Optimization of Ultrasound-Assisted Deep Eutectic Solvent (DES) Extraction Process for Total Flavonoids from Robinia pseudoacacia Flowers and Evaluation of Their Antioxidant Activity

Abstract

Objective: To establish a green and efficient extraction process for total flavonoids from Robinia pseudoacacia L. flos using ultrasound-assisted deep eutectic solvents (DESs), optimize the process using response surface methodology, and systematically evaluate their in vitro antioxidant activities, providing experimental evidence for the development and utilization of active components in Robinia pseudoacacia flowers. Methods: Choline chloride was used as the hydrogen bond acceptor (HBA), and 1,2-propanediol, ethylene glycol, and lactic acid were used as hydrogen bond donors (HBD) to prepare three DES systems. Single-factor experiments were conducted to investigate the effects of HBA: HBD molar ratio, water content, liquid-to-solid ratio, ultrasonic time, and temperature on the extraction rate of total flavonoids. The extraction conditions were then optimized using Box-Behnken response surface design. High-performance liquid chromatography (HPLC) was used to determine the content of major flavonoid components (rutin, quercetin, and luteolin). The antioxidant capacity was evaluated by DPPH· and ABTS⁺ free radical scavenging rates. Results: Single-factor analysis showed that the highest extraction rate was achieved when the DES was choline chloride-1,2-propanediol (molar ratio 1:3), water-to-DES volume ratio was 3:1, liquid-to-solid ratio was 35:1 (mL/g), ultrasonic temperature was 65 ℃, and ultrasonic time was 40 min. Response surface optimization indicated that the theoretical extraction rate under these conditions was 4.26%, and the average value of the validation experiment was 4.22%, with an error of less than 1%. HPLC analysis revealed that the contents of rutin, quercetin, and luteolin were 2.06, 1.32, and 0.84 mg/g, respectively. Antioxidant experiments showed that when the extract concentration was 0.4 mg/mL, the scavenging rates of DPPH· and ABTS⁺ were 81.45% and 92.30%, respectively, with no significant difference compared to the vitamin C group (P>0.05). Conclusion: The established DES-ultrasound-assisted extraction method is green, environmentally friendly, and reproducible, significantly improving the extraction rate of flavonoids from Robinia pseudoacacia flowers. The extract has strong antioxidant capacity, providing a scientific basis for its application in the development of natural antioxidants.