In vitro Investigation of Metabolism, Bioactivation, and Botanical-Drug Interactions of Licorice

The dried roots of licorice have been consumed for the past 6000 years as a flavoring and sweetening agent in the western world and in Asian countries for their anti-allergic and anti-inflammatory agent properties. Among the constituents identified in popular licorice species, few have been studied for metabolism by human cytochrome P450 enzymes. Glabridin in vitro metabolism was studied with respect to metabolic stability, metabolite identification, and identification of metabolizing enzymes. Glabridin was metabolized with low hepatic clearance. In the incubations of glabridin with human liver microsomes and rat liver microsomes, six glabridin metabolites were observed. The D-ring diols formed via unstable epoxides were the major glabridin metabolites, and this finding was confirmed by treating glabridin treated with the epoxide-forming reagent mCPBA. Cytochrome P450 isozyme 1A2, 2C9, and 3A4 were found to be the major enzymes responsible for the formation of the diol metabolites while CYP3A4 and CYP2C9 were predominantly responsible for the other metabolites. A fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advance knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The method was used to study the bioactivation of a licorice extract from Glycyrrhiza glabra. The licorice extracts showed significant inhibition of CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 when the extract concentration was increased from 1 µg/mL to 20 µg/mL. Significant inhibition of CYP2C8, CYP2C9 and CYP2C19 was observed, while CYP2B6 and CYP3A4 were moderately inhibited. CYP2C9 was inhibited the most with IC50 values of 0.6 µg/mL for licorice extract and 2.2 µM for glabridin. As a systematic approach to identify the CYP2C9 and CYP3A4 inhibitors, 13 sub-fractions of the crude licorice extract were tested. Fractions that showed significant inhibition were selected for further compound isolation and characterization. Diligent database searching and dereplication of known licorice compounds were used to determine possible chemical structures of the most potent inhibitors of CYP2C9 and CYP3A4. Due to lack of standards for these licorice compounds, the proposed chemical structures could not be identified at this time.