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Article(id=1198656147881292651, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198656143976399200, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2022-1437, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1672243200000, receivedDateStr=2022-12-29, revisedDate=1684080000000, revisedDateStr=2023-05-15, acceptedDate=null, acceptedDateStr=null, onlineDate=1763711495608, onlineDateStr=2025-11-21, pubDate=1694448000000, pubDateStr=2023-09-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763711495608, onlineIssueDateStr=2025-11-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763711495608, creator=13701087609, updateTime=1763711495608, updator=13701087609, issue=Issue{id=1198656143976399200, tenantId=1146029695717560320, journalId=1189982191388893191, year='2023', volume='58', issue='9', pageStart='2541', pageEnd='2834', issueExtLink='null', onlineDate='null', pubDate='1694448000000', pubDateStr='2023-09-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763711494677, creator='13701087609', updateTime=1763711620095, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1198656670072144034, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198656143976399200, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1198656670072144035, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1198656143976399200, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2771, endPage=2776, ext={EN=ArticleExt(id=1198656150737613702, articleId=1198656147881292651, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Molecular quantification for quality control of Jinqian Baihuashe in Jinlong Capsule, columnId=null, journalTitle=Acta Pharmaceutica Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=

The quality control of Chinese patent medicines containing animal-derived crude drugs is relatively difficult, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are relevant methods, they are usually qualitative, and quantitative indicators are either lacking or have poor specificity. This paper has proposed to use molecular quantitative technology to control the quality of Chinese patent medicines containing animal-derived crude drugs. In this study, a molecular quantitative method based on fluorescence quantitative PCR was established for the determination of Jinqian Baihua She in Jinlong Capsule. The method has good specificity, sensitivity, and