Enzyme pretreatment combined with ultrasound-assisted aqueous biphasic extraction of flavonoids from Abelmoschus manihot (L.) leaves: Process optimization, antioxidant activity, and molecular docking.

In this study, we developed a comprehensive strategy involving enzyme pretreatment combined with ultrasound-assisted aqueous biphasic extraction (EPUAABE) for extracting flavonoids from Abelmoschus manihot (L.) leaves (AMLF). Under optimized conditions (2.4 % enzyme concentration, 1:3 cellulase-to-xylanase ratio, pH 5.0, 37.5 °C, 2.5 h, 1:40 solid-liquid ratio, ultrasonic treatment of 180 W, 16 min, and (NH4)2SO4 mass fraction of 20 %), the maximum recovery of flavonoids recorded was 66.81 %. The yield was 30.90 mg/g in the top phase. HPLC analysis revealed significantly higher contents of myricetin and quercetin with EPUAABE than with 80 % ethanol solvent extraction (SE). The EPUAABE extracts presented better radical-scavenging activity, which was strongly correlated with quercetin, myricetin, and quercetin-3-O-glucoside. Elevated superoxide dismutase (SOD) and catalase (CAT) activities, alongside reduced malondialdehyde (MDA) levels in the serum, were detected in the mice treated with AMLF. Molecular docking demonstrated high binding affinities of these flavonoids to SOD and CAT. Overall, this study highlights that EPUAABE, a green, scalable process, effectively enhances flavonoid yield and has strong antioxidant activity in enriched flavonoid fractions, indicating promising application in developing functional foods and nutraceuticals.