Unraveling the Mechanisms of Active Saponins from <i>Rhizoma Anemarrhenae</i> Against Ferroptosis in Alzheimer’s Disease: Integrating Network Pharmacology, Cell Membrane Chromatography, and Experimental Validation

<i>Rhizoma Anemarrhenae</i>, in which the primary active components are saponins, has shown potential in treating Alzheimer's disease (AD). However, the specific mechanisms of action and the active saponins responsible remain unclear. This study aimed to explore the mechanisms of action and identify the active components of <i>Rhizoma Anemarrhenae</i> saponins (RAS). First, 24 saponin components in RAS and eight absorbed saponins in rats were identified. Then, a component-target interaction network between eight saponins and 83 targets was constructed after target refinement and SPR validation. Bioinformatics analysis indicated that these targets were closely related to lipid metabolism, iron metabolism, and the AMPK signaling pathway. In addition, differentially expressed genes from RAS intervention were significantly enriched in the ferroptosis pathway. <i>In vitro</i> and <i>in vivo</i> assays demonstrated that RAS could inhibit neuronal ferroptosis and alleviate cognitive impairment. Notably, the ferroptosis inducer markedly reversed the neuroprotective effects of RAS. Moreover, silencing AMPK or Nrf2 using the siRNA or AMPK inhibitor abolished the neuroprotective and ferroptosis-inhibitory effects of RAS <i>in vivo</i> or <i>in vitro</i>. Silencing LKB1 reversed the RAS-induced activation of the AMPK/Nrf2 pathway, and co-immunoprecipitation assay revealed that RAS could promote the LKB1-AMPK interaction. Finally, a 2D comprehensive NC/CMC system was used to screen out four potential saponins that inhibit neuronal ferroptosis, with Timosaponin B-III, Timosaponin A-I, and Timosaponin A-III being validated. In conclusion, RAS exerts anti-AD effects by enhancing the LKB1-AMPK interaction, and activating the AMPK/Nrf2 pathway, inhibiting neuronal ferroptosis as a result. Three saponins are identified as the active components potentially responsible for this effect.