repeatability. There is a good linear relationship between the content of DNA fragments and the CT (cycle threshold) value. The content of the Bungarus multicinctus-specific fragment in Jinlong Capsule is 24.1-46.6 IU·mg-1. It is suggested that the content of the specific fragment of Jinqian Baihua She should not be less than 19.3 IU·mg-1 as one of the quality control criteria of Jinlong Capsule. The study can provide a reference for the quality control of Chinese patent medicines containing animal-derived crude drugs.

, authors=null, authorsList=Hui LIN, Chan LI, Zhi CHAO, authorCompany=null, correspAuthors=Zhi CHAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2023 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=1198656151706497973, articleId=1198656147881292651, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=金龙胶囊中金钱白花蛇的分子质量控制, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

中成药中动物类药材的质量控制相对比较困难, 因为大多数动物药的有效成分仍不清晰。即使有相关的方法, 通常也是定性的, 而定量的指标或缺少, 或特异性比较差。因此, 本文提出运用分子定量技术对中成药中动物类药材进行质量控制。本研究以金龙胶囊为例, 建立了其中金钱白花蛇基于荧光定量PCR的分子定量法。该方法特异性好、灵敏度高、重复性好, DNA片段含量与CT (cycle threshold)值呈良好线性关系; 金龙胶囊中金钱白花蛇特异片段含量在24.1~46.6 IU·mg-1, 建议将金钱白花蛇特异片段含量不低于19.3 IU·mg-1作为金龙胶囊质控标准之一。研究可以为含动物类药材的中成药的质量控制提供参考。

, authors=null, authorsList=林慧, 李婵, 晁志, authorCompany=null, correspAuthors=晁志, authorNote=null, correspAuthorsNote=
*晁志, Tel: 86-20-61648764, E-mail:
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No. Species Collection site
A1 Bungarus multicinctus Guangzhou, Guangdong
A2 B. multicinctus Zhongshan, Guangdong
A3 B. multicinctus Zhongshan, Guangdong
A4 B. fasciatus Zhongshan, Guangdong
A5 Deinagkistrodon acutus Jiangxi
A6 Dinodon rufozonatum Hunan
A7 D. flavozonatum Shaoguan, Guangdong
A8 Orthriophis moellendorffi Guangxi
A9 Euprepiophis mandarinus Zhongshan, Guangdong
A10 Naja atra Taishan, Guangdong
A11 Protobothrops mangshanensis Conghua, Guangdong
A12 Lycodon ruhstrati Hunan
A13 Ptyas mucosus Zhongshan, Guangdong
A14 Enhydris plumbea Zhongshan, Guangdong
A15 Zaocys dhumnades Conghua, Guangdong
A16 Coelognathus radiatus Zhongshan, Guangdong
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Samples information of Bungarus multicinctus and its adulterants

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No. Species Collection site
A1 Bungarus multicinctus Guangzhou, Guangdong
A2 B. multicinctus Zhongshan, Guangdong
A3 B. multicinctus Zhongshan, Guangdong
A4 B. fasciatus Zhongshan, Guangdong
