Ultrasound extraction and bioactive flavonoid profiling of Alsophila spinulosa leaves across maturity stages

Alsophila spinulosa leaves are consumed as an herbal infusion, but the flavonoid-rich products obtainable from leaves at different maturity stages and their major bioactive constituents remain unclear. In this study, flavonoids from A. spinulosa leaves at three maturity stages were investigated by combining ultrasound-assisted extraction optimization, purification using AB-8 macroporous adsorption resin, structural characterization, targeted LC-MS/MS profiling, molecular docking, and cell-based validation in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. A four-factor Box-Behnken design optimized extraction time, ultrasonic power, liquid-to-solid ratio, and ethanol concentration, and the practical optimum yielded 61.9 ± 1.73 mg rutin equivalents/g dry weight total flavonoids. The enriched extract was characterized by Fourier-transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis, indicating a polyhydroxylated aromatic, largely amorphous product that was sensitive to light and strong alkali. Targeted LC-MS/MS annotated 166 flavonoid metabolites and revealed clear maturity-dependent remodeling, mainly involving flavones and flavonols; isoorientin, vitexin, nicotiflorin, and orientin were the most abundant constituents. Molecular docking highlighted sophoricoside and sieboldin as candidate ligands for Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor kappa B (NF-κB). In LPS-stimulated RAW 264.7 macrophages, the purified extract at 50 μg/mL and six representative monomers, each tested at 10 μM, reduced pro-inflammatory transcript levels and modulated cytoprotective defense-related genes. Overall, these results provide a practical basis for maturity-guided utilization and development of bioactive flavonoid products from A. spinulosa leaves.