UPLC-Q-TOF-MS结合网络药理学探讨威灵仙水提物抗炎的药效物质及作用机制

doi:10.16431/j.cnki.1671-7236.2025.06.037

摘要/Abstract

摘要： 【目的】利用超高效液相色谱-飞行时间质谱(UPLC-Q-TOF-MS)技术分析威灵仙水提物的主要化学成分，并结合网络药理学和细胞试验验证其抗炎作用机制。【方法】通过UPLC-Q-TOF-MS技术分析鉴定威灵仙水提物化学成分，结合网络药理学方法预测主要成分及相应靶点，并构建“成分-靶点-通路”网络，进一步利用AutoDock软件对主要活性成分与核心靶点进行分子对接，验证核心成分与关键靶点之间的结合能力。采用脂多糖(LPS)诱导RAW264.7细胞构建炎症模型，进行核心靶点和通路的验证。【结果】从威灵仙水提物中共鉴定出19个成分，其中酚酸类8个、皂苷类11个。网络药理学分析筛选出5个主要活性成分，主要通过调控磷脂酰肌醇3-激酶催化亚单位α(PIK3CA)、信号转导与转录激活子3(STAT3)、磷脂酰肌醇3-激酶催化亚单位β(PIK3CB)、肿瘤坏死因子(TNF)等核心靶点，并通过介导PI3K/Akt/STAT3等关键信号通路发挥抗炎作用。ELISA试验结果显示，与对照组相比，模型组细胞炎症因子TNF-α、白细胞介素-6(IL-6)、IL-1β含量显著升高(P＜0.05)；与模型组相比，威灵仙水提物各剂量组细胞中TNF-α、IL-6、IL-1β含量显著降低(P＜0.05)。Western blotting结果显示，与对照组相比，模型组细胞中PI3K、p-PI3K、Akt、p-Akt蛋白表达量显著上调(P＜0.05)；与模型组相比，威灵仙水提物各剂量组p-PI3K、p-Akt蛋白表达量均显著下调(P＜0.05)。【结论】威灵仙可通过多成分、多靶点、多途径发挥抗炎作用，且作用靶点与PI3K/Akt信号通路有关。本研究结果为深入研究威灵仙的药效物质基础及药理活性提供了参考。

关键词: 威灵仙; UPLC-Q-TOF-MS; 网络药理学; 抗炎; 炎性因子

Abstract: 【Objective】 This study aimed to analyze the main chemical components of Clematidis Radix decoction by ultra performance liquid chromatography with time-of-flight mass spectrometry (UPLC-Q-TOF-MS),and verify its anti-inflammatory mechanism by combining network pharmacology and cellular experiments.【Method】 The chemical composition of Clematidis Radix decoction was analyzed and identified by UPLC-Q-TOF-MS,the main components and corresponding targets were predicted by network pharmacology method,and the "component-target-pathway" network was constructed.The molecular docking between the main active ingredients and the core targets was carried out by AutoDock software to verify the binding ability between the core components and the key targets.The inflammation model of RAW264.7 cells was constructed by lipopolysaccharide (LPS) induction,and the core target and pathway were verified.【Result】 A total of 19 components were identified from Clematidis Radix decoction,including 8 phenolic acids and 11 saponins.Network pharmacological analysis screened 5 main active ingredients mainly modulating the PI3K/Akt/STAT3 signaling pathway mediated by core targets including phosphatidylinositol 3-kinase catalytic subunit α (PIK3CA),signal transduction and transcriptional activator 3 (STAT3),phosphatidylinositol 3-kinase catalytic subunit β (PIK3CB),and tumor necrosis factor (TNF) to exert anti-inflammatory effects.ELISA test results showed that compared with control group,the contents of inflammatory cytokines TNF-α,interleukin-6 (IL-6) and IL-1β in model group were significantly increased (P＜0.05).Compared with model group,the contents of TNF-α,IL-6 and IL-1β in each dose group of Clematidis Radix decoction were significantly decreased (P＜0.05). Western blotting results showed that compared with control group,the protein expressions of PI3K,p-PI3K,Akt and p-Akt in model group were significantly up-regulated (P＜0.05).Compared with model group,the expression levels of p-PI3K and p-Akt were significantly decreased in each dose group of Clematidis Radix decoction (P＜0.05). 【Conclusion】 Clematidis Radix could exert anti-inflammatory effects through multi-component,multi-target and multi-pathway,and the target was related to PI3K/Akt signaling pathway.The results of this study provided a reference for further study of the pharmacological material basis and pharmacological activity of Clematidis Radix.