A5 Deinagkistrodon acutus Jiangxi
A6 Dinodon rufozonatum Hunan
A7 D. flavozonatum Shaoguan, Guangdong
A8 Orthriophis moellendorffi Guangxi
A9 Euprepiophis mandarinus Zhongshan, Guangdong
A10 Naja atra Taishan, Guangdong
A11 Protobothrops mangshanensis Conghua, Guangdong
A12 Lycodon ruhstrati Hunan
A13 Ptyas mucosus Zhongshan, Guangdong
A14 Enhydris plumbea Zhongshan, Guangdong
A15 Zaocys dhumnades Conghua, Guangdong
A16 Coelognathus radiatus Zhongshan, Guangdong
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Number Shougong (30 mg) Jinqian Baihua She and its adulterants (15 mg) Qi She (15 mg)
H1 Gekko swinhonis Bungarus multicinctus Deinagkistrodon acutus
H2 G. swinhonis B. multicinctus D. acutus
H3 G. swinhonis B. multicinctus D. acutus
H4 G. swinhonis B. multicinctus: Dinodon rufozonatum 1∶1 D. acutus
H5 G. swinhonis B. multicinctus: D. rufozonatum 1∶1 D. acutus
H6 G. swinhonis B. multicinctus: D. rufozonatum 1∶1 D. acutus
H7 G. swinhonis B. multicinctus: D. rufozonatum 1∶4 D. acutus
H8 G. swinhonis B. multicinctus: D. rufozonatum 1∶4 D. acutus
H9 G. swinhonis B. multicinctus: D. rufozonatum 1∶4 D. acutus
H10 G. swinhonis D. rufozonatum D. acutus
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The composition of self-made Jinlong Capsule

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Number Shougong (30 mg) Jinqian Baihua She and its adulterants (15 mg) Qi She (15 mg)
H1 Gekko swinhonis Bungarus multicinctus Deinagkistrodon acutus
H2 G. swinhonis B. multicinctus D. acutus
H3 G. swinhonis B. multicinctus D. acutus
H4 G. swinhonis B. multicinctus: Dinodon rufozonatum 1∶1 D. acutus
H5 G. swinhonis B. multicinctus: D. rufozonatum 1∶1 D. acutus
H6 G. swinhonis B. multicinctus: D. rufozonatum 1∶1 D. acutus
H7 G. swinhonis B. multicinctus: D. rufozonatum 1∶4 D. acutus
H8 G. swinhonis B. multicinctus: D. rufozonatum 1∶4 D. acutus
H9 G. swinhonis B. multicinctus: D. rufozonatum 1∶4 D. acutus
H10 G. swinhonis D. rufozonatum D. acutus
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Sample No. Lot number CT RSD/% Concentration /IU·μL-1 Content /IU·mg-1
J1 190202 26.97 0.85 4.71×102 46.6
J2 190203 27.86 0.89 2.70×102 26.8
J3 190204 27.79 0.62 2.82×102 28.0
J4 190205 27.40 0.48 3.61×102 35.9
J5 190933 28.03 0.40 2.43×102 24.1
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Content of Jinqian Baihua She determined using fluorescence quantitative PCR test in Jinlong Capsule. CT: Cycle threshold

