片质量的优劣评价提供一种快速准确、绿色友好的新方法, 支撑市场流通的优质优价与高质量青黛饮片的开发转化。
材料与方法
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仪器  Turbiscan Lab稳定性分析仪(法国Formulaction公司); LC-20AT高效液相色谱仪(日本岛津公司); 马弗炉(北京中兴伟业仪器有限公司); BSA 224SA型1/1万分析天平和BT25S型1/10万分析天平(德国Sartorius公司); Mili-Q型超纯水仪(美国Milipore公司); KQ-500DE超声波清洗机(昆山市超声仪器有限公司)。
药材与试剂  靛蓝对照品(批号AF21040652)、靛玉红对照品(批号AF20070606) 均购于成都埃法生物科技有限公司, 纯度≥ 98%; 色谱甲醇(批号34860-4L-R) 购自Sigma-Aldrich西格玛奥德里奇贸易有限公司; N, N-二甲基甲酰胺(批号2022032301)、三氯甲烷(批号2021122101)、水合氯醛(批号2019071601)、无水硫酸钠(批号2020091401) 均购于成都市科隆化学品有限公司。
26批青黛(含靛花) 药材样品(S1~S26) 均购自于四川、福建等地。样品信息见表 1。
检测原理  Turbiscan技术基于多重光散射原理。Turbiscan Lab仪器的测量探头是由一个脉冲近红外光源(λ = 880 nm) 和两个同步检测器组成: 其中一个为透射光检测器, 用来接收穿过样品池的光(与入射光成0°), 用于测定清澈的样品; 另一个为背散射光检测器, 用来接收样品的背散射光(与入射光成45°), 用于测定高浓度样品, 浓度可高达95%[9]。测量探头从底部到顶部扫描整个样品池, 每40 μm扫描一次, 收集样品的透射光(T) 和背散射光(BS) 数据。通过设定测量次数、扫描时间, 使样品被重复扫描, 由Turbiscan电流-电压转换器处理信号, 微处理器软件可进行数据采集、数模转换、数据存储及机器控制等, 从而得到一张表征产品稳定性或不稳定特征的指纹图谱。
指标选择  青黛在水中的稳定性是由Turbiscan Lab中的TSI值来评估的。TSI值反映的是在测量时间内, 每次测量相较前一次测量其背散射光或透射光强度变化值的累加, 反映样品在整个测量时间内体积浓度和颗粒粒径的综合变化[10], 如式1所示。TSI值越高, 体系越不稳定, 样品变化幅度越大[11]。根据青黛疏水性强的特性, 可以推断青黛的质量越好, 靛蓝、靛玉红含量越高, 样品几乎完全漂浮于液面, 不浸入水中, TSI值则越小。在本研究中, 将20 mL纯水装进圆柱形透明玻璃瓶中, 装入一定量的青黛粉末, 再放进Turbiscan仪器测量池。在25 ℃下持续10 min, 沿垂直方向扫描。
$ \mathrm{T}\mathrm{S}\mathrm{I}=\sum _{i}\frac{{\sum }_{h}\left|\mathrm{s}\mathrm{c}\mathrm{a}{\mathrm{n}}_{i}-\mathrm{s}\mathrm{c}\mathrm{a}{\mathrm{n}}_{i-1}\right|}{H} $
式1中: i为测量次数; h为仪器扫描高度; scan为背散射光或透射光强度; H为测量最大高度。
供试品制备方法的考察
加样方式的优化  取青黛样品(编号S9) 各0.4 g, 分别以漏斗加样方式(漏斗固定于铁架台上, 将装有纯水的样品瓶放置于漏斗正下方, 漏斗下缘与瓶口齐平。加样时, 固定在距漏斗下缘80 cm高处释放样品, 使样品能沿漏斗自由滑落)、手动加样方式(将样品直接倾入装有纯水的样品瓶中) 加入含20 mL纯水的样品瓶中, 将样品瓶置于Turbiscan Lab的测量池, 分别用稳定性分析仪在25 ± 1 ℃下进行检测, 每30 s扫描一次, 共扫描10 min。
