高效液相色谱结合化学指纹图谱进行多模式识别，鉴定金线莲真伪
Summary
在这里，我们提出了一种建立高效液相色谱（HPLC）的协议，结合化学指纹多模式识别，为有效鉴定铁 线莲 及其掺杂物的真正品种提供了一种新策略。
Abstract
以 铁线莲 为例，构建了中药材及其相关掺杂物的鉴别方法。采用高效液相色谱（HPLC）指纹图谱结合化学计量学方法，对10批次真品川木通品种和5批次相关掺杂物进行分析比较，包括聚类分析（CA）、主成分分析（PCA）和正交偏最小二乘判别分析（OPLS-DA）。此外，测定了β-谷甾醇的含量。建立川木通对照化学指纹图谱，鉴定出12个共同峰。10批正品川木通品种指纹与对照指纹的相似度为0.910-0.989，而5批次掺杂物的相似度仅为0.133-0.720。基于色谱图中的常见峰，将15批次样品通过PCA分为3个含量等级，并按CA归集为4类，明确区分正品川木通和川木通掺杂物。此外，通过OPLS-DA发现了7种能够有效识别川木桐正宗和川木掺杂物的差异成分。10批次纯川木通品种的β-谷甾醇含量为97.53-161.56 μg/g，而5批次掺杂物的β-谷甾醇含量差异较大，其中 铁线莲 的β-谷甾醇含量为马兹。和 铁线莲 gouriana Roxb。Var. finetii Rehd.等威尔斯。明显低于川木通正宗品种。本研究建立的HPLC指标组分含量和化学指纹多模式识别方法为有效鉴定正宗中药材及相关掺杂物提供了新的策略。
Introduction
川木通，铁线莲的干花。或铁线莲蒙大拿州Buch.-Ham.，是诊所1，2，3常用的传统中药。它用于治疗泌尿问题，水肿，舌头和口腔溃疡，乳汁分泌减少，关节僵硬和湿热引起的肌肉疼痛4。川木通一直从野生品种获得，主要分布在西南地区，尤其是四川，那里的品质最好5，6。由于正宗品种具有相似的特征，因此很难区分正宗品种及其密切相关的掺杂物7，8，9，10。2020年版《中国药典》中川木通的质量标准仅规定了性质、显微鉴别、薄层鉴定等，没有进行含量测定，不能有效鉴别掺杂物，因此存在潜在风险。此外，很少有报告比较和鉴定川木通和相关植物。因此，一种确保川木通真伪的质量控制方法值得进一步研究。
川木通的化学成分主要由桫椤类五环三萜类化合物及其糖苷、黄酮类化合物和有机酸11，12，13，14组成。其中齐墩果酸、β-谷甾醇、豆甾醇、麦角甾醇具有不同强度的利尿作用，可能是促进利尿、缓解纹尿的潜在药效学物质15，16。化学指纹图谱是通过高效液相色谱（HPLC）、气相色谱（GC）等方法分离和检测样品中所含的许多化学成分而获得的。采用适当的统计分析方法分析川木通的特性，可以确定中药的整体质量控制和科学鉴定17、18、19。
本研究共收集了10批次川木桐正宗品种和5批次掺杂物。采用HPLC指纹图谱法结合多模式识别技术对它们的质量进行比较和分析，包括聚类分析（CA）、主成分分析（PCA）、正交偏最小二乘判别分析（OPLS-CA）和药效学成分含量测定。该方案建立了高特异性正宗品种鉴定方法，是科学鉴定中药材正宗品种和掺杂物的新策略。
Protocol
1. 化学指纹检测方法 色谱条件制备乙腈（A）/水（B）流动相。在HPLC软件中设置梯度程序如下：0-20分钟，3%A-10%A;20-25分钟，10%A-13%A;25-65分钟，13%A-25%A;65-75分钟，25%A-40%A;75-76分钟，40%A-3%A;76-85分钟，3%A-3%A。 将流动相的流速保持在1.0 mL/min。 在保持在30°C的C18色谱柱（250 mm x 4.6 mm，5 μm）上进行色谱分离。 将进样体积设置为 10 μL。 检测波长为…
Representative Results
川木通色谱指纹图谱及相似度分析（SA）精密度、重复性和稳定性相对保留时间的RSD值分别低于0.46%、1.65%和0.53%;相对峰面积的RSD值分别低于4.23%、3.56%和3.96%。 如图1A，B所示，10份真品川木通样品的HPLC指纹图中有12个不同的共同峰（从峰1到峰12）。由于10号峰面积比较大，分辨率较好;由于它是每个样品中存在的组分，因此将其用作参考峰来研究指?…
Discussion
用于研究的样本采集是构建多模式识别识别中药材真伪的第一步。通过市场调研，我们发现四川雅安、凉山、乐山是川木通野生资源的主要产区。同一属的相关品种也具有相同的地理分布6，20;CC、DC、DE、XS和SMT经常被误用为创木通16，21;因此，本研究在上述产地采集了10批次正宗川木桐和5批次混合样品，并确?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了四川省中医药管理局项目（编号：2020JC0088，编号：2021MS203）的支持。
Materials
| Acetic acid | Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China | 2017381038 | |
| Acetonitrile | Sigma-Aldrich Trading Co., Ltd., Shanghai, China | WXBD5243V | |
| β-Sitosterol | Meisai Biological Technology Co., Ltd., Chongqing, China | 20210201 | |
| C18 column | Yuexu Material Technology Co., Ltd., Shanghai, China | Welch Ultimate LP | |
| Chuanmutong | Guoqiang Chinese Herbal Pieces Co., Ltd., Sichuan, China | 19020103 | CMT-1 |
| Chuanmutong | Hongya Wawushan Pharmaceutical Co., Ltd., Sichuan, China | 200701 | CMT-2 |
| Chuanmutong | Hongpu Pharmaceutical Co., Ltd., Sichuan, China | 200701 | CMT-3 |
| Chuanmutong | Hongpu Pharmaceutical Co., Ltd., Sichuan, China | 200901 | CMT-4 |
| Chuanmutong | Xinrentai Pharmaceutical Co., Ltd., Sichuan, China | 210701 | CMT-5 |
| Chuanmutong | Haobo Pharmaceutical Co., Ltd., Sichuan, China | 210401 | CMT-6 |