Key words: Clematidis Radix; UPLC-Q-TOF-MS; network pharmacology; anti-inflammatory; inflammatory factor

中图分类号:

S853.74

陈海燕, 张景正, 许晨新, 刘元芬, 周咏梅, 韩瑾. UPLC-Q-TOF-MS结合网络药理学探讨威灵仙水提物抗炎的药效物质及作用机制[J]. 中国畜牧兽医, 2025, 52(6): 2851-2864.

CHEN Haiyan, ZHANG Jingzheng, XU Chenxin, LIU Yuanfen, ZHOU Yongmei, HAN Jin. Exploration of the Pharmacodynamic Substances of Anti-inflammatory and Mechanism of Clematidis Radix Decoction Using UPLC-Q-TOF-MS Combined with Network Pharmacology[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2851-2864.

参考文献

[1] 国家药典委员会.中华人民共和国药典(一部)[M].北京:中国医药科技出版社,2020.STATE PHARMACOPOEIA COMMISSION.Phamacopoeia of the People’s Republic of China (Vol.1)[M].Beijing:China Pharmaceutical Science and Technology Press，2020.(in Chinese)
[2] 杜希扬,孙兆祥,公素琴,等.威灵仙药用历史沿革研究[J].山东中医杂志,2020,39(8):874-878.DU X Y,SUN Z X,GONG S Q,et al.Medicinal history of Weilingxian (Clematidis Radix et Rhizoma)[J].Shandong Journal of Traditional Chinese Medicine,2020,39(8):874-878.(in Chinese)
[3] 付强,王萍,杜宇凤,等.威灵仙化学成分及其药理活性最新研究进展[J].成都大学学报(自然科学版),2018,37(2):113-119.FU Q,WANG P,DU Y F,et al.Latest research progress on chemical constituents of Clematidis Radix and their pharmacological effects[J].Journal of Chengdu University (Natural Science Edition),2018,37(2):113-119.(in Chinese)
[4] LIN T F,WANG L,ZHANG Y,et al.Uses,chemical compositions,pharmacological activities and toxicology of Clematidis Radix et Rhizome—A review[J].Journal of Ethnopharmacology,2021,270:113831.
[5] LI R,GUO L X,LI Y,et al.Dose-response characteristics of Clematidis Radix triterpenoid saponins and clematichinenoside AR in rheumatoid arthritis rats by liquid chromatography/mass spectrometry-based serum and urine metabolomics[J].Journal of Pharmaceutical and Biomedical Analysis,2017,136:81-91.
[6] YAN S,ZHANG X,ZHENG H,et al.Clematichinenoside inhibits VCAM-1 and ICAM-1 expression in TNF-alpha-treated endothelial cells via NADPH oxidase-dependent Ⅰ kappa B kinase/NF-kappa B pathway[J].Free Radical Biology and Medicine,2015,78:190-201.
[7] HAN W,XIONG Y,LI Y,et al.Anti-arthritic effects of clematichinenoside (AR-6) on PI3K/Akt signaling pathway and TNF-alpha associated with collagen-induced arthritis[J].Pharmaceutical Biology,2013,51(1):13-22.
[8] 魏岚,马亚雄.发酵中药在兽医临床的应用[J].中兽医学杂志,2023,12:67-69.WEI L,MA Y X.Application of fermented Chinese medicine in veterinary clinic[J].Chinese Journal of Traditional Veterinary Science,2023,12:67-69.(in Chinese)
[9] 肖世忠.家畜风湿性关节炎的新疗法[J].中兽医医药杂志,1994,5:38.XIAO S Z.A new therapy for rheumatoid arthritis in livestock[J].Journal of Traditional Chinese Veterinary Medicine,1994,5:38.(in Chinese)
