Ultrasound-assisted deep eutectic solvent green extraction, mechanistic insight, and molecular antifungal mechanism of flavonoids from Begonia fimbristipula against Microsporum canis.

Abstract

In this work, an eco-friendly ultrasound-assisted deep eutectic solvent (DES) extraction method combined with UPLC-Q-TOF-MS was developed for the efficient enrichment of flavonoids from Begonia fimbristipula. Among 45 screened DESs, betaine-propionic acid (1:1) was selected as the optimal system. The maximum total flavonoid content reached 82.45 mg rutin/g dry weight under optimized conditions (solid-liquid ratio 52 mL/g, 30 min, 57 °C, 240 W). Mechanistic analysis demonstrated that the DES system disrupted plant cell walls and formed strong hydrogen-bond interactions with flavonoids, thereby improving extraction efficiency. Subsequently, UPLC-Q-TOF-MS analysis was performed to profile the phytochemical composition of the purified extract. Ten major phenolic and flavonoid components were identified in the purified flavonoid extract (Bf-FE), clarifying its material basis of bioactivity. In vitro assays showed that Bf-FE exerted potent antifungal activity against Microsporum canis. Mechanistically, Bf-FE destabilized fungal membrane integrity, inhibited ergosterol biosynthesis, impaired mitochondrial function, and induced excessive ROS accumulation, which was further confirmed by transcriptomic analysis. In vivo results validated the antifungal efficacy and biosafety of Bf-FE, which reduced fungal load and modulated epidermal barrier protein expression. This study systematically elucidates the DES-based green extraction mechanism and multi-target molecular antifungal mechanism of B. fimbristipula flavonoids, providing a theoretical basis for developing these flavonoids as natural antifungal lead compounds.