title: Screening for selective inhibitors of xanthine oxidase from Flos Chrysanthemum using ultrafiltration LC-MS combined with enzyme channel blocking.
abstract: In this study, a new method based on ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) combined with enzyme channel blocking (ECB) was developed to discover bioactive components from herbal medicines. Xanthine oxidase (XOD), a critical enzyme for treating gout, was employed as the target protein for screening. By comparing chromatographic profiles of the compounds binding to XOD before and after the ECB experiment, the selective ligands could be distinguished from the non-selective binders. In this experiment, febuxostat bound to the channel entering into the active site of the enzyme and prevented potential ligands from binding. Finally, four compounds, namely, luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin and apigenin were screened and identified as the candidate XOD inhibitors based on the ultrafiltration chromatogram of Flos Chrysanthemum, a famous traditional Chinese medicine used in many prescriptions for gout treatment. To verify the compounds screened further, a microplate method was applied to evaluate their enzyme inhibitory activities. The IC50 values of the above 4 compounds were 23.61, 38.80, 1.54 and 1.96μM, respectively. The structure-function relationship was also estimated according to the in vitro assay. The results were in favor of the hypothesis that the Flos Chrysanthemum extract might be used for gout treatment by inhibiting XOD.
authors.affiliation: State Key Laboratory of Natural Medicines (China Pharmaceutical University) , No. 24, Tongjia Lane, Jiangsu, Nanjing 210009, China.
authors.forename: Hui-Peng
authors.initials: HP
authors.lastname: Song
authors.affiliation: State Key Laboratory of Natural Medicines (China Pharmaceutical University) , No. 24, Tongjia Lane, Jiangsu, Nanjing 210009, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural Medicines (China Pharmaceutical University) , No. 24, Tongjia Lane, Jiangsu, Nanjing 210009, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Fu
authors.affiliation: State Key Laboratory of Natural Medicines (China Pharmaceutical University) , No. 24, Tongjia Lane, Jiangsu, Nanjing 210009, China.
authors.forename: Hua-yan
authors.initials: HY
authors.lastname: Mo
authors.affiliation: State Key Laboratory of Natural Medicines (China Pharmaceutical University) , No. 24, Tongjia Lane, Jiangsu, Nanjing 210009, China.
authors.forename: Mu
authors.initials: M
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural Medicines (China Pharmaceutical University) , No. 24, Tongjia Lane, Jiangsu, Nanjing 210009, China. Electronic address: chenj2002@gmail.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Natural Medicines (China Pharmaceutical University) , No. 24, Tongjia Lane, Jiangsu, Nanjing 210009, China. Electronic address: liping2004@126.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Li
mesh_terms: D047310:Apigenin / Q000302:isolation & purification / Q000494:pharmacology; D020134:Catalytic Domain / Q000187:drug effects*; D002853:Chromatography, Liquid / Q000379:methods; D031202:Chrysanthemum / Q000737:chemistry*; D004365:Drugs, Chinese Herbal / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D000069465:Febuxostat; D047309:Flavones / Q000302:isolation & purification / Q000494:pharmacology; D035264:Flowers / Q000737:chemistry; D005960:Glucosides / Q000302:isolation & purification / Q000494:pharmacology; D006074:Gout Suppressants / Q000302:isolation & purification*; D047311:Luteolin / Q000302:isolation & purification / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D013844:Thiazoles / Q000378:metabolism; D014462:Ultrafiltration / Q000379:methods; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors*
chemical_list: D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D047309:Flavones; D005960:Glucosides; D006074:Gout Suppressants; D013844:Thiazoles; D000069465:Febuxostat; C057792:apigetrin; D047310:Apigenin; C066408:luteolin-7-glucoside; D014969:Xanthine Oxidase; D047311:Luteolin
keywords: Febuxostat; Flos Chrysanthemum; Liquid chromatography/mass spectrometry; Ultrafiltration; Xanthine oxidase