二苓化湿降酸颗粒治疗痛风的网络药理学分析及其主要成分的含量测定

Network Pharmacology and Content Determination of Main Components of Erling Huashi Jiangsuan Granules in Treating Gout

目的以网络药理学分析二苓化湿降酸颗粒治疗痛风的作用机制,并采用高效液相色谱(HPLC)法测定其主要活性成分。方法借助中药系统药理学分析平台(TCMSP),筛选二苓化湿降酸颗粒的主要活性成分及靶点,通过Genecards、OMIM、CTD数据库获取痛风主要靶点,利用STRING平台进行蛋白质相互作用分析;通过Metascape数据库进行GO和KEGG富集分析,采用Cytoscape软件构建“活性成分-核心靶点-信号通路”网络,并通过Autodock完成关键化合物-靶点的模拟对接。采用HPLC法同时测定二苓化湿降酸颗粒主要活性成分槲皮素、木犀草素、山奈酚。色谱柱为Hypersil GOLD^(TM)(250 mm×4.6 mm,5μm);以甲醇(A)-0.4%磷酸溶液(B)为流动相进行梯度洗脱;检测波长为360 nm;柱温为30℃;流速为1.0 mL·min-1;进样量为10μL。结果从二苓化湿降酸颗粒中获得木犀草素、槲皮素、山奈酚、β-谷甾醇、汉黄芩素等80个活性成分,预测二苓化湿降酸颗粒治疗痛风的核心靶点有86个;调节通路中,与痛风相关有4条、与细胞凋亡相关有2条、与免疫炎症相关有9条;分子对接结果显示,二苓化湿降酸颗粒的主要活性成分与痛风核心靶点蛋白均有较好的结合活性。HPLC法测定结果显示,二苓化湿降酸颗粒中主要活性成分槲皮素、木犀草素、山奈酚的含量分别为0.0764、0.0151、0.0175 mg·g^(-1),3种成分在各自线性范围内线性关系良好,平均回收率分别为99.01%、98.12%、98.27%,RSD分别为1.66%、2.13%、2.60%。结论初步揭示二苓化湿降酸颗粒治疗痛风可通过多成分、多途径降低尿酸、抑制炎症等的作用机制;在网络药理学研究的基础上建立了二苓化湿降酸颗粒的主要活性成分含量测定方法,为其药理学研究和质量控制提供了一定的依据。

Objective To analyze the action mechanism of Erling Huashi Jiangsuan Granules (EHJG)in treating gout by network pharmacology,and to determine its main active ingredients by high performance liquid chromatography (HPLC).Methods The main active ingredients and targets of EHJG were screened from TCMSP.The main targets of gout were obtained by Genecards,OMIM and CTD databases,and the protein interaction was analyzed by STRING platform.GO and KEGG enrichment analysis were conducted using Metascape database,and Cytoscape software was used to construct the"active ingredient-core target-action pathway"network,and Autodock was used to conduct docking between the core compound and target.Finally,the main active ingredients in EHJG included quercetin,luteolin and kaempferol were determined simultaneously by HPLC.The separation was applied on Hypersil GOLD^(TM)column (250 mm×4.6 mm,5μm) by gradient elution with methanol (A) and 0.4%phosphoric acid solution (B) as mobile phase.The detection wavelength was 360 nm,column temperature was30℃,the flow rate of the mobile phase was 1.0 mL·min^(-1)and the injection volume was 10μL.Results A total o80 main active ingredients of EHJG,including luteolin,quercetin,kaempferol,β-sitosterol and wogonin were obtained,and 86 core targets of EHJG were predicted for the treatment of gout disease.Among the main regulatory pathways,4 items were related to gout,2 items were related to apoptosis and 9 items were related to immune inflammation.Molecular docking results showed that the main active ingredients of EHJG had good binding activity with the core target protein of gout.The results of HPLC suggested that the contents of quercetin,luteolin and kaenferol in EHJG were 0.076 4,0.015 1,0.017 5 mg·g^(-1).The above three ingredients had good linearity within their respective linear range,and the average recoveries were 99.01%,98.12%and 98.27%with RSD of 1.66%(n=3),2.13%(n=3)and 2.60%(n=3).Conclusion This study preliminarily revealed that EHJG could be used to treat gout by lowering uric acid in the manner of multi-component and multi-pathway and inhibiting inflammation.On the foundation of network pharmacology,the analytical method of the main active ingredients in EHJG was established,which provided a certain research basis for pharmacological research and quality control of EHJG.