| Chuanmutong | Xinrentai Pharmaceutical Co., Ltd., Sichuan, China | 200901 | CMT-7 |
| Chuanmutong | Wusheng Pharmaceutical Group Co., Ltd., Sichuan, China | 201201 | CMT-8 |
| Chuanmutong | Limin Chinese Herbal Pieces Co., Ltd., Sichuan, China | 201001 | CMT-9 |
| Chuanmutong | Yuhetang Pharmaceutical Co., Ltd., Sichuan, China | 210501 | CMT-10 |
| Clematis argentilucida (Levl. et Vant.) W. T. Wang | Madzi Bridge, Sanlang Township, Tianquan County, Sichuan, China | – | CC |
| Clematis apiifolia var. obtusidentata Rehd. et Wils. | Heilin Village, Qiliping Township, Hongya County, Sichuan, China | – | DC |
| Clematis peterae Hand.-Mazz. | Huangmu Village, Huangmu Township, Hanyuan County, Sichuan, China | – | DE |
| Clematis gouriana Roxb. Var. finetii Rehd. et Wils | Mixedang Mountain, Huangwan Township, Emei County, Sichuan, China | – | XS |
| Clematis finetiana Levl. et Vaniot. | Wannian Village, Huangwan Township, Emei County, Sichuan, China | – | SMT |
| Electronic balance | Haozhuang Hengping Scientific Instrument Co., Ltd., Shanghai, China | FA1204 | |
| Ergosterol | Meisai Biological Technology Co., Ltd, Chongqing, China | 20210201 | |
| Ethanol | Kelon Chemical Co., Ltd., Chengdu, China | 2021112602 | |
| Ethyl acetate | Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China | 2017042043 | |
| Formic acid | Kelon Chemical Co., Ltd., Chengdu, China | 2016062901 | |
| High performance liquid chromatography | Agilent, USA. | 1260 | |
| IBM SPSS Statistics version 26.0 | International Business Machines Corporation, USA | – | |
| Methanol | Sigma-Aldrich Trading Co., Ltd., Shanghai, China | WXBD6409V | |
| Methanol | Kelon Chemical Co., Ltd., Chengdu, China | 202010302 | |
| n-butyl alcohol | Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China | 2020071047 | |
| Petroleum ether | Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China | 2020090125 | |
| Phosphoric acid | Comeo Chemical Reagent Co., Ltd., Tianjin, China | 20200110 | |
| SESCF-TCM version 2012 | National Pharmacopoeia Commission, China | – | http://114.247.108.158:8888/login |
| Stigmasterol | Meisai Biological Technology Co., Ltd., Chongqing, China | 20210201 | |
| Trichloromethane | Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China | 20200214 | |
| Umetrics SIMCA version 14.1.0.2047 | Umetrics, Sweden | – | https://www.sartorius.com/en/products/process-analytical-technology/data-analytics-software/mvda-software/simca/simca-free-trial-download |
| Ultrapure water machine | Youpu Ultrapure Technology Co., Ltd., Sichuan, China | UPH-II-10T | |
| Ultrasonic cleaner | Kunshan Hechuang Ultrasound Instrument Co., Ltd., Jiangsu, China | KH3200E |
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Wang, F., Qian, Z., Liao, G., Zeng, J., Huang, D., Liu, Q., Xie, X. HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis. J. Vis. Exp. (189), e64690, doi:10.3791/64690 (2022).