[10] 汪永忠,邓龙飞,韩燕全,等.威灵仙总皂苷对佐剂性关节炎(AA)大鼠IL-6、IL-10及滑膜中p-JAK2、p-STAT3表达的影响[J].中药药理与临床,2015,31(1):86-90.WANG Y Z,DENG L F,HAN Y Q,et al.Effects of total saponins of Clematis on the levels of IL-6，IL-10 and the expression of p-JAK2，p-STAT3 in adjuvant-induced arthritis rats[J].Pharmacology and Clinics of Chinese Materia Medica,2015,31(1):86-90.(in Chinese)
[11] 李丹,李爱平,李科,等.液质联用技术在中药化学成分定性分析中的研究进展[J].药物评价研究,2020,43(10):2112-2119.LI D,LI A P,LI K,et al.Research progress on qualitative analysis of chemical composition of traditional Chinese medicine by liquid chromatography-mass spectrometry[J].Drug Evaluation Research,2020,43(10):2112-2119.(in Chinese)
[12] 王迪,俞佳,詹固,等.液质联用技术在中药研究中的应用进展[J].中华中医药学刊,2022,40(2):68-71.WANG D,YU J,ZHAN G,et al.Advances in application of liquid chromatography-mass spectrometry technology in study of traditional Chinese medicine[J].Chinese Archives of Traditional Chinese Medicine,2022,40(2):68-71.(in Chinese)
[13] 解静,高杉,李琳,等.网络药理学在中药领域中的研究进展与应用策略[J].中草药,2019,50(10):2257-2265.XIE J,GAO S,LI L,et al.Research progress and application strategy on network pharmacology in Chinese materia medica[J].Chinese Traditional and Herbal Drugs,2019,50(10):2257-2265.(in Chinese)
[14] 宋雨,孙彦刚,张艺媛,等.基于网络药理学和试验验证探讨茵陈蒿汤治疗糖尿病的作用机制[J].中国畜牧兽医,2024,51(9):4106-4119.SONG Y,SUN Y G,ZHANG Y Y,et al.Study on the mechanism of Yinchenhao decoction in treating diabetes mellitus based on network pharmacology and experimental verification[J].China Animal Husbandry & Veterinary Medicine,2024,51(9):4106-4119.(in Chinese)
[15] 许晨新.威灵仙水溶性成分及其体外透皮行为研究[D].淮安：淮阴工学院,2020.XU C X.Study on water-soluble constituents of Clematidis Radix and their transdermal behavior in vitro[D].Huai’an：Huaiyin Institute of Technology,2020.(in Chinese)
[16] 胡萌,付娟,胡军华,等.基于HPLC-Q-TOF-MS/MS与网络药理学小儿佛芍和中颗粒成分解析及治疗功能性腹痛药效物质基础及机制预测[J].药物评价研究,2023,46(5):997-1011.HU M,FU J,HU J H,et al.Component analysis and pharmacological mechanism prediction of Xiaoer Foshao Hezhong granules in treatment of functional abdominal pain based on HPLC-Q-TOF-MS/MS and network pharmacology[J].Drug Evaluation Research,2023,46(5):997-1011.(in Chinese)
[17] 赵文燕,向茜,王蝉,等.UPLC-Q-TOF-MS联合网络药理学和实验验证分析黄连水炒吴茱萸治疗慢性结肠炎的作用机制[J].中国中药杂志,2022,47(19):5316-5326.ZHAO W Y,XIANG Q,WANG C,et al.Mechanism of Euodiae Fructus stir-fried with water decoction of Coptidis Rhizoma in treatment of chronic colitis based on UPLC-Q-TOF-MS,network pharmacology and experimental verification[J].China Journal of Chinese Materia Medica,2022,47(19):5316-5326.(in Chinese)
