Natural dual inhibitor isorhamnetin-3-O-neohespeidoside targets tyrosinase and MC1R for skin pigmentation management

Skin pigmentation disorders involve complex biological regulation, with tyrosinase (TYR) and melanocortin 1 receptor (MC1R) serving as key therapeutic targets. Through molecular docking screening of 389 natural compounds, we identified isorhamnetin-3-O-neohespeidoside as a potent dual inhibitor, demonstrating superior binding affinities (-8.001 kcal/mol for TYR and - 7.342 kcal/mol for MC1R) compared to arbutin (reference compound). Subsequent in vitro validation revealed that isorhamnetin-3-O-neohespeidoside (8 µM) significantly inhibited TYR activity by 44.42% (p < 0.0001) and reduced MC1R expression by 33.39% (p < 0.0001) in B16 melanoma cells, while maintaining > 85% cell viability (IC₅₀ = 52.22 µM). The compound also decreased melanin content by 38.7% (p < 0.0001) and upregulated LC3-II expression (2.1-fold vs. control, p < 0.0001), indicating enhanced autophagy. These results demonstrate that isorhamnetin-3-O-neohespeidoside, a flavonoid glycoside from Typhae Pollen, acts through multiple mechanisms - direct enzyme inhibition, receptor downregulation, and autophagy induction - making it a promising natural candidate for hyperpigmentation treatment. Our integrated approach combining computational screening with experimental validation provides a robust framework for identifying multi-target depigmenting agents.