title: Methylation, Glucuronidation, and Sulfonation of Daphnetin in Human Hepatic Preparations In Vitro: Metabolic Profiling, Pathway Comparison, and Bioactivity Analysis.
abstract: Our previous study demonstrated that daphnetin is subject to glucuronidation in vitro. However, daphnetin metabolism is still poorly documented. This study aimed to investigate daphnetin metabolism and its consequent effect on the bioactivity. Metabolic profiles obtained by human liver S9 fractions and human hepatocytes showed that daphnetin was metabolized by glucuronidation, sulfonation, and methylation to form 6 conjugates which were synthesized and identified as 7-O-glucuronide, 8-O-glucuronide, 7-O-sulfate and 8-O-sulfate, 8-O-methylate, and 7-O-suflo-8-O-methylate. Regioselective 8-O-methylation of daphnetin was investigated using in silico docking calculations, and the results suggested that a close proximity (2.03 Å) of 8-OH to the critical residue Lysine 144 might be the responsible mechanism. Compared with glucuronidation and sulfonation pathways, the methylation of daphnetin had a high clearance rate (470 μL/min/mg) in human liver S9 fractions and contributed to a large amount (37.3%) of the methyl-derived metabolites in human hepatocyte. Reaction phenotyping studies showed the major role of SULT1A1, -1A2, and -1A3 in daphnetin sulfonation, and soluble COMT in daphnetin 8-O-methylation. Of the metabolites, only 8-O-methyldaphnetin exhibited an inhibitory activity on lymphocyte proliferation comparable to that of daphnetin. In conclusion, methylation is a crucial pathway for daphnetin clearance and might be involved in pharmacologic actions of daphnetin in humans.
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China; Second Affiliated Hospital of Dalian Medical University, Dalian, China; University of Chinese Academy of Sciences, Beijing, China.
authors: Si-Cheng
authors: SC
authors: Liang
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China; Second Affiliated Hospital of Dalian Medical University, Dalian, China.
authors: Yang-Liu
authors: YL
authors: Xia
authors: Dalian Medical University, Dalian, China.
authors: Jie
authors: J
authors: Hou
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China. Electronic address: geguangbo@dicp.ac.cn.
authors: Guang-Bo
authors: GB
authors: Ge
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors: Jiang-Wei
authors: JW
authors: Zhang
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors: Yu-Qi
authors: YQ
authors: He
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China; Second Affiliated Hospital of Dalian Medical University, Dalian, China.
authors: Jia-Yue
authors: JY
authors: Wang
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China; Second Affiliated Hospital of Dalian Medical University, Dalian, China. Electronic address: 7xiao1@163.com.
authors: Xiao-Yi
authors: XY
authors: Qi
authors: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors: Ling
authors: L
authors: Yang
mesh_terms: D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D020719:Glucuronides / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D053858:Metabolic Networks and Pathways / Q000187:drug effects / Q000502:physiology*; D055442:Metabolome / Q000187:drug effects / Q000502:physiology*; D008745:Methylation; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D014468:Umbelliferones / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D020719:Glucuronides; D013431:Sulfates; D014468:Umbelliferones; C039952:daphnetin
keywords: 8-O-methyldaphnetin; anti-inflammatory activity; daphnetin; glucuronidation; methylation; sulfonation