加样粒径的优化  取青黛药材粉末(编号S9), 使其依次通过一、三、五、七、九号筛, 收集每个筛号间的样品, 依次标记为一到三号筛、三到五号筛、五到七号筛、七到九号筛。分别取一到三号筛、三到五号筛、五到七号筛、七到九号筛之间的青黛样品各0.4 g, 以漏斗加样方式加入含20 mL纯水的样品瓶中, 将样品瓶置于Turbiscan Lab的测量池, 分别用稳定性分析仪在25 ± 1 ℃下进行检测, 每30 s扫描一次, 共扫描10 min。
加样量的优化  取七到九号筛之间的青黛样品(编号S9) 0.1、0.2、0.4、0.6 g, 以漏斗加样方式分别加入含20 mL纯水的样品瓶中, 将样品瓶置于Turbiscan Lab的测量池, 用稳定性分析仪在25 ± 1 ℃下进行检测, 每30 s扫描一次, 共扫描10 min。
检测温度的优化  取七到九号筛之间的青黛样品(编号S9) 各0.2 g, 以漏斗加样方式加入含20 mL纯水的样品瓶中, 将样品瓶置于Turbiscan Lab的测量池, 用稳定性分析仪分别在25 ± 1、35 ± 1、45 ± 1、55 ± 1 ℃的温度下进行检测, 每30 s扫描一次, 共扫描10 min。
方法学考察
重复性  取同一批七到九号筛之间的青黛样品(编号S9) 0.2 g, 以漏斗加样方式加入圆柱形透明玻璃瓶, 用稳定性分析仪在25 ± 1 ℃下进行检测, 平行6份, 获取10 min处的TSI值, 计算RSD值。
日间重复性  取同一批七到九号筛之间的青黛样品(编号S9) 0.2 g, 以漏斗加样方式加入圆柱形透明玻璃瓶, 用稳定性分析仪在每日同一时间、25 ± 1 ℃下进行检测, 连续三天, 获取10 min处的TSI值, 计算RSD值。
稳定性  取同一批七到九号筛之间的青黛样品(编号S9) 0.2 g, 以漏斗加样方式加入圆柱形透明玻璃瓶, 用稳定性分析仪在25 ± 1 ℃下进行检测, 检测20 min。获取8、9、10、11、12、13、14 min处的TSI值, 计算RSD值。
靛蓝含量测定
色谱条件  参照现行版中国药典[2], Welchrom®-C18色谱柱(250 mm × 4.6 mm, 5 μm), 流动相甲醇-水(75∶25), 流速1.0 mL·min-1, 检测波长606 nm, 柱温30 ℃, 进样量10 μL。
对照品溶液的制备  精密称定靛蓝对照品2.67 mg, 置250 mL量瓶中, 加2%水合氯醛的三氯甲烷溶液(取水合氯醛, 置硅胶干燥器中放置24 h, 称取2.0 g, 加三氯甲烷至100 mL, 放置, 出现浑浊, 以无水硫酸钠脱水, 滤过, 即得) 约220 mL, 超声1.5 h, 放冷, 加2%水合氯醛的三氯甲烷溶液至刻度, 摇匀, 得每1 mL中含靛蓝10.68 μg的对照品溶液。
供试品溶液的制备  取青黛样品约2 mg, 精密称定, 置10 mL量瓶中, 加2%水合氯醛的三氯甲烷溶液约8 mL, 超声30 min, 放冷, 加2%水合氯醛的三氯甲烷溶液至刻度, 摇匀, 滤过, 取续滤液, 即得。
靛玉红含量测定
色谱条件  参照现行版中国药典[2], Welchrom®-C18色谱柱(250 mm × 4.6 mm, 5 μm), 流动相甲醇-水(70∶30), 流速1.0 mL·min-1, 检测波长292 nm, 柱温30 ℃, 进样量10 μL。
对照品溶液的制备  精密称定靛玉红对照品2.5 mg, 置50 mL量瓶中, 加N, N-二甲基甲酰胺约45 mL, 超声使溶解, 放冷, 加N, N-二甲基甲酰胺至刻度, 摇匀, 得每l mL中含靛玉红50 μg的对照品溶液。
供试品溶液的制备  取青黛样品约20 mg, 精密称定, 置10 mL量瓶中, 加N, N-二甲基甲酰胺约8 mL, 超声处理(功率250 W, 频率33 kHz) 30 min, 放冷, 加N, N-二甲基甲酰胺至刻度, 摇匀, 滤过, 取续滤液, 即得。
总灰分测定  参照中国药典现行版(四部) 通则灰分测定法, 分别取各批次的青黛2~3 g, 置炽灼至恒重的坩埚中, 称定质量(准确至0.01 g), 缓缓炽热, 至完全炭化时, 逐渐升高温度到500~600 ℃, 使完全灰化并至恒重。根据残渣质量, 分别计算各批次总灰分的质量分数(%)。
