Process optimization and antidepression multi-target mechanisms of Total Flavonoids from Hemerocallis citrina Baroni: An integrated approach combining DES-UAE, network pharmacology, and experimental validation

ObjectiveThis study aimed to establish a green and efficient ultrasonic-assisted deep eutectic solvent (DES) extraction method for total flavonoids from Hemerocallis citrina Baroni (TFHC) and to elucidate its multi-target antidepressant mechanism using an integrated strategy of network pharmacology, molecular docking, and in vitro validation. MethodsAn ultrasonic-assisted DES extraction using choline chloride-ethylene glycol was optimized via response surface methodology. The TFHC extract was profiled by UPLC-ESI-MS/MS. A network pharmacology approach was employed to predict core targets and pathways of TFHC constituents against depression, with key interactions validated by molecular docking. The neuroprotective effects of TFHC were evaluated in vitro using a corticosterone (CORT)-induced injury model in PC-12 cells, assessing cell viability (MTT assay) and levels of 5-HT, BDNF, TNF-, and CORT (ELISA). ResultsThe optimized DES extraction yielded 16.63 {+/-} 0.13 mg/g of TFHC. UPLC-ESI-MS/MS identified fourteen flavonoids, with quercetin, kaempferol, and rutin being most abundant. Network pharmacology revealed six core targets (AKT1, TNF, IL6, IL1{beta}, TP53, PTGS2) and implicated pathways including PI3K-AKT and TNF. Molecular docking confirmed strong binding affinities ([≤] -4.25 kcal/mol) between major flavonoids and core targets. In vitro, TFHC significantly alleviated CORT-induced cytotoxicity, restored 5-HT and BDNF levels, and suppressed TNF- and CORT elevation, with effects comparable or superior to fluoxetine. ConclusionsTFHC exerts antidepressant-like effects through a multi-target mechanism involving neuroinflammation suppression, neurotrophic support, and HPA axis modulation. The green DES extraction combined with mechanistic insights positions TFHC as a promising candidate for phytotherapeutic antidepressant development.