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Sample No. Lot number CT RSD/% Concentration /IU·μL-1 Content /IU·mg-1
J1 190202 26.97 0.85 4.71×102 46.6
J2 190203 27.86 0.89 2.70×102 26.8
J3 190204 27.79 0.62 2.82×102 28.0
J4 190205 27.40 0.48 3.61×102 35.9
J5 190933 28.03 0.40 2.43×102 24.1
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金龙胶囊中金钱白花蛇的分子质量控制
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林慧 , 李婵 , 晁志 *
药学学报 | 研究论文 2023,58(9): 2771-2776
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药学学报 |研究论文 2023 , 58 (9) : 2771 -2776
金龙胶囊中金钱白花蛇的分子质量控制
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林慧, 李婵, 晁志*
作者信息
  • 南方医科大学中医药学院, 广东 广州 510515
通讯作者:
*晁志, Tel: 86-20-61648764, E-mail:
Molecular quantification for quality control of Jinqian Baihuashe in Jinlong Capsule
Hui LIN, Chan LI, Zhi CHAO*
Affiliations
  • School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
出版时间: 2023-09-12 doi: 10.16438/j.0513-4870.2022-1437
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摘要
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中成药中动物类药材的质量控制相对比较困难, 因为大多数动物药的有效成分仍不清晰。即使有相关的方法, 通常也是定性的, 而定量的指标或缺少, 或特异性比较差。因此, 本文提出运用分子定量技术对中成药中动物类药材进行质量控制。本研究以金龙胶囊为例, 建立了其中金钱白花蛇基于荧光定量PCR的分子定量法。该方法特异性好、灵敏度高、重复性好, DNA片段含量与CT (cycle threshold)值呈良好线性关系; 金龙胶囊中金钱白花蛇特异片段含量在24.1~46.6 IU·mg-1, 建议将金钱白花蛇特异片段含量不低于19.3 IU·mg-1作为金龙胶囊质控标准之一。研究可以为含动物类药材的中成药的质量控制提供参考。

金钱白花蛇  /  银环蛇  /  金龙胶囊  /  分子定量  /  质量控制  /  实时荧光定量PCR

The quality control of Chinese patent medicines containing animal-derived crude drugs is relatively difficult, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are relevant methods, they are usually qualitative, and quantitative indicators are either lacking or have poor specificity. This paper has proposed to use molecular quantitative technology to control the quality of Chinese patent medicines containing animal-derived crude drugs. In this study, a molecular quantitative method based on fluorescence quantitative PCR was established for the determination of Jinqian Baihua She in Jinlong Capsule. The method has good specificity, sensitivity, and repeatability. There is a good linear relationship between the content of DNA fragments and the CT (cycle threshold) value. The content of the Bungarus multicinctus-specific fragment in Jinlong Capsule is 24.1-46.6 IU·mg-1. It is suggested that the content of the specific fragment of Jinqian Baihua She should not be less than 19.3 IU·mg-1 as one of the quality control criteria of Jinlong Capsule. The study can provide a reference for the quality control of Chinese patent medicines containing animal-derived crude drugs.

