br Material and methods br Results The Vmax
Material and methods
Results The Vmax (nmol/min/mg) and Km (μM) values for PNPH and BFC activities in each microsomal pool were generated from the Michaelis–Menten non-linear regression equation (Table 1). Quercetin, but not myricetin or isorhamnetin, inhibited PNPH activity in human recombinant cDNA-expressed CYP2E1 in a concentration-dependent manner (Fig. 2). The remaining activity was 46.7% in the presence of 128μM of quercetin. Further investigations to explore the inhibition mechanism of quercetin using substrate concentrations from 0.0025 to 0.4mM revealed a competitive mode of inhibition with the Ki=52.1±6.31μM. Overall, the degree of inhibition of PNPH activity by quercetin in the human microsomes slightly differed between genders (i.e., higher in the microsomes from male pools) (Fig. 3). However, the degree of inhibition did not reach 50%. The average remaining PNPH activity in the presence of quercetin ranged from 56.5 to 65.5% in male pools and from 65.4 to 96.5% in female pools. Including a pre-incubation step did not further decrease PNPH activity indicating reversible nature of this Tunicamycin slight inhibition (Fig. 3). Quercetin competitively inhibited BFC activity in human recombinant cDNA-expressed CYP3A4 with Ki=15.4±1.52μM, and myricetin noncompetitively with Ki=74.6±7.99μM (Fig. 4). The degree of inhibition by quercetin was not affected by a pre-incubation step and was similar in the microsomes from both genders (Fig. 5). Analysis of enzyme kinetics in the presence of quercetin indicated uncompetitive inhibition concentrations with alpha Ki values below 100μM (Fig. 5). Alpha Ki values did not differ significantly between pools from male and female donors (P=0.113). Myricetin showed somewhat stronger inhibition in female pools (Fig. 6). However, more specific analysis of enzyme kinetics in the presence of myricetin indicated that Ki values were above 100μM which is much higher than physiological concentrations (Conquer et al., 1998) and was not considered as relevant. Isorhamnetin did not affect BFC activity either in human recombinant cDNA-expressed CYP3A4 (Fig. 5) or in the microsomes (Fig. 7). Thus no further analyses with isorhamnetin were performed.
Discussion Modulations of CYP450 activity are a primary cause of herb–drug interactions. Occurrence of herb–drug interactions highlighted the importance of understanding the mechanism and factors responsible for variations in the activities of drug-metabolizing enzymes. Potency of several flavonoid compounds as an inhibitor of human CYP450 has been demonstrated, but investigations on gender-related differences are lacking. The anatomical and physiological differences between males and females influence the action of many drugs and thus interactions between drugs and bioactive compounds. There are also gender-related differences in expression and activity of some CYP450 isoforms. A number of studies showed that CYP3A activity is higher in females than in males (El-Eraky and Thomas, 2003, Waxman and Holloway, 2009). The purpose of the present study was to determine the inhibitory effects of quercetin, myricetin, and isorhamnetin on human CYP2E1 and CYP3A in the microsomes from male and female donors. Both CYP2E1 and CYP3A are involved in the metabolism of pharmaceuticals; thus, modulation of these enzyme activities can generate effects of pharmacological and toxicological importance.