畲药小香勾香豆素类成分RP-HPLC含量测定及抗炎靶点预测

Determination of Coumarin Ingredients in Xiaoxianggou of She Herb by RP-HPLC and Prediction of the Corresponding Anti-inflammatory Targets

[目的]以反相高效液相色谱(reverse phase high-performance liquid chromatography,RP-HPLC)法测定畲药小香勾香豆素类成分补骨脂素和佛手柑内酯的含量,并结合网络药理学和动物实验验证小香勾的抗炎作用。[方法]色谱柱为Waters C18柱(4.6 mm×250 mm,5.0μm),流动相A为乙腈,流动相B为0.1%甲酸溶液(体积比),梯度洗脱,流速为0.8 mL·min^(-1),补骨脂素检测波长245 nm,佛手柑内酯320 nm,柱温25℃。利用网络药理学平台收集小香勾香豆素类成分及抗炎相关靶点,利用Cytoscape 3.8.2软件建立成分-靶点网络,借助网络拓扑分析插件(Cytoscape network centrality analysis,CytoNCA)筛选抗炎核心靶点,进一步采用Metascape软件进行京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomics,KEGG)通路富集分析。通过二甲苯致小鼠耳肿胀法进一步验证小香勾的抗炎作用。[结果]补骨脂素和佛手柑内酯的质量浓度分别在0.51~102.00μg·mL^(-1)(r=0.9996)和0.24~48.00μg·mL^(-1)(r=0.9998)时与峰面积呈良好的线性关系。通过筛选获得小香勾补骨脂素和佛手柑内酯的相关基因57个,抗炎靶点2148个,交集靶点31个。KEGG通路富集分析发现,这些靶点主要涉及化学致癌作用-活性氧、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路、癌症途径、催乳素信号通路和叶酸生物合成等信号通路。与模型对照组比较,小香勾高剂量组(400 mg·kg^(-1))能显著抑制二甲苯所致小鼠耳肿胀(P<0.01),显示小香勾具有良好的抗炎作用。[结论]建立的RPHPLC法操作简单,灵敏度高,可用于小香勾质量控制。小香勾香豆素类成分通过多靶点多通路表现出良好的抗炎作用,为后续深入探讨小香勾抗炎机制提供了有利的科学依据。

[Objective]To simultaneously determinate coumarin ingredients psoralen and bergapten in Xiaoxianggou(Radix et Chulis Fici Angustifoliae)of She herb by reverse phase high-performance liquid chromatography(RP-HPLC),and validate the anti-inflammatory effect of Xiaoxianggou with network pharmacology and animal experiment.[Methods]The RP-HPLC system consisted of a Waters C18 column(4.6 mm×250 mm,5.0μm)by gradient elution using a mixture of acetonitrile-0.1%HAC as the mobile phase at a flow rate of 0.8 mL·min^(-1).The UV detection wavelengths of psoralen and bergapten were monitored at 245 nm and 320 nm respectively and the column temperature was 25℃.The targets of Xiaoxianggou ingredients and anti-inflammatory genes were collected using network pharmacology platform.The herb ingredient-target network was constructed using Cytoscape 3.8.2 software and the core anti-inflammatory targets were further screened by Cytoscape network centrality analysis(CytoNCA).The Kyoto Encyclopedia of Genes and Genomics(KEGG)pathway enrichment analysis of the targets were analyzed with Metascape software.Dimethylbenzene-induced auricle edema in mice was respectively performed to investigate the anti-inflammatory effect of Xiaoxianggou.[Results]There was good linearity and the response ranges of psoralen and bergapten in 0.51~102.00μg·mL^(-1)(r=0.9996),0.24~48.00μg·mL^(-1)(r=0.9998),respectively.A total of 57 potential targets of psoralen and bergapten,2148 inflammatory genes and 31 intersection targets were screened.The targets were involved in chemical carcinogenesis-reactive oxygen species,mitogen-activated protein kinase(MAPK)signaling pathway,pathways in cancer,prolactin signaling pathway and folate biosynthesis.Compared with model control group,the high dose group of Xiaoxianggou(400 mg·kg^(-1))could significantly inhibit the ear swelling induced by dimethylbenzene in mice(P<0.01),indicating that Xiaoxianggou had good antiinflammatory effect.[Conclusion]The method was simple,sensitive and rapid,and could be used for simultaneous determination of psoralen and bergapten in Xiaoxianggou.Coumarin ingredients of Xiaoxianggou existed good anti-inflammatory effect through multi-target and multi-pathway.The studies provided a scientific basis for future exploring the anti-inflammatory mechanism of Xiaoxianggou.