摘要
目的运用网络药理学方法分析消炎退热颗粒清热抗炎的作用机制,并采用超高效液相色谱-质谱联用(UPLC-MS/MS)技术测定其8种主要活性成分。方法通过Swiss Target Prediction数据库筛选退热颗粒入血成分的靶点,利用CTD数据库获取符合发热的疾病主要靶点,借助String平台进行蛋白相互作用(PPI)分析;通过DAVID数据库进行GO功能与KEGG通路富集分析,然后用Cytoscape软件构建成分-靶点-通路网络,并对关键化合物-靶点进行分子对接验证。采用UPLC-MS/MS法测定消炎退热颗粒中靛玉红、异甘草素、甘草素、甘草酸、甘草苷、秦皮乙素、咖啡酸、绿原酸的含量。采用WATERS ACQUITY UPLC?BEH C18色谱柱(2.1 mm×100 mm,1.7μm);以0.1%甲酸乙腈(A)-0.1%甲酸-5 mmol·L^(-1)甲酸铵(B)为流动相进行梯度洗脱,以多反应监测(MRM)模式进行测定。结果网络药理学及分子对接筛选出消炎退热颗粒中靛玉红、秦皮乙素和咖啡酸等16个潜在的活性成分,获得转录因子(JUN)、黏着连接蛋白β1(CTNNB1)和半胱氨酸天冬氨酸蛋白酶3(CASP3)等10个核心靶点,通路富集分析发现其清热抗炎作用主要涉及白细胞介素17(IL-17)和缺氧诱导因子1(HIF-1)等信号通路;分子对接实验表明主要活性成分与关键靶点对接呈现良好的亲和力。含量测定结果表明,各成分在各自的浓度范围内具有良好的线性关系(r>0.999),精密度、准确度和稳定性良好。16批样品中8种成分的含量分别为0.94~3.41、0.99~5.61、0.80~5.84、85.48~141.11、4.30~10.09、152.35~271.80、11.31~26.94、1.99~5.58μg·g-1,其中,2个厂家样品中靛玉红、甘草素、异甘草素、甘草苷和绿原酸的含量具有显著性差异。结论该研究初步揭示了消炎退热颗粒多成分、多靶点、多途径发挥清热抗炎的作用机制;建立了消炎退热颗粒多成分的含量测定方法,为其药理学研究和质量控制提供了参考依据。
Objective To analyze the heat-clearing and anti-inflammatory mechanism of Xiaoyan Tuire Granules by network pharmacology,and to determine the 8 main active components of Xiaoyan Tuire Granules by ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS/MS).Methods Swiss Target Prediction database was used to screen the target of components absorbed in the blood of Xiaoyan Tuire Granules.The main target of disease was obtained by CTD database,and the protein-protein interaction(PPI)was analyzed by String platform.GO and KEGG enrichment analysis were conducted using the DAVID database,followed by the construction of the components-targets-pathways network using Cytoscape software.Finally,the molecular docking verification of the key compound-target was conducted.The main active components in Xiaoyan Tuire Granules,including indirubin,isoliquiritigenin,liquiritigenin,glycyrrhizinic acid,liquiritin,esculetin,caffeic acid and chlorogenic acid,were determined by UPLC-MS/MS method.Analysis was performed on a WATERS ACQUITY UPLC^?BEH C_(18)column(2.1 mm×100 mm,1.7μm)with gradient elution of 0.1%formate in acetonitrile(A)-0.1% formate-5 mmol·L^(-1)ammonium formate(B),and the determination was carried out in multiple reaction monitoring(MRM)mode.Results Sixteen potential active components,such as indirubin,esculetin,and caffeic acid were identified by network pharmacology and molecular docking,and 10 core targets including transcription factor AP-1(JUN),adhesive connexinβ1(CTNNB1)and cysteine aspartic protease-3(CASP3)were predicted.The pathway enrichment analysis revealed that heat-clearing and anti-inflammatory effects of Xiaoyan Tuire Granules were mainly related to signaling pathways such as interleukin-17(IL-17)and hypoxia-induced cause-1(HIF-1).Molecular docking experiments showed that its main active components docked well with core targets.The results of content determination exhibited that all components had good linear relationship within certain concentration range(r>0.999),good precision,repeatability,and stability.The contents of 8 components in 16 batches of samples were0.94-3.41,0.99-5.61,0.80-5.84,85.48-141.11,4.30-10.09,152.35-271.80,11.31-26.94,1.99-5.58μg·g^(-1),respectively.The contents of indirubin,isoliquiritigenin,liquiritigenin,liquiritin,and chlorogenic acid in Xiaoyan Tuire Granules from two manufacturers were significantly different.Conclusion This study preliminarily revealed the mechanism of Xiaoyan Tuire Granules,which exerted heat-clearing and anti-inflammatory effects through multiple components,targets and pathways.A method for the determination of multiple components in Xiaoyan Tuire Granules was established.This study may provide a reference for its pharmacological research and quality control.
作者
王冠楠
王宏进
苑楠楠
针涛
王佳雪
孙立新
WANG Guannan;WANG Hongjin;YUAN Nannan;ZHEN Tao;WANG Jiaxue;SUN Lixin(Shenyang Pharmaceutical University,Benxi 117004 Liaoning,China;Liaoning Institute for Food Control,Shenyang 110168 Liaoning,China)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2023年第12期1753-1766,共14页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
辽宁特聘教授滚动资助项目[辽教函发(2018)35号]。
