Novel ternary deep eutectic solvent coupled with in-situ-ultrasound synergistic extraction of flavonoids from Epimedium wushanense: machine learning, mechanistic investigation, and antioxidant activity

This study innovatively developed a novel ternary deep eutectic solvent coupled with in-situ-ultrasound synergistic extraction (TDES-ISUSE)method for efficient extraction of total flavonoids (TF) fromEpimedium wushanense. Among 20 prepared DESs, the ternary system composed of choline bitartrate, urea, and glycerol (Chb:Ure:Gly) exhibited the highest extraction efficiency. Response surface methodology-artificial neural network-genetic algorithm (RSM-ANN-GA) optimization identified the optimal extraction parameters: water content of 32 %, vortex time of 10 min, liquid/solid ratio of 34:1 mL/g, ultrasound time of 30 min, and ultrasound power of 270 W. Under these conditions, the TF yield reached26.39 ± 0.61 mg/g, which was significantly higher than that of conventional extraction methods. Phytochemical profiling ofE. wushanensevia UHPLC-Q-TOF-MS (positive/negative ion modes) unambiguously annotated 15 compounds, including 9 flavonoids; concurrently, an HPLC method was established for quantitative determination of 4 key flavonoids. FT-IR spectroscopy and nuclear magnetic resonance (NMR)confirmed the successful prepared of the novel TDES, while scanning electron microscopy (SEM) and molecular dynamics (MD) simulations elucidated the underlying extraction mechanism, revealing enhanced cell wall disruption and improved solubility of flavonoids in TDES-ISUSE. Antioxidant assays further demonstrated the potential bioactivity of the TF extract. Overall, this work provides aneco-friendly, efficient, and reproducible approachfor extracting bioactive flavonoids fromE. wushanense, with implications for sustainable utilization of medicinal plant resources and advancement of green extraction technologies.