[18] 武博文,颜培宇,武密山.基于网络药理学探讨麻黄活性成分治疗血管痉挛的作用机制[J].中国畜牧兽医,2024,51(6):2680-2696.WU B W,YAN P Y,WU M S,et al.Mechanism of the active ingredients of Epherdra sinica Stapf dried grass stems in the treatment of vasospasm based on network pharmacology[J].China Animal Husbandry & Veterinary Medicine,2024,51(6):2680-2696.(in Chinese)
[19] 王辉钦,段莹莹,杨彦平,等.基于网络药理学探究地锦草防治溃疡性结肠炎的作用机制[J].中国畜牧兽医,2024,51(5):2154-2168.WANG H Q,DUAN Y Y,YANG Y P,et al.Study on the mechanism of Euphorbiae humifusae herba in the treatment of ulcerative colitis based on network pharmacology[J].China Animal Husbandry & Veterinary Medicine,2024,51(5):2154-2168.(in Chinese)
[20] 孙凤,蔡铮,杨得坡,等.液相色谱-质谱联用定量分析威灵仙及其同属植物中4种三萜皂苷[J].分析化学，2010,38(9):1293-1298.SUN F,CAI Z,YANG D P,et al.Determination of triterpenoid saponins in Radix Clematidis and related Clematis species by high performance liquid chromatography-mass spectrometry[J].Chinese Journal of Analytical Chemistry,2010,38(9):1293-1298.(in Chinese)
[21] DEEPAK M,HANDA S S.Anti inflammatory activity and chemical composition of extracts of Verbena officinalis[J].Phytotherapy Research,2000,14(6):463-465.
[22] LIU J,ZHENG L,MA L,et al.Oleanolic acid inhibits proliferation and invasiveness of Kras-transformed cells via autophagy[J].The Journal of Nutritional Biochemistry,2014,25(11):1154-1160.
[23] MARTÍN R,CORDOVA C,SAN ROMÁN J A,et al.Oleanolic acid modulates the immune-inflammatory response in mice with experimental autoimmune myocarditis and protects from cardiac injury.Therapeutic implications for the human disease[J].Journal of Molecular and Cellular Cardiology,2014,72:250-262.
[24] 邓莉,冯健,何成松.齐墩果酸抑制肿瘤坏死因子-α诱导的成纤维细胞样滑膜细胞炎症因子表达及其机制研究[J].天然产物研究与开发,2018,30(7):1138-1142.DENG L,FENG J,HE C S.Oleanolic acid inhibit tumor necrosis factor alpha-induced inflammatory cytokines production of synovial cells and its mechanism[J].Natural Product Research and Development,2018,30(7):1138-1142.(in Chinese)
[25] 谷慧敏,孟庆良,左瑞庭,等.β-谷甾醇对类风湿性关节炎滑膜成纤维细胞功能的影响及机制[J].中国药房,2023,34(15):1847-1852.GU H M,MENG Q L,ZUO R T,et al.Effects of β-sitosterol on the function of synovial fibroblasts in rheumatoid arthritis and its mechanism[J].China Pharmacy,2023,34(15):1847-1852.(in Chinese)
[26] DILSHARA M G,LEE K T,JAYASOORIYA R G,et al.Downregulation of NO and PGE₂ in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1[J].International Immunopharmacology,2014,18(1):203-211.
[27] 吴力超,李俊峰,张婷婷,等.基于网络药理学和细胞实验探讨豆甾醇抗炎作用[J].中成药,2022,44(2):609-615.WU L C,LI J F,ZHANG T T,et al.The anti-inflammatory effect of stigmasterol was studied based on network pharmacology and cell experiments[J].Chinese Traditional Patent Medicine,2022,44(2):609-615.(in Chinese)
