Inhibition of cytochrome P450 activities by juglone in human and rat liver microsomes: kinetic characterization and implications for natural product–drug interaction assessment

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Abstract
Introduction: Juglone is a redox-active constituent of walnut green husks, but its effects on drug-metabolizing cytochrome P450 (CYP) enzymes remain incompletely characterized. Methods: We evaluated juglone against six CYP activities in human and rat liver microsomes (HLMs and RLMs) using cocktail probe assays, time-dependent inhibition experiments, kinetic analyses, and molecular docking. Results: In HLMs, CYP2C9 was the most susceptible activity (IC 50 = 8.12 μM; K i = 4.97 μM) and showed competitive inhibition. CYP2E1 was the next most susceptible activity (IC 50 = 12.67 μM; K i = 19.59 μM; αK i = 8.74 μM) and showed mixed inhibition. In RLMs, CYP1A2 (IC 50 = 12.03 μM) and CYP2E1 (IC 50 = 18.50 μM) met the prespecified criterion for further kinetic analysis. Subsequent analyses identified mixed inhibition of CYP1A2 ( K i = 12.31 μM) and competitive inhibition of CYP2E1 (Ki = 19.89 μM). No consistent progressive loss of activity was observed during preincubation for up to 20 min under the tested conditions. CYP2E1 showed numerically similar apparent K i values but different fitted inhibition models in HLMs and RLMs. However, differing assay conditions precluded attributing these findings solely to species. Molecular docking provided qualitative structural insight into the potential interactions between juglone and CYP2C9. Conclusion: Juglone inhibited CYP activities in vitro , with CYP2C9 showing the greatest susceptibility among the six human CYP activities tested. These findings characterize biochemical inhibition and provide a basis for evaluating potential metabolic interactions involving juglone-containing products.

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