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Aim Luteolin (3, 4, 5, 7-tetrahydroxyflavone) is a typical flavonoid widely distributed in plants, which possesses many biological effects.Due to its catecholic ring, luteolin could be methylated at meta-or para-hydroxyl in B ring.Moreover, the methylated luteolin still displayed bioactivities.But there was lack of information about the methylation of luteolin in human.The present study aims to elucidate the regioselective methylation ofluteolin in vitro and in vivo.Methods Recombinant human COMT (rhCOMT) and human liver S9 (HLS9) were utilized to study the kinetics of meta (3)-and para (4)-methylation of luteolin, and urine samples from volunteers after giving a luteolin containing formulation were collected to determine the ratio of meta-/para-production in vivo.Furthermore, metabolism stability of the meta-and para-methylated luteolin was evaluated with human liver microsomes (HLM) and recombinant human CYP450s (rhCYP450s), and a series of typical inhibitors of CYP450s were utilized to identify the CYP450 isoforms contributing to further metabolism of meta-and para-methylated luteolin.Results The kinetics study showed that, Vmax values (in rhCOMT/HLS9) for meta-and para-methylation were 0.784± 0.044/0.0673+0.0022 and 1.17+0.082/0.0979±0.0024 nmol mg protein-1min-1, respectively; Km values for meta and para-methylation were 0.673+0.097/0.769±z0.13 and 0.706a±0.147/0.75 1L±0.63 μM, respectively.The ratio of meta-/para-methylation in Clint (Vmax/Km) for rhCOMT and HLS9 were 1.43 and 1.49, respectively.Both meta-and para-methylated luteolin were also detected in human urine after dosing of luteolin, but the ratio of meta-/ para-mathylated luteolin was 0.528±0.14, which contrasted with the in vitro result.Furthermore, CYP450s, especially CYP1A2 and CYP3A4, preferentially demethylate the para-methylated-luteolin, which might result in different ratios of meta-/para-production between in vivo and in vitro.Conclusion Luteolin could be meta-and para-methylated by human COMT, which displayed a preference of para-methylation.However, further demethylation by human CYP1A2 and CYP3A4/5 caused a preference of accumulation in meta-methylated luteolin in human in vivo.