不同批次的青黛TSI值的测定  取不同批次下七到九号筛之间的青黛样品0.2 g, 以漏斗加样方式加入含20 mL纯水的样品瓶中, 将样品瓶置于Turbiscan Lab的测量池, 用稳定性分析仪分别在25 ± 1 ℃的温度下进行检测, 每30 s扫描一次, 共扫描10 min, 测量3次。记录10 min处的TSI平均值。
结果
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1 供试品制备方法的考察
1.1 加样方式的优化
漏斗加样、手动加样的结果显示, 10 min时的TSI值分别为0.98 ± 0.11、1.02 ± 0.18。加样方式对TSI值影响不大。由于测量人员的不同, 手动加样方式由于操作误差不可避免, 会对结果产生影响。为了建立更加标准的供试品制备方法, 选择漏斗加样方式对测定结果影响更小。
1.2 加样粒径的优化
结果显示, 一到三号筛、三到五号筛、五到七号筛、七到九号筛之间的样品在10 min时的TSI值依次为1.41 ± 0.13、1.13 ± 0.00、1.01 ± 0.05、0.79 ± 0.10。由结果可知, 随着青黛粉末粒径减小, TSI值也逐渐降低。低粒径范围的青黛疏水性更强, 不易润湿浸入水中, 是优质青黛的水试表现。因此选择七到九号筛之间的青黛作为最佳测量粒径范围。
1.3 加样量的优化
0.1、0.2、0.4、0.6 g的加样量结果显示, 10 min时的TSI值分别为0.63 ± 0.08、0.56 ± 0.00、0.82 ± 0.10、0.72 ± 0.11。由结果可知, 青黛入水后, 重量对其稳定性的影响较小。加样量过小时, 样品代表性稍差; 但加样量过大时, 样品堆积在液面上, 重力可能影响测量结果。因此选择0.2 g作为最佳加样量。
1.4 检测温度的优化
25、35、45、55 ℃的结果显示, 10 min时的TSI值分别为0.58 ± 0.09、1.04 ± 0.11、1.74 ± 0.18、2.26 ± 0.23。温度越高, 微粒的运动速度越快。因此, 为减少温度因素的影响, 选择25 ℃作为最佳检测温度。
2 方法学考察
2.1 重复性
结果显示, 6份样品在10 min时的TSI值分别为0.62、0.65、0.58、0.60、0.64、0.58。RSD值为4.63%, 表明该方法重复性较好。
2.2 日间重复性
结果显示, 10 min时的TSI值分别为0.58、0.60、0.57。RSD值为2.68%, 表明该方法日间重复性较好。
2.3 稳定性
结果显示, 第8~14 min内的TSI值分别为0.60、0.64、0.65、0.63、0.61、0.63、0.63。RSD值为2.47%, 表明供试品在8~14 min内基本稳定。因此, 选择中间时间10 min为测量时长。
3 青黛靛蓝和靛玉红的含量测定、总灰分测定及TSI值测定
靛蓝、靛玉红是青黛中主要的活性物质, 也是主要的有机物成分。由于青黛中含大量石灰, 因此石灰含量的高低会影响其总灰分数值。靛蓝、靛玉红的含量及总灰分数值可在整体上反映青黛的质量。26批靛蓝、靛玉红含量测定结果和总灰分测定结果见表 2。由结果可知, 26批青黛样品中靛蓝、靛玉红、灰分值均波动较大。S9、S11~S19的靛蓝含量在4.5%以上, 是靛蓝规定质量分数的2.2~4.5倍; 靛玉红含量在0.29%以上, 是靛玉红规定质量分数的2.2~4.6倍; 总灰分值在52%~62%, 与普通青黛的灰分值70%~90%相比, 这些批次下石灰含量更低。S1、S3、S6~S8、S20~S23、S26的靛蓝含量在2%~3.5%之间, 是靛蓝规定质量分数的1~1.8倍; 靛玉红含量在0.14%~0.28%以上, 是靛玉红规定质量分数的1~2.2倍; 总灰分值在60%~84%, 属于普通青黛。其余批次S2、S4为伪品, 均无法检测出靛蓝、靛玉红; S5、S10、S25的青黛质量不佳, 只能检测出靛蓝。