Jinqian Baihu She  /  Bungarus multicinctus  /  Jinlong capsule  /  molecular quantification  /  quality control  /  real time fluorescent quantitative PCR
林慧, 李婵, 晁志. 金龙胶囊中金钱白花蛇的分子质量控制. 药学学报, 2023 , 58 (9) : 2771 -2776 . DOI: 10.16438/j.0513-4870.2022-1437
Hui LIN, Chan LI, Zhi CHAO. Molecular quantification for quality control of Jinqian Baihuashe in Jinlong Capsule[J]. Acta Pharmaceutica Sinica, 2023 , 58 (9) : 2771 -2776 . DOI: 10.16438/j.0513-4870.2022-1437
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中成药是我国中医药的组成部分, 在临床治疗中发挥着不可替代的作用, 其质量直接关系到临床用药的安全[1, 2]。随着中成药品种的不断增加, 大量的中成药进入我国的医疗市场[3, 4], 而由于中成药其成分复杂, 药材市场上各种混伪品层出不穷, 导致中成药在临床上存在一定的用药风险, 同时使其质量控制难度变大[5]。近年来, 在中成药的国家药品抽验工作中, 对从生产企业收集的原料药进行了质量考察, 发现主要问题有掺伪、染色、非药用部位使用、外源性有毒有害物质残留等[5]。质量控制对于确保中成药的有效性和安全性至关重要且不可或缺, 目前, 以化学标志物检测为重点的质量检测技术体系已逐步建立[6]。
这种以化学标记物作为质量标准的方法, 对于大多数含有植物原料药的中成药来说是可行的, 但对于含有动物原料药的中成药, 通常无法对其中的动物药成分进行准确的定量与定性。因为动物药和植物药在化学成分上存在很大差异, 对动物原料药化学成分的研究还很薄弱。大多数动物原料药的化学成分不明确, 在含有动物原料药的质量标准中往往缺少含量测定项目[7]。2010~2014年, 国家药品抽验共抽取了46个含动物类成分的中成药品种, 23个品种对处方中任何一味动物药均未设置含量检测项目, 仅10个品种对处方中所有动物药规定了含量检测项目[5]。即使有含量测定项, 大多数指标成分是常见的氨基酸、糖、核苷酸等, 缺乏专属性[8]。
最近研究提出了采用分子定量法对中成药中的动物药进行质量控制的新策略[9-11]。中成药制备过程中, 各组分的DNA会残留在成品中, 这对中成药中的组分进行分子鉴定奠定了基础。如果某动物药组分足量正确投料, 并且生产过程稳定, 最终产品中该动物药的物种特异性DNA片段含量应在固定范围内。中成药中动物组分特定DNA片段的含量, 可以反映成药中各原料药的投料量和占比, 进而保证成药品质。如果含量低于一定值, 则产品不合格。
本研究以金龙胶囊为例进行了一些尝试。金龙胶囊是由鲜守宫、鲜金钱白花蛇、鲜蕲蛇以2∶1∶1的比例组合而成的动物药制剂, 其功效是破瘀散结、解郁通络, 在临床上可用于治疗毒邪伤肝、脉络瘀阻所导致的原发性肝癌[12]。其现行质量标准主要对各原料药及其混合原料中氨基酸、蛋白质、游离单糖和多糖、小分子类物质尿嘧啶及次黄嘌呤含量进行测定[13]。这类指标成分专属性不高, 无法确定是否存在其他掺杂物, 不能解决物种鉴别问题, 因此寻求一种适用于金龙胶囊的质量控制方法已成为迫切的需求。
作者前期通过荧光定量PCR法测定DNA片段含量, 首先证实了药材投料量与特异片段的含量呈正相关, 然后成功建立了金龙胶囊中蕲蛇的分子定量法, 可同时完成其定性与定量[9]。本研究进一步针对其中的金钱白花蛇, 设计了特异引物和探针, 建立和优化了金钱白花蛇特异片段的荧光定量PCR测定法, 对市售样品进行了测定, 就质量控制标准提出了建议。
材料与方法
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材料  如表 1所示, 从全国各地收集了银环蛇及其混伪品共16份, 保存于95%乙醇中, 存放在南方医科大学中医药学院标本馆中。各样品均经华南濒危动物研究所张亮助理研究员协助鉴定。其中样品A1~A3与银环蛇鉴别特征完全相符, 即头部椭圆而略扁, 体背黑褐色, 有约50个白色环纹, 环纹宽1~2行鳞片; 背脊突起成棱脊状, 脊鳞扩大呈六角形; 尾下鳞单行。
收集金龙胶囊5批, 均为北京建生药业有限公司生产, 批号分别为190202~190205、190933, 保存于4 ℃冰箱中。
试剂  乙二胺四乙酸(ethylenediaminetetraacetic acid, EDTA) (美国Sigma-Aldrich公司, 批号E5134); 三羟甲基氨基甲烷[tris(hydroxymethyl)aminomethane, Tris]、氯化钠(NaCl)、十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB) (上海麦克林生化科技股份有限公司, 批号C13231085、C12198134、C14635683); 血液/细胞/组织基因组DNA提取试剂盒(天根生化科技有限公司, DP304-03)。
