目的 采用混合人肝微粒体和重组人源细胞色素酶P450（CYP）同工酶，研究小檗碱的代谢特性，明确参与小檗碱代谢的CYP酶亚型及其贡献度，并确证相关代谢产物的结构。方法 将混合人肝微粒体分别与20、100、200、400、600、800、1 200 ng/mL的小檗碱共同孵育后，计算小檗碱酶反应动力学米氏常数（K_m）、最大反应速率（V_max）、体外肝微粒体对药物的固有清除率（CL_int）；采用CYP酶的特异性抑制剂研究小檗碱的代谢表型；将重组人源CYP同工酶CYP3A4、CYP1A2、CYP2D6、CYP2C9与一定质量浓度的小檗碱孵育，UPLC法测定孵育液中原形药物的剩余量和代谢产物的生成量；整体归一化法计算各酶的代谢贡献度；LC-MS/MS法鉴定相关代谢产物。结果 小檗碱在混合人肝微粒体中的V_max为1.51 nmol?mg^?1?h^?1；K_m为2.69 nmol/mL；CL_int为0.56 mL?mg^?1?h^?1。CYP2D6的特异性抑制剂奎尼丁和CYP1A2的特异性抑制剂呋喃茶碱对小檗碱的代谢有显著抑制作用，其他CYP酶抑制剂对小檗碱的代谢无显著影响。CYP2D6、CYP1A2对小檗碱代谢产物M₁（去亚甲基小檗碱）的贡献度分别为75.253 9%、23.323 6%；对M₂产物（唐松草分定或小檗红碱）的贡献度分别为46.893 8%、8.679 5%。小檗碱在体外混合人肝微粒体温孵体系中的主要代谢途径为O-去甲基化，可生成去亚甲基小檗碱和唐松草分定或小檗红碱。结论 小檗碱在肝脏中主要被CYP2D6和CYP1A2代谢，分别生成去亚甲基小檗碱、唐松草分定或小檗红碱。

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 (K_m), maximum velocity (V_max), and clearance (CL_int) 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 V_max, K_m, and CL_int 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.

国家重大新药创制专项（2009ZX09103-312，2011ZX09102-011-08）