Ultrasound-assisted deep eutectic solvent extraction of bioactive flavonoids from Anchusa italica retz.: optimization, characterization, and evaluation of antidepressant potential - targeting oxidative stress and neurotrophic pathways.

This study optimized the extraction of flavonoids from Anchusa italica Retz. using ultrasound-assisted deep eutectic solvent (DES) technology to maximize yield. A Box-Behnken design identified the optimal extraction parameters: solid-liquid ratio of 32.97, temperature of 68.74 °C, extraction time of 101 min, and ultrasonic power of 480.68 W. Flavonoid components were identified by liquid chromatography-mass spectrometry and quantified using high-performance liquid chromatography. In vitro, the total flavonoid extract significantly reduced oxidative stress markers-malondialdehyde, reactive oxygen species, and iron-and enhanced antioxidant defense markers, including superoxide dismutase, glutathione, glutathione peroxidase, and brain-derived neurotrophic factor (BDNF) in lipopolysaccharide-stimulated BV2 microglia. These protective effects were potentially mediated through the Toll-like receptor 4/nuclear factor kappa B/glutathione peroxidase 4 (TLR4/NF-κB/GPX4) signaling pathway. In vivo, oral administration of the extract for four weeks ameliorated depression-like behaviors in C57BL/6 mice exposed to chronic unpredictable mild stress. Treatment with the extract elevated serum levels of the neurotransmitters dopamine and serotonin, as well as those of BDNF, while upregulating hippocampal protein expression in the BDNF/tyrosine receptor kinase B/cAMP response element-binding protein (BDNF/TrkB/CREB) signaling pathway, thereby confirming its antidepressant efficacy. This study establishes an efficient DES-based extraction method for bioactive flavonoids and provides compelling evidence for the therapeutic potential of A. italica flavonoids in treating depression by targeting oxidative stress and neurotrophic signaling.