ABSTRACT
Objective: To study the metabolic characteristics of berberine using the pooled human liver microsomes and recombinant human cytochrome enzymes P450 (CYP) isozymes, to identify CYP isozymes responsible for berberine metabolism and its contribution, and to determine the structures of metabolism. Methods: Pooled human liver microsomes were incubated with berberine (20, 100, 200, 400, 600, 800, and 1 200 ng/mL). The Michaelis-Menten parameters (Km), maximum velocity (Vmax), and clearance (CLint) of pooled liver microsomes were initially estimated by analyzing Lineweave-Brurk plot. Various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of berberine and the certain concentration of berberine was incubated with recombinant human CYP isozymes (CYP3A4, CYP1A2, CYP2D6, and CYP2C9). The concentration of berberine and metabolites in the incubation pool was determined by UPLC method. The P450 isozymes were ranked with the method of total normalized rate (TNR) and the related metabolites were identified by LC-MS/MS. Results: The Vmax, Km, and CLint of berberine in pooled human liver microsomes were 1.51 nmol·mg-1·h-1, 2.69 nmol/mL, and 0.56 mL·mg-1·h-1, respectively. Quinidine (the specific inhibitor of CYP2D6) and Furafylline (the specific inhibitor of CYP1A2) could significantly inhibit the berberine metabolism, and the other CYP inhibitors had no significant effect on the metabolism of berberine. CYP2D6 and CYP1A2 were responsible for 75.253 9% and 23.323 6% of the berberine metabolite M1 (demethyleneberberine), and responsible for 46.893 8% and 8.679 5% of M2 (thalifendine or berberrubine). The major metabolic pathway of berberine in pooled human liver microsomes incubation system is O-demethylated, demethyleneberberine, thalifendine, or berberrubine could be generated in vitro. Conclusion: Bererine is metabolized by CYP2D6 and CYP1A2 in human liver, the metabolites of berberine are demethyleneberberine and thalifendine or berberrubine.