Unraveling anti-inflammatory metabolic signatures of Glycyrrhiza uralensis and isoliquiritigenin through multiomics

Glycyrrhiza uralensis, a key component of over 70% of traditional herbal medicines (Kampo) in Japan, exhibits diverse pharmacological effects, including immunoregulation, anti-tumor, and antioxidant properties. Despite over 300 identified compounds, the molecular mechanisms remain unclear due to the chemical diversity. Here, we performed a multiomics analysis incorporating untargeted hydrophilic metabolomics, lipidomics, and phosphoproteomics to elucidate the mechanisms distinguishing the G. uralensis extract (GU) from a single bioactive compound, isoliquiritigenin (ILG). Time-course analyses of lipopolysaccharide (LPS)-stimulated RAW264.7 cells under four conditions (control, LPS(+), LPS(+)/ILG(+), and LPS(+)/GU(+)) quantified 182 hydrophilic metabolites, 381 lipids, and 13,211 phosphopeptides. Both ILG(+) and GU(+) attenuated inflammatory signatures characterized by elevated glycolytic intermediates, succinate, citrulline, triacylglycerols, and cholesteryl esters. A multiset partial least squares technique identified sirtuin (SIRT) 1/2 phosphorylation and altered nicotinamide adenine dinucleotide metabolism specific to ILG(+). SIRT2 inhibition abolished ILG's suppression of interleukin-6 (IL-6). Furthermore, GU(+) uniquely increased γ-aminobutyric acid (GABA) and 4-guanidinobutyric acid via endogenous synthesis by glutamic acid decarboxylase. Exogenous GABA reduced IL-6 and IL-1β expression, and its co-administration with ILG enhanced anti-inflammatory effects. This study demonstrates that multiomics can elucidate the synergistic anti-inflammatory actions of G. uralensis, highlighting endogenous GABA production as a key contributor to ILG-mediated immunomodulation.