26批青黛样品的TSI值测量结果见表 2。由Turbiscan多重光散射法测试原理可知, 稳定性指数越高, 样品变化幅度越大, 体系稳定性越差。TSI值测量结果表明, 所有批次的TSI值在0.6~75内波动。大部分批次的TSI值均较低, 可知其内部微粒沉降幅度小, 体系较稳定。而S2、S4、S5、S10、S24、S25的TSI值异常高于其他批次, 证明这些批次的稳定性极差, 其内部变化幅度极大, 推测样品发生明显的沉降过程。同时结合其靛蓝、靛玉红含量来看, 这些批次均为伪劣品, 不符合质量标准规定, 表明稳定性分析仪适用于青黛的简单鉴别。但结果显示, 劣伪品的TSI值具有随机性, 因此这些批次不考虑纳入后续一致性及相关性分析。
此外, 本课题组在实验过程中发现, 部分批次间的TSI值存在差异。为此, 选取TSI变异系数较大的两个批次S16 (RSD 10.31%)、S26 (RSD 10.03%), 通过选取6个不同部位(将青黛粉末叠成锥形, 分别在顶部取一个样、在腰部取两个样、在底部取三个样) 的粉末重复进行含量测定。结果表明, S16靛蓝含量的RSD为19.13%, 靛玉红含量的RSD为2.47%; S26靛蓝含量的RSD为16.22%, 靛玉红含量的RSD为3.24%, 说明样品不均一, 主要由于靛蓝含量不均匀导致。该含量测定结果验证了青黛样品自身TSI值存在不均一性的测定结果。
4 一致性和相关性分析
采用Origin 2021软件对符合中国药典规定青黛样品的靛蓝、靛玉红、总灰分含量与稳定性分析仪测得的TSI值进行简单的一致性分析, 如图 2所示。气泡面积代表青黛靛蓝、靛玉红、总灰分含量的高低, 气泡越大, 有效成分的含量越高。从气泡大小的排列顺序来看, 除S3、S6、S7、S26稍有偏差, 靛蓝、靛玉红的含量排序与TSI值总体上呈现相反趋势。总灰分含量排序与TSI值总体上呈现一致的趋势, 除S3、S6、S9、S21、S26稍有偏差。不同批次样品的自身差异是影响TSI值的最大因素。但供试品的前处理过程也是TSI值的重要影响因素, 其中影响较大的有检测温度、加样粒径、加样量, 对该过程严格控制才能保证结果的一致性。随后, 为了进一步阐述该质量快检方法的准确性, 利用SPSS 26.0对符合药典规定青黛样品的2种成分含量、总灰分含量与TSI值进行相关性分析。结果发现, 靛蓝、靛玉红含量与TSI值呈负相关, 相关系数分别为-0.850、-0.801, R2分别为0.72、0.64。总灰分值与TSI值存在正相关, 相关系数为0.724, R2为0.52。由此, 可以验证, TSI值越低, 青黛中靛蓝、靛玉红含量越高, 总灰分越低。在生产中通过实时检测青黛入水后一段时间的沉浮变化, 可预测青黛化学成分的含量情况, 特别是快速预测有机物含量情况。
5 青黛质量快速评价的等级划分研究
TSI值的大小与青黛靛蓝、靛玉红、总灰分的含量密切相关。因此可通过TSI值快速评定青黛饮片质量优劣。本文根据26批样品入水后的TSI值构建了青黛饮片等级划分的范围及其对应成分含量, 后续用于分级评定, 如图 3。一等品为优质青黛, 靛蓝含量大于4.5%, 靛玉红含量大于0.3%, TSI值低于1.0, 如图 4A (编号S9); 二等品为普通青黛, 靛蓝含量在2.1%~3.5%之间, 靛玉红含量在0.14%~0.3%之间, TSI介于1.0~2.0, 如图 4B (编号S23); 三等品为伪劣青黛, 靛蓝含量小于2%, 靛玉红含量小于0.14%, TSI值高于20, 如图 4C (编号S25)、图 4D (编号S4)。从图 3可以明显看出, 一等品均为靛花。靛花为青黛原药材经浸泡发酵、石灰打靛、水飞精制后获得的上层浮沫, 靛蓝、靛玉红含量高, 也是传统青黛主要的药用来源。而现代药用青黛多为“粗靛”, 靛蓝、靛玉红含量较靛花低, 灰分较靛花高。因此, 可通过稳定性分析仪的TSI参数将优质青黛(靛花) 与普通青黛(粗靛) 区分, 避免以次充好的现象。
讨论
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中国药典现行版规定青黛的含量测定方法为高效液相色谱法。该法周期长, 工作量大, 且应用较多有机试剂, 成本高; 而多重光散射技术作为一种全新的分析方法, 近年来在液体制剂(乳状液、混悬液) 稳定性研究中已被广泛应用, 具有操作方便、分析快速、灵敏度高、实时检测等特点。此外, 多重光散射技术运用于青黛粉末的质量评价将大大减少有机试剂的运用, 缩短测定时间。因此, 本研究建立的是一种易于实现、简便快速、成本低廉的青黛质量鉴定与评价方法。
