Ultrasound-Assisted Enzymatic Extraction of Flavonoids from Pouteria caimito Leaves: Process Optimization, Mechanistic Investigation, and Bioactivity Evaluation

Abstract To enhance the resource utilization value of Pouteria caimito leaves, this study employed ultrasound-assisted enzymatic extraction (UAE) to extract flavonoids from Pouteria caimito leaves (PCL). The UAE process was optimized using single-factor experiments combined with response surface methodology. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) were used to investigate structural changes in leaf tissues and the synergistic extraction effects of ultrasound and enzymatic hydrolysis. UHPLC-HRMS/MS was used to characterize extracts obtained by different extraction methods, and seasonal variations in flavonoid content and biological activity were also evaluated. The optimal extraction conditions were as follows: solid–liquid ratio of 1:50, ethanol concentration of 50%, pH 3.6, enzyme addition of 1.6%, enzymatic hydrolysis temperature of 50 °C, enzymatic hydrolysis time of 51 min, ultrasonic temperature of 50 °C, and ultrasonic time of 39 min. Under these conditions, the flavonoid extraction yield by UAE was higher than that achieved by ultrasonic extraction (UE) or enzymatic extraction (EE) alone. SEM, FT-IR, and XRD results demonstrated more extensive cell wall disruption and higher extraction efficiency with UAE. After purification using AB-8 macroporous resin, flavonoid purity increased from 32.84% to 73.15%. Activity evaluation showed that the purified product (MRP) exhibited strong antioxidant and multienzyme inhibitory activities: DPPH IC50 = (0.0197 ± 0.0014) mg/mL, ABTS+• IC50 = (0.0560 ± 0.0016) mg/mL, tyrosinase IC50 = (0.2911 ± 0.0022) mg/mL, acetylcholinesterase IC50 = (0.5303 ± 0.0101) mg/mL, and α-amylase IC50 = (0.3328 ± 0.0100) mg/mL. Network pharmacology further identified catechin, dihydrotricetin, and kaempferol 3,5,7-trimethyl ether as key candidate compounds and suggested that their potential effects against Alzheimer’s disease may involve AChE-related regulation within the “Neuroactive ligand signaling” pathway. January was identified as the optimal harvest period. This study demonstrates the potential of UAE for the high-value utilization of PCL flavonoids.