目的: 建立UPLC-MS/MS法同时测定清热解毒口服液中的栀子苷、绿原酸、新绿原酸、哈巴苷、龙胆苦苷、黄芩苷6个成分含量,并对以上成分展开网络药理学研究,揭示其潜在药理作用机制。方法: 色谱柱采用Phenomenex Kinetex C18柱(50 mm×2.1 mm,2.6 μm),流动相采用0.3%甲酸水溶液-0.3%甲酸乙腈梯度洗脱,流速为0.4 mL·min-1,柱温40 ℃,进样体积为2 μL。质谱方法选择电喷雾离子源(ESI),负离子模式,多反应检测模式(MRM);方法学验证包括专属性、精密度、重复性、稳定性、准确度等;网络药理学研究基于Swiss Target Prediction、Drugbank、TTD、KEGG、Cytoscape等数据库或平台,收集成分-靶点-代谢通路-疾病信息,并绘制药理作用网络图。结果: 方法学验证均符合方法学要求。栀子苷、绿原酸、新绿原酸、哈巴苷、龙胆苦苷、黄芩苷分别在0.050~5.000、0.049~4.900、0.050~5.000、0.010~1.020、0.052~5.150、0.052~5.150 μg·mL-1范围内线性关系良好。2个厂家各3个不同批号清热解毒口服液黄芩苷含量均符合药典要求(>1.0 mg·mL-1),然而栀子苷、绿原酸、新绿原酸、哈巴苷等成分呈现较大含量差异,必要对其进行质量控制。以上成分通过IL-17信号通路、自然杀伤细胞介导的细胞毒性、NF-κB信号通路、抗叶酸耐药通路、TRP通道的炎症介质调节等通路,发挥其抗炎、抗流感、治疗上呼吸道疾病、发热、烦躁口渴等方面的作用。结论: 建立的UPLC-MS/MS快速、准确可用于清热解毒口服液质量控制,网络药理学研究工作初步揭示了其潜在作用机制。本研究为清热解毒口服液的质量评价和临床应用提供了参考。
Objective: To establish an UPLC-MS/MS method for quantitative analysis of geniposide, chlorogenic acid, neochlorogenic acid, harpagide, gentiopicroside and baicalin in Qingre Jiedu oral liquid. And to investigate potential mechanism of pharmacology effect by approach of network pharmacology. Methods: Components were separated by a Phenomenex Kinetex C18 column (50 mm×2.1 mm, 2.6 μm), with mobile phase consisted of0.3% formic acid aqueous solution and 0.3% formic acid acetonitrile, using gradient elution. The flow rate was 0.4 mL·min-1. Column temperature was set as 40 ℃ and injection volume was 2 μL. Mass data acquisition was carried out in ESI- mode on an AB Sciex 4500 MD mass spectrometer (AB Sciex, Framinghan, MA, USA), using multiple reaction detection mode (MRM). The developed method was validated by aspects of specificity, precision, repeatability, stability, and accuracy. Network pharmacology analysis was conducted based on platform of Swiss Target Prediction, Drugbank, TTD, KEGG, Cytoscape software and so on. Results: The established method was well validated, with linearity range of geniposide, chlorogenic acid, neochlorogenic acid, harpagide, gentiopicroside and baicalin of 0.050-5.000 (r=0.998 1), 0.049-4.900 (r=0.998 4), 0.050-5.000 (r=0.998 3), 0.010-1.020 (r=0.997 3), 0.052-5.150(r=0.999 3) and 0.052-5.150 (r=0.997 9) μg·mL-1, respectively. The contents of baicalin in Qingrejiedu oral liquid of 3 different batches from 2 manufacturers all met the requirements of the Pharmacopoeia (>1.0 mg·mL-1). However,thecontents of geniposide, chlorogenic acid, neochlorogenic acid, and harpaside were quite different,presenting necessity for multicomponents quality control of Qingre Jiedu oral liquid. Network pharmacology study was carried out to explore potential pathways under effect of anti-inflammatory, anti-influenza, treatment of upper respiratory tract diseases, fever, dysphasia and thirst. Involved KEGG pathways were revealed as follows: IL-17 signaling pathway, cytotoxicity mediated by natural killer cells, NF-κB signaling pathway, anti-folic acid resistance pathway, inflammatory mediator regulation of TRP channel and others. Conclusion: The established UPLC-MS/MS method is fast and accurate for quality evaluation of Qingre Jiedu oral liquid. Network pharmacology analysis reveals its potential mechanism of action, verifying reliability of established quality method and benefitting clinical application of Qingre Jiedu oral liquid.
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