Abstract
OBJECTIVE: To characterize the oxidative metabolic pathway(s) of pectolinarigenin (PEC) and reveal the effect of PEC oxidative metabolism on its biological activities including peroxisome proliferator-activated receptors (PPAR) and nuclear factor erythroid 2-related factor 2 (Nrf2) agonist effects, as well as the anti-oxidative and hepatoprotective activities. METHODS: The oxidative metabolites of PEC were identified by liquid chromatography-time of flight-mass spectrometry (LC-TOF-MS/MS). The key enzymes involved in oxidative metabolism of PEC were assigned by P450 reaction phenotyping assays and enzymatic kinetics assays. Luciferase reporter assays and western blotting analysis were used to evaluate the Nrf2 and PPAR agonist effects of PEC and its oxidative metabolites. The intracellular levels of total reactive oxygen species (ROS), mitochondrial membrane potential (MMP), lactate dehydrogenase (LDH) and glutathione (GSH) in acetaminophen (APAP)-challenged hepatocytes were also tested. RESULTS: PEC could be readily metabolized to form two O-demethylated metabolites including hispidulin (HIS, 4'-O-demethylated PEC) and 6-O-demethylated PEC in human liver microsomes (HLM) in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), while HIS was identified as the major oxidative metabolite of PEC. At least nine human cytochrome P450 enzymes (CYP) enzymes could catalyze PEC-4'-O-demethylation, while CYP1A2 and CYP2D6 showed the highest binding affinities and rapid metabolic clearance rates in the oxidative metabolism of PEC. Biological assays showed that PEC 4'-O-demethylation slightly decreased the PPAR agonist effects of PEC, while HIS showed more potent Nrf2 agonist effect. Compared with PEC, HIS showed more efficacious hepatoprotective effect against APAP-induced hepatocyte injury, evidenced by more potent ability to reduce intracellular ROS and LDH levels, as well as more effective ability to elevate the intracellular levels of both MPP and GSH in APAP-challenged hepatocytes. CONCLUSION: CYPs catalyze PEC-4'-O-demethylation to generate a more active Nrf2 agonist (HIS), which shows more efficacious hepatoprotective effects against APAP-induced hepatocyte injury.