民间药材降真香抗炎质量标志物的研究

Identification of quality markers associated with the anti-inflammatory effects of Jiangzhenxiang,a folk medicine

筛选海南、广西的民间药材降真香的抗炎质量标志物(quality marker,Q-marker)。采用质谱分析、鉴定降真香的化学成分。通过网络药理学分析降真香抗炎活性成分、作用靶点和信号通路,筛选降真香抗炎的潜在质量标志物。用高效液相色谱法验证潜在质量标志物,体外实验验证潜在质量标志物的抗炎活性、作用靶点。实验方案经由广西中医药大学实验动物福利伦理委员会审查通过。结果从降真香提取物中分析、鉴定出31个化学成分。通过网络药理学筛选得到成分靶点727个、疾病靶点422个以及包括前列腺素G/H合酶2(prostaglandin G/H synthase 2,PTGS2)在内的作用靶点110个,靶点主要富集在炎症反应、对脂多糖的反应等498个生物过程,TNF信号通路、Toll样受体信号通路等101条信号通路。通过高效液相色谱法可检测出降真香提取物中含有异鼠李素、芒柄花素、柚皮素、黄豆黄素、熊果酸和齐墩果酸。降真香含药血清以及6个成分可显著降低脂多糖诱导的RAW264.7炎症细胞的一氧化氮、白介素-6(interleukin 6,IL-6)和肿瘤坏死因子-α的含量(P<0.01或P<0.05)。异鼠李素等6个成分可视为潜在质量标志物。通过分子对接和体外环氧合酶2(cyclooxygenase-2,COX-2)活性实验,从6个潜在质量标志物中筛选、验证异鼠李素是PTGS2的抑制剂,半数抑制浓度为9.55μmol·L^(-1)。综上,降真香提取物具有显著体外抗炎作用,其机制可能与调控IL-6、丝氨酸/苏氨酸蛋白激酶1、PTGS2等靶点介导的TNF信号通路、Toll样受体信号通路有关。异鼠李素、芒柄花素、柚皮素、黄豆黄素、熊果酸和齐墩果酸可作为降真香的质量标志物,其中异鼠李素可能是COX-2的抑制剂。

We have identified anti-inflammatory quality markers(Q-markers)of Jiangzhenxiang.The chemical components of Jiangzhenxiang were identified by mass spectrometry and the substances that contribute to its antiinflammatory activity,their targets and signaling pathways were analyzed by network pharmacology to identify potential Q-markers of the anti-inflammatory action of Jiangzhenxiang.The potential Q-markers were verified by high performance liquid chromatography,and in vitro experiments verified the anti-inflammatory activity and the target of the potential Q-markers.The experimental scheme was approved the Guangxi University of Chinese Medicine Institutional Animal Ethical and Welfare Committee.The results show that 31 chemical components were identified by mass spectrometry from the Jiangzhenxiang extract.Through network pharmacological screening,727 component targets,422 disease targets and 110 targets including prostaglandin G/H synthase 2(PTGS2)were obtained.These targets were mainly enriched in 498 biological processes including inflammatory response and response to lipopolysaccharide,with 101 pathways that included the TNF signaling pathway,toll-like receptor signaling pathway and others.Isorhamnetin,formononetin,naringenin,glycitein,ursolic acid and oleanolic acid were detected by high performance liquid chromatography.Jiangzhenxiang medicated serum and 6 components thereof could significantly reduce the content of nitric oxide,interleukin-6(IL-6)and tumor necrosis factor-αin RAW264.7 cells induced by lipopolysaccharide(P<0.01 or P<0.05).These six components are regarded as the potential anti-inflammatory Q-markers of Jiangzhenxiang.Isorhamnetin was screened and verified from the 6 potential Q-markers as an inhibitor of PTGS2 by molecular docking and in vitro cyclooxygenase 2(COX-2)activity assay.The half-inhibitory concentration of isorhamnetin was 9.55μmol·L^(-1).In summary,extracts of Jiangzhenxiang showed significant in vitro anti-inflammatory actions.The anti-inflammatory mechanism of Jiangzhenxiang appears to be related to regulation of TNF signaling pathway and Toll-like receptor signaling pathway meditated by IL-6,RAC alpha serine/threonine protein kinase,PTGS2 and other targets.Isorhamnetin,formononetin,naringenin,glycitein,ursolic acid and oleanolic acid could be regarded as the Q-markers of Jiangzhenxiang.Isorhamnetin appears to act as a COX-2 inhibitor.