Integration of ANN-GA optimization and multi-scale mechanistic analysis for ultrasound-assisted enzymatic extraction...

Cortex Mori (CM), a valuable medicinal plant with abundant flavonoids, exhibits diverse biological and pharmacological activities. This work aims to develop an environmentally friendly and efficient ultrasound-assisted enzymatic extraction (UAEE) strategy for extracting flavonoids from CM, while also elucidating the mechanism underlying this process, characterizing major flavonoids and evaluating their bioactivities. First, UAEE was utilized for flavonoid extraction, and the critical parameters were determined using Plackett-Burman design (PBD), followed by optimization by integrating response surface methodology (RSM) with the Artificial Neural Network (ANN)-genetic algorithm (GA) approach. In addition, FT-IR, SEM and molecular dynamics simulations (MDS) were employed to elucidate the extraction mechanisms. UHPLC-HRMS was utilized to identify flavonoids in CM extracts, and the antioxidant and anti-proliferation activities were assessed. Results demonstrated that the ANN-GA model optimization outperformed RSM, yielding a maximum total flavonoid content (TFC) of 21.18 ± 0.94 mg/g under optimized conditions using an ultrasonic bath system, with ultrasonic parameters of 40 kHz and 200 W, combined with a solid-to-liquid ratio of 1: 24 g/mL, ultrasonic temperature of 55 ℃, ethanol volume fraction of 67 % and ultrasonic time of 60 min. Mechanistic analysis revealed that ultrasound promoted structural disruption of cell walls, altered intermolecular interactions, and enhanced solvent accessibility, thereby facilitating enzyme-assisted flavonoid release. A total of 41 flavonoids were tentatively characterized in CM extracts. CM extracts exhibited antioxidant capacity, including ABTS⁺ scavenging activity (0.8940 ± 0.0010 mmol/L), DPPH scavenging activity (96.78 ± 4.28 %) and ferric reducing antioxidant power (FRAP, 1.3074 ± 0.1234 mmol/L). CM extracts also showed antiproliferative activity against SW620, 4 T1, A2780, LOVO, and MCF-7 cell lines. Overall, this research provides multi-scale optimization strategies and mechanistic insights into the UAEE process, and offers theoretical guidance for the efficient utilization of CM and other botanical resources.