Ultrasound-assisted extraction of flavonoids from Blumea balsamifera using deep eutectic solvents: optimization, characterization, and antioxidant evaluation.

Blumea balsamifera, a traditional medicinal herb rich in flavonoids, is recognized for its strong antioxidant potential. In this study, a sustainable extraction strategy combining deep eutectic solvents (DES) and ultrasound-assisted extraction (UAE) was developed to enhance flavonoid recovery. Extraction parameters were optimized using response surface methodology (RSM) for DES screening, and further refined by an artificial neural network-genetic algorithm model (ANN-GA). Choline chloride-glycerol (ChCl-Gly, 1:2, 40% water) was selected as the optimal solvent. Under the ANN-GA conditions of 40:1 mL/g, 77 °C, 45 min, and 300 W, the total flavonoid yield reached 3.52 ± 0.01%, which was 73.40% higher than that obtained by 50% ethanol heat-assisted extraction. Factorial, kinetic, rated-power-based operational, and SEM analyses were consistent with ultrasound-associated matrix disruption and mass-transfer intensification, while FTIR and 1H NMR supported hydrogen-bond network reorganization in the DES. Antioxidant evaluation through DPPH, ABTS, and reducing power assays showed superior activity of DES extracts, with DES-3 achieving a DPPH IC50 of 0.1443 ± 0.0063 mg/mL. Total flavonoid content was positively correlated with DPPH (r = 0.82, p < 0.01) and ABTS (r = 0.75, p < 0.05) radical-scavenging activities. A cell-based H2O2-injury model in L02 hepatocytes further demonstrated cytoprotective effects of the DES-UAE extract. UPLC-Q-Orbitrap HRMS identified fifteen flavonoids, several of which were reported in this species for the first time. After three recovery and reuse cycles, the regenerated DES retained 90.57% of its initial extraction yield, with limited changes in viscosity, density, and principal 1H NMR signals. These findings demonstrate that DES-UAE provides a green and efficient approach for extracting bioactive compounds from B. balsamifera, offering a sustainable foundation for natural antioxidant production.