仪器  LightCycler® 96实时荧光定量PCR仪(瑞士Roche公司); ABI 2720型PCR仪(美国Applied Biosystems公司); NanoDrop 2000c超微量紫外分光光度计(美国Thermo Fisher Scientific公司); Tanon-4100凝胶成像系统(上海天能科技有限公司); DYY-6C型电泳仪(北京市六一仪器厂)。
DNA提取  取表 1所示样品的肌肉组织, 用血液/细胞/组织基因组DNA提取试剂盒进行提取, 实验操作按照试剂盒说明书进行, 将收集的DNA放在-20 ℃冰箱保存。
对Cheng等[14]提出的DNA提取方法进行改良, 操作如下: 取700 mg金龙胶囊内含物于离心管中, 加入2.5 mL预热的1 mol·L-1 Tris盐酸盐[tris(hydroxymethyl)aminomethane hydrochloride, Tris-HCl] (pH 8.0) 溶液, 预热前加入千分之二的1 mol·L-1二硫苏糖醇(DTT) 溶液, 再加入125 mg聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP40) 和100 μL β-巯基乙醇, 旋涡震荡1 min。65 ℃水浴2 h, 冷却后, 12 000 r·min-1离心15 min, 取上清液。上清液中加入甲醇2.5 mL, -20 ℃沉淀1 h。8 000 r·min-1离心5 min, 在沉淀中加入600 μL预热的1 mol·L-1 Tris-HCl (pH 8.0) 溶液及16 μL β-巯基乙醇, 65 ℃水浴20 min使沉淀溶解。加入等体积的氯仿/异戊醇(24∶1) 混合液, 12 000 r·min-1离心15 min, 取上清液, 加入等体积预冷的无水乙醇和十分之一体积的3 mol·L-1醋酸钠(NaAc) 溶液(pH 5.2), 于-20 ℃沉降至少1 h。将上一步得到的液体和沉淀分多次收集于血液/细胞/组织基因组DNA提取试剂盒的吸附柱中, 剩余操作步骤根据试剂盒说明书进行。收集的DNA保存在-20 ℃冰箱。
荧光定量PCR扩增  运用Primer5软件设计银环蛇特异性鉴别引物对, Bmforw (5′-ATCGGAGCCTGTC TAAGC-3′) 和Bmrev (5′-GTTCAACCTGTGCCGGCA-3′), 并利用Oligo6软件设计了TaqMan特异探针, Bmpro (5′-CGCATAGAGTTAACCCAACCCGGCTCGC-3′)。
荧光定量PCR扩增体系为: 上下游引物各0.4 μL (终浓度为200 nmol·L-1), 探针Bmpro 0.2 μL (终浓度为100 nmol·L-1), DNA模板用量为2 μL, Bester® qPCR Master Mix 10 μL, 最后加入灭菌ddH2O补足至20 μL。荧光定量PCR扩增程序为: 95 ℃预变性2 min; 95 ℃变性10 s, 50 ℃退火30 s, 72 ℃延伸30 s, 设置40个循环(在每次循环结束后收集荧光信号)。每次试验均设置空白对照组, 并且每个样品均平行测定三次。
标准品的制备  提取银环蛇样品的DNA, 用金钱白花蛇的特异引物进行扩增, 将扩增的目标片段作为标准品。PCR扩增体系为20 μL, 体系中包括10×Buffer Mg2+ (-) 2 μL (终浓度为1×), 2.5 mmol·L-1的dNTP 2 μL (终浓度为0.25 mmol·L-1), 25 mmol·L-1的MgCl2 2 μL (终浓度为2.5 mmol·L-1), 10 μmol·L-1的Bmforw、Bmrev各1 μL (终浓度为0.5 μmol·L-1), 5 U·μL-1 rTaq酶0.2 μL, DNA模板2 μL, 加灭菌ddH2O补足至20 μL。PCR扩增程序为: 94 ℃预变性5 min; 94 ℃变性30 s, 50 ℃复性45 s, 72 ℃延伸45 s (共35个循环); 72 ℃延伸7 min; 4 ℃保存。
纯化后, 用NanoDrop 2000c超微量紫外分光光度计测定纯化后的PCR扩增产物的浓度, 并根据公式[15] N = (6.02×1023)×(DNA concentration×10-9) / (DNA length× 660×5.6)。其中N单位为IU·μL-1, DNA浓度单位为ng·μL-1, 计算得到该特异性扩增产物(标准品) 的国际单位浓度。
标准曲线的绘制  将纯化后的PCR扩增产物作为标准品, 用灭菌ddH2O进行10倍比稀释, 将浓度以10倍变化的标准品DNA作为模板, 进行荧光定量PCR。将荧光定量PCR中得到的CT (cycle threshold) 值与对应浓度的对数值进行线性拟合, 不同浓度的标准品均平行测量三次, 并在实验中设置空白对照组。
荧光定量PCR法的特异性  将提取得到的金钱白花蛇及其混伪品的DNA, 进行荧光定量PCR, 根据荧光扩增曲线和荧光信号判断引物的特异性。每个样品平行测量三次, 并设置空白对照组。
荧光定量PCR法的灵敏度  在标准曲线的建立过程中, 对标准品DNA进行了10倍比稀释, 不同稀释倍数的标准模板得到检测, 根据荧光定量PCR仪所能检测到的最低浓度, 得到该方法的检测灵敏度。