[28] GABAY O,SANCHEZ C,SALVAT C,et al.Stigmasterol:A phytosterol with potential anti-osteoarthritic properties[J].Osteoarthritis and Cartilage,2010,18(1):106-116.
[29] 苏清君,李鹏,边朝辉,等.补骨脂素通过炎症和Ras/Raf1介导对类风湿关节炎大鼠的保护作用[J].世界中医药,2021,16(21):3180-3184.SU Q J,LI P,BIAN C H,et al.Protective effects of psoralen on rheumatoid arthritis rats by reducing inflammation and inhibiting the expression of Ras/Raf1[J].World Chinese Medicine,2021,16(21):3180-3184.(in Chinese)
[30] 潘起晨,班允超,刘源,等.PIK3CA、PIK3CB、PIK3CD在胶质瘤中的表达及其意义[J].解剖科学进展,2020,26(2):162-165.PAN Q C,BAN Y C,LIU Y,et al.Expression and significance of PIK3CA,PIK3CB and PIK3CD in glioma[J].Progress of Anatomical Sciences,2020,26(2):162-165.(in Chinese)
[31] 郭雪冬,仪慧兰.黄芩苷通过苦味受体促进哮喘小鼠呼吸道炎性细胞凋亡[J].中国生物化学与分子生物学报,2022,38(5):680-688.GUO X Y,YI H L.Baical in promotes the apoptosis of respiratory inflammatory cells in asthmatic mice through bitter taste receptors[J].Journal of Biochemistry and Molecular Biology,2022,38(5):680-688.(in Chinese)
[32] BLACK A R,BLACK J D.The complexities of PKCα signaling in cancer[J].Advances in Biological Regulation,2021,80:100769.
[33] 耿云峰,杜鸿斌,刘琳琳,等.NF-κB家族成员RelA的翻译后修饰及其生理病理作用的研究进展[J].生命科学,2020,32(5):431-438.GENG Y F,DU H B,LIU L L,et al.Research progress on post-translational modification of NF-κB family member RelA and its physiological and pathological effects[J].Chinese Bulletin of Life Sciences,2020,32(5):431-438.(in Chinese)
[34] 陈永安,黄雅阳,苏安宇,等.基于网络药理学与分子对接探讨岭南药材三桠苦茎叶治疗类风湿性关节炎的作用特点[J].中药新药与临床药理,2023,34(8):1105-1116.CHEN Y A,HUANG Y Y,SU A Y,et al.Exploration on action characteristics of the stems and leaves of Melicope ptelefolia on the treatment of rheumatoid arthritis based on network pharmacology and molecular docking[J].Traditional Chinese Drug Research and Clinical Pharmacology,2023,34(8):1105-1116.(in Chinese)
[35] 王素杰.PRKCA调控食管鳞癌增殖迁移侵袭的分子机制及临床意义[D].新乡：新乡医学院,2021.WANG S J.Molecular mechanism and clinical significance of PRKCA in regulating the proliferation,migration and invasion of esophageal squamous cell carcinoma[D].Xinxiang：Xinxiang Medical University,2021.(in Chinese)
[36] 戴伟平,齐彬,周平安,等.非小细胞肺癌MET、KRAS、PIK3CA和BRAF基因突变状态与临床特征的关系分析[J].中国现代药物应用,2024,18(8):48-51.DAI W P,QI B,ZHOU P A,et al.Correlation analysis of mutation status of MET,KRAS,PIK3CA and BRAF genes with clinical characteristics in non-small cell lung cancer[J].Chinese Journal of Modern Drug Application,2024,18(8):48-51.(in Chinese)