Ultrasonic-assisted extraction (UAE) of flavonoids (JMBF) from Juglans mandshurica Maxim., an important industrial crop in China, was investigated in the present study. To improve the extraction efficiency of JMBF, suitable UAE was proposed after optimization using a hybrid response surface methodology–artificial neural network–genetic algorithm approach (RSM–ANN–GA). The maximum extraction yield (6.28 mg·g⁻¹) of JMBF was achieved using the following optimum UAE conditions: ethanol concentration, 62%; solid–liquid ratio, 1:20 g·mL⁻¹; ultrasonic power, 228 W; extraction temperature, 60 °C; extraction time, 40 min; total number of extractions, 1. Through the investigation of extraction kinetics, UAE offered a higher saturated concentration (C_s) for JMBF in comparison to traditional solvent extraction (TSE). Scanning electron microscopy (SEM) images showed that deeper holes were generated in J. mandshurica powder under the action of ultrasound, indicating that ultrasound significantly changed the structure of the plant materials to facilitate the dissolution of active substances. Extracts obtained using UAE and TSE were compared by Fourier-transform infrared spectroscopy analysis, the results of which revealed that the functional group of bioactive compounds in the extract was unaffected by the ultrasonication process. Moreover, JMBF was further shown to exhibit significant antioxidant properties in vitro. This study provides a basis for the application of JMBF as a natural antioxidant.

Flavonoids are important secondary metabolites found in Juglans mandshurica Maxim., which is a precious reservoir of bioactive substances in China. To explore the antitumor actions of flavonoids (JMFs) from the waste branches of J. mandshurica, the following optimized purification parameters of JMFs by macroporous resins were first obtained. The loading concentration, flow rate, and loading volume of raw flavonoid extracts were 1.4 mg/mL, 2.4 BV/h, and 5 BV, respectively, and for desorption, 60% ethanol (4 BV) was selected to elute JMFs-loaded AB-8 resin at a flow rate of 2.4 BV/h. This adsorption behavior can be explained by the pseudo-second-order kinetic model and Langmuir isotherm model. Subsequently, JMFs were identified using Fourier transform infrared combined with high-performance liquid chromatography and tandem mass spectrometry, and a total of 156 flavonoids were identified. Furthermore, the inhibitory potential of JMFs on the proliferation, migration, and invasion of HepG2 cells was demonstrated. The results also show that exposure to JMFs induced apoptotic cell death, which might be associated with extrinsic and intrinsic pathways. Additionally, flow cytometry detection found that JMFs exposure triggered S phase arrest and the generation of reactive oxygen species in HepG2 cells. These findings suggest that the JMFs purified in this study represent great potential for the treatment of liver cancer.