荧光定量PCR法的稳定性  取6个在标准曲线浓度范围内的理论浓度值, 每个浓度分别制备3份样品, 并进行荧光定量PCR检测, 且每个样品平行测定3次, 通过检测中得到的CT值的相对偏差RSD来分析组内差异。在不同时间段(间隔3 h) 分别进行3次独立重复试验, 根据每组在不同时间段所检测CT值的相对标准偏差RSD来分析组间差异。
荧光定量PCR法的准确度  取理论浓度为3.61×106 IU·μL-1标准品所算得的浓度对数值定为理论值, 采用空白加标回收法, 即取该标准品进行DNA提取且最后加入等体积的TE溶液进行溶解, 将得到的DNA样品进行荧光定量PCR扩增, 测得的CT值所算得的浓度对数值为待测值, 最后计算回收率(待测值/理论值×100%)。
自制金龙胶囊的制备  根据金龙胶囊的配方, 按照守宫、金钱白花蛇、蕲蛇的组方比例2∶1∶1制备供试品, 并在保持总量(约为60 mg)不变的情况下, 制备掺入金钱白花蛇常见伪品的供试品。实验中供试品分为4组, 分别为全正品组、正伪1∶1组、正伪1∶4组、全伪品组。如表 2所示, 前三组分别设置了三次平行实验。
自制供试品中金钱白花蛇成分的定量检测  提取供试品的DNA, 以供试品DNA为模板, 进行荧光定量PCR。每个反应均进行三次平行测定, 且设置空白对照组。根据荧光定量检测结果, 计算每毫克供试品含有的DNA特异片段含量。
市售金龙胶囊中金钱白花蛇成分定量检测  将提取的金龙胶囊DNA, 进行荧光定量PCR, 各样本平行测定三次, 且设置空白对照(双蒸水替代DNA模板), 根据得到的CT值计算含量。
结果
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1 荧光定量PCR法的特异性
金钱白花蛇及13种混伪品的荧光定量PCR扩增曲线如图 1所示, 3个正品金钱白花蛇出现特异性的S形荧光扩增曲线, CT值为12.69~16.91。在循环数超过32时, 部分伪品出现了扩增。
2 荧光定量PCR法的标准曲线与灵敏度
将浓度为4.6×100~4.6×107 IU·μL-1 (10倍比梯度变化) 的标准品进行荧光定量PCR扩增, 如图 2所示, 各不同浓度的标准品扩增正常, 各浓度间间距相等。当标准品稀释至4.6 IU·μL-1时, 仍有扩增信号, 平均CT值为35.5, 说明该方法具有高灵敏度。结合特异性扩增结果(图 1), 为了防止混伪品扩增信号的干扰, 将金钱白花蛇的最低检测浓度设置为4.6×101 IU·μL-1。
将得到的CT值与浓度的对数值进行线性拟合, 在4.6×101~4.6×107 IU·μL-1线性范围内, 标准曲线回归方程为y = -3.672x + 39.532 (R² = 0.999 1), 扩增效率为87.2%, CT值和标准品浓度的对数值间呈良好的线性关系, 线性范围是4.6×101~4.6×107 IU·μL-1。
3 荧光定量PCR法的稳定性
一共设置了6组标准品, 浓度在7.55×101~7.55×106 IU·μL-1之间, 各组分别设置了三个平行样品, 组内重复测定的RSD值为0.35%~2.26%, 组间重复测定的RSD值在0.63%~1.64%之间, 均在可接受范围内, 说明建立的金钱白花蛇的荧光定量PCR法稳定性好。
4 荧光定量PCR法的准确度
取理论浓度为3.61×106 IU·μL-1的标准品, 计算得到回收率为96.52%~98.02%, 均在定量检测方法可接受的回收率范围内, 说明建立的金钱白花蛇的荧光定量PCR法准确度良好。
5 自制供试品中金钱白花蛇的定量检测
当供试品中正伪品的投料比为100∶0时, 其平均含量为2.45×105 IU·mg-1; 正伪品投料比为50∶50时, 其平均含量为8.82×104 IU·mg-1; 正伪品投料比为20∶80时, 其平均含量为3.54×104 IU·mg-1; 未加入金钱白花蛇的全伪品组及空白对照组, 均未出现荧光扩增反应。结果显示, 自制样品浓度的对数值与金钱白花蛇肌肉组织重量成正比, 说明DNA片段含量可以反映出实际投料量, 可用于估算原始投料量。
6 市售金龙胶囊中金钱白花蛇组分的定量检测
如表 3所示, 5批金龙胶囊的DNA经过荧光定量PCR扩增, CT值在26.97~28.03之间; RSD值在0.40%~0.89%之间, 说明测定结果稳定准确; 市售金龙胶囊中金钱白花蛇组分的特异片段的含量在24.1~46.6 IU·mg-1之间。
讨论
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目前, 中成药中的动物类组分, 多因指标成分不明而缺乏质控标准。本研究提出了采用分子定量技术对中成药中动物类药材进行质量控制的新思路, 即用物种特异DNA片段的浓度反映中成药中动物类药材的含量。本研究以金龙胶囊中金钱白花蛇为例验证了其可行性。
DNA的提取是分子质量控制最关键的一环, 对提取方法的研究是分子质量控制方法成功建立的必经环节。从中成药中获得高质量的基因组DNA比从生药材中获得要困难得多。在中成药的制备过程中, 通常会加入大量的糊精、蔗糖和其他辅料[16], 并且伴有加热、沉淀、过滤等过程, 这些都会导致DNA降解。金龙胶囊在其制备过程中, 对原料进行超低温破碎处理, 并经过过滤、高速离心、超滤等过程, 这导致了DNA片段的大量丢失。由于在制备胶囊内含物的过程中进行了加工, 动物材料的残留物很少, 使得DNA提取非常困难。为了从金龙胶囊中获得高质量的DNA, 本研究优化了提取工艺, 提高了提取DNA时所需胶囊内含物的含量。为了消除β-环糊精包裹和淀粉掩盖的影响, 应调整提取缓冲液的配方, 使缓冲液具有更好的消化能力。通过提高十二烷基硫酸钠(sodium dodecyl sulfate, SDS) 的浓度、CTAB的浓度等对提取配方进行调整, 以提高消化能力。样品用量与提取液的用量比为1 mg样品使用10 μL的提取液, 在这个比例用量下, 提取消化过程中, 样品溶液甚至会达到澄清状态, 实现样品被完全消化的效果, 大大提高了提取效率。
