Hepatic Metabolism of Licochalcone A, a Chalcone from the Licorice Species Glycyrrhiza inflata
Authors: Huang Lingyi , Nikolic Dejan , van Breemen Richard B 
Published: November 2023

Introduction

Licochalcone A is a chalcone natural product that has been isolated from roots of the licorice species, Glycyrrhiza inflata. It shows antimalarial, anticancer, antibacterial, and antiviral (specifically against influenza neuraminidase) properties in vitro. Since little information is available concerning the human bioavailability and metabolism of licochalcone A, we carried out preclinical in vitro hepatic metabolism studies. Several Phase I and phase II metabolites were observed and characterized using high resolution LC‐MS/MS with accurate mass measurement. Based on fragmentation pathways during high resolution tandem mass spectrometry and LC‐UV analysis, the structures of licochalcone A metabolites were characterized and in some cases identified.

Methods

For phase I metabolism studies, licochalcone A was incubated with human liver microsomes or recombinant cytochrome P450 enzymes and NADPH in phosphate buffer. After reaction, samples were analyzed using high resolution LC‐MS/MS with accurate mass measurement on a Waters Synapt G1 QToF mass spectrometer. High resolution MS was used to determine the elemental compositions of the phase I metabolites, and product ion tandem mass spectrometry was used for structural elucidation.

Incubations were also carried out using cryopreserved human hepatocytes to investigate not only phase I metabolism but also phase II conjugation with glucuronic acid, sulfate and glutathione. Deconjugation by using beta‐glucuronidase and sulfatase followed by HPLC‐UV and high resolution tandem mass spectrometry confirmed the formation of phase II conjugates.

Preliminary Data

After incubation with human liver microsomes, five (5) mono‐oxygenated or di‐oxygenated licochalcone A phase I metabolites were detected. When microsomes or NADPH was omitted from the incubation, no metabolites were detected, indicating that oxidative metabolism occurred via cytochrome P450 enzymes. The biotransformation pathway of each phase I metabolite was determined using recombinant cytochrome P450 enzymes. Based on accurate mass MS/MS data and application of Biemann's shift method of data interpretation, addition of oxygen to licochalcone A could be localized on either the A‐ring or on the B‐ring.

Incubation of licochalcone A produced several glucuronide metabolites, including glucuronic acid conjugates of phase I oxygenated metabolites. One sulfate conjugate and one GSH conjugate of licochalcone A were also found. Using a combination of MS/MS and UV analysis, the two most abundant mono‐glucuronide conjugates of licochalcone A were identified as 4‐O‐glucuronide and 4′‐O‐glucuronide. The in vitro metabolic stability of licochalcone A was investigated, and the half‐life was determined to be 83 min.

Conclusion

Licochalcone A, a chemoprevention agent in the licorice species G. inflate, is slowly metabolized by human hepatic cytochrome P450 enzymes to form several mono‐ and di‐oxygenated phase I metabolites. However, phase II glucuronidation followed by sulfation is expected to occur much more rapidly. These in vitro metabolism studies of licochalcone A will be useful in predicting its bioavailability and potential efficacy as a chemoprevention agent in vivo.

Support or Funding Information

NIH grant P50 AT000155 from the Office of Dietary Supplements and the National Center for Complementary and Integrative Health. NIH grant R01 AT007659 from the National Center for Complementary and Integrative Health and the Office of the Director.