Dihydroflavonoids outperform their flavonoid counterparts in lipid-lowering efficacy based on pairwise analyses

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease worldwide, affecting approximately 25% of the adult population, yet no approved pharmacotherapy is currently available. Flavonoids have shown promise in improving MASLD, but the structural determinants underlying their lipid-lowering activities remain unclear. This study aimed to systematically investigate whether the presence or absence of the C-2,3 double bond affects the efficacy of flavonoids in ameliorating MASLD. Two pairs of C-2,3 positional isomeric flavonoids-eriodictyol (Compound 1a, dihydro form, C-2,3 single bond)/luteolin (Compound 1b, non-dihydro form, C-2,3 double bond) and dihydromyricetin (Compound 2a, dihydro form, C-2,3 single bond)/myricetin (Compound 2b, non-dihydro form, C-2,3 double bond)-were compared using an FFA-induced HepG2 cell model and an HFD-induced MASLD mouse model. Lipid accumulation, gene expression, target protein thermal stabilization (via CETSA), and compound-target interactions (via molecular docking) were systematically assessed. Both in vitro and in vivo experiments consistently demonstrated that the dihydro forms (C-2,3 single bond) exhibited superior efficacy to their non-dihydro counterparts (C-2,3 double bond) in reducing lipid accumulation and modulating lipid metabolism-related genes. Mechanistically, the dihydro forms showed broader regulatory effects on genes involved in fatty acid oxidation, de novo lipogenesis, and fatty acid uptake. CETSA and molecular docking analyses further suggested that the dihydro forms exhibited greater thermal stabilization of the target proteins and generally lower predicted binding energies across all three targets (PPARγ, ACC1, and FASN). This study provides the first systematic evidence that the C-2,3 single-bond configuration may represent a structural feature associated with enhanced lipid-lowering activity of flavonoids, offering experimental evidence for dietary intervention strategies and flavonoid-based lead compound optimization in MASLD.