Cheng等[14]发现用甲醇沉淀可以有效克服淀粉对DNA提取的不良影响, 在这个基础上对提取方法进行改进, 可以得到质量更高的DNA。为进一步减少多糖的影响, 本研究尝试运用试剂盒中的吸附柱对DNA进行收集, 发现有很好的改善作用, DNA的纯度有明显的提高。
在前期研究中, Li[9]采用SYBR Green染料法, 对金龙胶囊配方中的蕲蛇组分进行定性及定量分析, Li等[10]运用TaqMan荧光探针检测尖吻蝮线粒体基因组Cytb区的特定片段。探针法与染料法相比, TaqMan荧光探针的应用使反应比SYBR Green染料法更具特异性, 并提高了灵敏度, 从1.3×103 IU·μL-1提高到1.6 IU·μL-1 [9, 10]。此外, 该测定方法显示了更宽的线性范围, 染料法的线性范围是1.3×104~1.3×109 IU·μL-1 [9], 探针法是1.6×101~1.6×106 IU·μL-1 [10]。因此本研究采用TaqMan荧光探针, 建立了荧光定量PCR检测方法, 对金龙胶囊中的金钱白花蛇进行了定性与定量。
应用该特异探针进行荧光定量PCR扩增时, 某些伪品出现扩增信号, CT值均超过32, 远大于银环蛇的CT值。若出现特异性S形荧光扩增曲线且CT值小于32, 则为阳性反应, 样品为正品金钱白花蛇; 若无荧光扩增或CT值大于32, 则为阴性反应, 样品为混伪品。
本研究对金钱白花蛇的分子定量方法进行了方法学考察。组内和组间实验的RSD值都在可接受的范围内(< 5%), 说明所建立的荧光定量PCR测定方法在用于金钱白花蛇分子定量时具有良好的重复性和稳定性。此外, 回收率实验中加标样品的回收率为96.52%~98.02%, 均在可接受的范围内, 说明了测定方法的准确性。
从自制金龙胶囊的荧光定量PCR结果中, 可以看到, 所有含有金钱白花蛇的样品都出现了荧光扩增反应, 而在未加入金钱白花蛇的全伪品组及空白对照组, 均未出现荧光扩增反应, 这说明该荧光定量PCR检测方法具有良好的再现性和特异性, 可用于金龙胶囊中金钱白花蛇的定性鉴别。
对自制金龙胶囊中金钱白花蛇进行定量检测, 结果显示自制样品中特异DNA片段的含量的对数值与正品的投料量成正比, 说明DNA片段含量可以反映出实际投料量。运用该方法, 成功检测出了5个批次市售金龙胶囊金钱白花蛇的组分, 可作为金龙胶囊中金钱白花蛇质量控制的一种新方法, 不仅可以定性识别中成药中的成分, 还可对其成分进行定量分析。5个批次市售金龙胶囊中金钱白花蛇组分的浓度是比较接近的, 说明制剂生产工艺比较稳定, 其DNA特异片段含量在24.1~46.6 IU·mg-1之间, 因此, 建议将金龙胶囊中金钱白花蛇含量最低限设定为不低于19.3 IU·mg-1, 即比检测到的最小值低20%。
含有动物类药材中成药的质量控制中, 存在指标成分缺少或特异性低的缺点。本研究提供了一种新的思路, 采用甲醇沉淀结合吸附柱的提取方法, 从金龙胶囊DNA中提取出质量较好的DNA, 运用荧光定量PCR法, 建立了一种DNA分子定量方法, 并将其应用于金钱白花蛇的定性和定量分析。该方法具有特异性好、灵敏度高、重复性好、准确度高等优点。研究对其他含动物药的中成药的质量控制有一定的借鉴意义。
作者贡献: 林慧、李婵负责主要实验操作、实验设计、结果分析与统计; 林慧负责论文撰写与修改; 晁志负责实验设计、指导和参与论文的写作与修改。
利益冲突: 所有作者均声明不存在利益冲突。
基金
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  • 国家自然科学基金(81573540)
  • 广州市重点研发计划(202206010111)
  • 建生鲜药创研基金(JSJC-2019-01-03-045)
  • 建生鲜药创研基金(JSJC-20200102-054)
  • 建生鲜药创研基金(JSJC-20210102-065)
文献
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2023年第58卷第9期
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文章信息
doi: 10.16438/j.0513-4870.2022-1437
  • 接收时间：2022-12-29
  • 首发时间：2025-11-21
  • 出版时间：2023-09-12
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出版历史
  • 收稿日期：2022-12-29
  • 修回日期：2023-05-15
基金
国家自然科学基金(81573540)
广州市重点研发计划(202206010111)
建生鲜药创研基金(JSJC-2019-01-03-045)
建生鲜药创研基金(JSJC-20200102-054)
建生鲜药创研基金(JSJC-20210102-065)
作者信息
    南方医科大学中医药学院, 广东 广州 510515

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2种不同金属材料的力学参数
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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