Highly efficient discovery of CYP1B1 inhibitors from licorice as novel anti-fibrotic agents via integrating interdisciplinary methodologies.

Aberrant CYP1B1 activity has been implicated in various metabolic disorders, while targeted inhibition on CYP1B1 has been validated as an emerging therapeutic strategy for hepatic fibrosis. Although a range of edible herbs including Licorice (the root of Glycyrrhiza glabra) have been widely used for treating liver diseases, identifying the key anti-CYP1B1 constituents in these herbs and elucidating their mechanisms of action remain challenging. This study presents a comprehensive interdisciplinary framework for discovering CYP1B1 inhibitors from herbal medicines (HMs) via integrating interdisciplinary methodologies. An initial fluorescence-based screening of 65 herbal extracts revealed that licorice aqueous extract (LAE) exhibited potent CYP1B1 inhibitory activity. Docking-based virtual screening predicted 23 phytochemicals in LAE as potential CYP1B1 inhibitors, 18 of which were experimentally confirmed as potent CYP1B1 inhibitors (IC50 < 1 μM). Further investigations showed that the nine most potent phytochemicals in LAE (IC50 < 260 nM) inhibited CYP1B1 in a competitive manner, with the Ki values below 560 nM. Cellular assays further demonstrated that three of these phytochemicals (glycyrol, licoflavonol, and licochalcone C) effectively blocked intracellular CYP1B1 activity in living cells (IC50 < 3 μM). Anti-fibrotic assays showed that glycyrol significantly reduced cell migration and downregulated the expression of key fibrotic genes in TGF-β1-stimulated JS-1 cells. Collectively, this work establishes a practical and efficient strategy for discovering potent anti-CYP1B1 agents from HMs against liver fibrosis, while glycyrol emerges as a promising anti-fibrotic therapeutic.