青黛的有机成分靛蓝、靛玉红受到马蓝等药材自身品质及生产炮制工艺等因素影响。靛蓝、靛玉红不溶于水, 在炮制时需加入石灰, 其中的Ca(OH)2与浸泡液中的CO32-生成的CaCO3作为靛蓝、靛玉红得以顺利沉降的载体[12, 13]。而青黛中若混有大量石灰会严重影响其临床疗效, 还会对皮肤、消化道产生一定的刺激作用。因此, 青黛的整体质量考察需要结合靛蓝、靛玉红和灰分含量。青黛入水后, 靛蓝、靛玉红含量与TSI值呈显著的负相关, 随着TSI值降低, 靛蓝、靛玉红含量呈上升趋势。总灰分与TSI值具有显著的正相关。推测原因可能是: 青黛的微观结构是由于靛蓝、靛玉红附着在碳酸钙石灰表面。青黛的亲水性差, 放于水的样品瓶时, 首先接触水面的是青黛的靛蓝、靛玉红, 支撑青黛与水面的浮力。因此, 靛蓝、靛玉红含量越多, 疏水性越强, 越易漂浮。而总灰分反映的是青黛的石灰含量, 总灰分越高, 质地偏重, 则越易下沉。本研究基于多重光散射光谱技术建立了不同批次青黛的定量水试模型, 实现了以TSI值为指标的快速鉴别, 为青黛的优劣评价提供一种客观、简便、快捷的判断方法。在企业饮片生产中, 利用TSI模型的简便性与快速性, 可降低含量测定带来的时间成本与经济成本, 可作为现有青黛质量控制的补充方法。
作者贡献: 刘雪梅、许润春、杨明和张定堃设计实验; 刘雪梅、贺亚男和王芳进行实验数据采集; 韩丽、张海和阳向波进行药材收集; 刘雪梅、许润春和张定堃撰写、修改论文。
利益冲突: 无利益冲突。
基金
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  • 国家重点研发计划项目(2018YFC1707205)
  • 国家自然科学基金项目(82173976)
  • 江西中医药大学创新药物与高效节能降耗制药设备国家重点实验室开放基金课题项目(GZSYS202003)
文献
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2022年第57卷第11期
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文章信息
doi: 10.16438/j.0513-4870.2022-0820
  • 接收时间：2022-07-05
  • 首发时间：2025-12-24
  • 出版时间：2022-11-12
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出版历史
  • 收稿日期：2022-07-05
  • 修回日期：2022-08-18
基金
国家重点研发计划项目(2018YFC1707205)
国家自然科学基金项目(82173976)
江西中医药大学创新药物与高效节能降耗制药设备国家重点实验室开放基金课题项目(GZSYS202003)
作者信息
    1.成都中医药大学药学院, 西南特色中药资源国家重点实验室, 四川 成都 611137
    2.江西中医药大学, 现代中药制剂教育部重点实验室, 江西 南昌 330004
    3.雅安迅康药业有限公司, 四川 雅安 625600

通讯作者:

*许润春, E-mail: ;
张定堃, E-mail:
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2种不同金属材料的力学参数
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Genus		 种数
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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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