Ultrasound-assisted deep eutectic solvent extraction of pine pollen flavonoids and their Antifatigue effects

This study optimized a green ultrasound-assisted extraction (UAE) method using deep eutectic solvents (DESs) to enhance flavonoid yield from pine pollen. Among eight tested DESs, choline chloride/citric acid (DES-3) demonstrated the highest efficiency. Single-factor and Box-Behnken design experiments identified optimal conditions: 30 % water content, 1:24 g/mL solvent-to-solute ratio, and 73 °C extraction temperature, achieving a yield of 13.95 mg/g—significantly surpassing conventional ethanol-based methods. Flavonoids were purified using NKA-9 macroporous resin, achieving a 75.72 % recovery rate and 52.78 % purity with gradient ethanol elution. In vitro, the flavonoid extract exhibited potent antioxidant activity, scavenging 96.58 % of DPPH radicals at 0.26 mg/mL. In vivo antifatigue effects were evaluated in mice: high-dose (300 mg/kg·d) and low-dose (150 mg/kg·d) groups showed prolonged exhaustive swimming times (18.73 min and 16.60 min vs. control 9.12 min), reduced serum lactate and urea nitrogen, elevated liver glycogen, and enhanced antioxidant enzyme activities (SOD, CAT, GSH-Px). These results validate pine pollen flavonoids as promising natural agents for functional foods targeting fatigue mitigation.