In Vitro Inhibitory Effects and Molecular Mechanism of Four Theaflavins on Isozymes of CYP450 and UGTs

Theaflavins, benzotropolone compounds formed during black tea processing via catechin condensation, have drawn attention for their potential health benefits and diverse biological effects. This study evaluated the inhibitory effects of four theaflavin monomers—theaflavin-3′-gallate, theaflavin-3,3′-digallate, theaflavin-3-gallate, and theaflavin—on eight CYP450 enzymes using pooled human liver microsomes and specific probe substrates, and seven UGT enzymes using human recombinant UGT enzymes and specific probe substrates. Theaflavin-3′-gallate moderately inhibited CYP1A2-catalyzed phenacetin metabolism and CYP2C8-mediated amodiaquine metabolism, with IC₅₀ values of 8.67 μM and 10–20 μM, respectively. Theaflavin-3,3′-digallate exhibited similar effects. Both compounds showed negligible inhibition with other CYP enzymes. In UGT assays, theaflavin-3′-gallate and theaflavin-3,3′-digallate moderately inhibited UGT1A1- and UGT1A3-mediated beta-estradiol glucuronidation (IC₅₀: 1.40–5.22 μM), with weak or no effects on other UGT enzymes. Molecular docking revealed that CYP1A2-theaflavin-3′-gallate and CYP2C8-theaflavin-3,3′-digallate interactions were non-competitive, primarily mediated by hydrogen bonding and π-interactions. UGT1A1-theaflavin interactions suggested non-competitive inhibition, while UGT1A3-theaflavin interactions indicated competitive inhibition. Other enzyme-theaflavin interactions exhibited minimal binding energy differences, implying mixed-type inhibition. These findings highlight the selective inhibitory effects of theaflavins on specific hepatic enzymes, with potential implications for nutrient interactions, particularly for nutrients metabolized by CYP1A2, CYP2C8, UGT1A1, and UGT1A3. Further research is needed to explore the in vivo relevance and assess the dietary implications of theaflavin-rich black tea in nutrition and metabolism.