Unveiling the Phytochemical and Bioactive Potential of Rhododendron anthopogonoides Flavonoids Through Optimization of Ultrasound-Assisted Enzymatic Extraction

Abstract

This study developed an integrated process combining ultrasound-assisted enzymatic extraction (UAEE) with purification for the efficient recovery of bioactive flavonoids from Rhododendron anthopogonoides Maxim. (RA). Extraction conditions were optimized via Plackett–Burman screening followed by response surface methodology (RSM). Optimal conditions (enzyme dosage 30 mg/g, liquid-to-solid ratio 30:1 mL/g, 70% ethanol, 70 °C, ultrasonic power 490 W, extraction time 40 min) produced a total flavonoid yield of 13.56%. RSM established a reliable quadratic model (R2 = 0.9800), with artificial neural network cross-validation further validating the robustness of the optimized parameters. Notably, UAEE substantially outperformed conventional ultrasound-assisted extraction. Purification using AB-8 resin raised flavonoid purity to 25.4 ± 0.48%. SEM and FT-IR analyses revealed effective cell wall disruption while maintaining flavonoid structural integrity. UPLC-Q-Orbitrap-HRMS identified 19 flavonoid compounds in the purified fraction. In vitro assays demonstrated that the purified flavonoids exhibited strong antioxidant activity, with IC50 values of 28 µg/mL (DPPH), 38 µg/mL (ABTS+), and 43 µg/mL (•OH), corresponding to scavenging rates of 98.4%, 98.1%, and 94.2% at 200 µg/mL, respectively. Furthermore, the purified flavonoids dose-dependently inhibited the release of NO, TNF-α, and IL-6 in LPS-induced macrophages at non-cytotoxic concentrations.