title: Bioactivities of berberine metabolites after transformation through CYP450 isoenzymes.
abstract: BACKGROUND
  Berberine (BBR) is a drug with multiple effects on cellular energy metabolism. The present study explored answers to the question of which CYP450 (Cytochrome P450) isoenzymes execute the phase-I transformation for BBR, and what are the bioactivities of its metabolites on energy pathways.
  
  
  METHODS
  BBR metabolites were detected using LC-MS/MS. Computer-assistant docking technology as well as bioassays with recombinant CYP450s were employed to identify CYP450 isoenzymes responsible for BBR phase-I transformation. Bioactivities of BBR metabolites in liver cells were examined with real time RT-PCR and kinase phosphorylation assay.
  
  
  RESULTS
  In rat experiments, 4 major metabolites of BBR, berberrubine (M1), thalifendine (M2), demethyleneberberine (M3) and jatrorrhizine (M4) were identified in rat's livers using LC-MS/MS (liquid chromatography-tandem mass spectrometry). In the cell-free transformation reactions, M2 and M3 were detectable after incubating BBR with rCYP450s or human liver microsomes; however, M1 and M4 were below detective level. CYP2D6 and CYP1A2 played a major role in transforming BBR into M2; CYP2D6, CYP1A2 and CYP3A4 were for M3 production. The hepatocyte culture showed that BBR was active in enhancing the expression of insulin receptor (InsR) and low-density-lipoprotein receptor (LDLR) mRNA, as well as in activating AMP-activated protein kinase (AMPK). BBR's metabolites, M1-M4, remained to be active in up-regulating InsR expression with a potency reduced by 50-70%; LDLR mRNA was increased only by M1 or M2 (but not M3 and M4) with an activity level 35% or 26% of that of BBR, respectively. Similarly, AMPK-α phosphorylation was enhanced by M1 and M2 only, with a degree less than that of BBR.
  
  
  CONCLUSIONS
  Four major BBR metabolites (M1-M4) were identified after phase-I transformation in rat liver. Cell-free reactions showed that CYP2D6, CYP1A2 and CYP3A4 seemed to be the dominant CYP450 isoenzymes transforming BBR into its metabolites M2 and M3. BBR's metabolites remained to be active on BBR's targets (InsR, LDLR, and AMPK) but with reduced potency.
authors.affiliation: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.forename: Gang
authors.initials: G
authors.lastname: Ren
authors.forename: Yan-Xiang
authors.initials: YX
authors.lastname: Wang
authors.forename: Wei-Jia
authors.initials: WJ
authors.lastname: Kong
authors.forename: Peng
authors.initials: P
authors.lastname: Yang
authors.forename: Yue-Ming
authors.initials: YM
authors.lastname: Wang
authors.forename: Ying-Hong
authors.initials: YH
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Yi
authors.forename: Zhuo-Rong
authors.initials: ZR
authors.lastname: Li
authors.forename: Dan-Qing
authors.initials: DQ
authors.lastname: Song
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Jiang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000818:Animals; D001599:Berberine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics*; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D050216:Metabolic Detoxication, Phase I; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011973:Receptors, LDL; C055785:jatrorrhizine; D001599:Berberine; C115958:berberrubine; C477156:thalifendine; C477155:demethyleneberberine; D003577:Cytochrome P-450 Enzyme System; D011972:Receptor, Insulin; D055372:AMP-Activated Protein Kinases