Multi-target neuroprotection of Atractylodes macrocephala ethyl acetate extract against Alzheimer's disease: from bioactivity-guided screening to mechanistic validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes macrocephala (AM) has traditionally been used for treating inflammatory conditions, edema, and digestive issues. It is now being explored for its potential in treating Alzheimer's disease (AD), which is characterized by cognitive decline and impairments in learning and memory. While AM shows promise as a potential treatment for AD, further research is needed to identify its specific bioactive components and understand its mechanisms of action.

AIM OF THE STUDY: This study aim to identify and evaluate bioactive fractions from AM for their potential to treat AD. The research focused on assessing the antioxidant, anti-inflammatory, and neuroprotective properties of these fractions to determine the most effective one. The ultimate goal was to find a basis for isolating active neuroprotective components from AM that could be used to develop new AD therapies.

MATERIALS AND METHODS: To identify AM fractions with potential against AD, the study began with in vitro assessments of their ability to inhibit amyloid-β (Aβ) aggregation and their antioxidant capacity. The neuroprotective effects of these fractions were then evaluated against Aβ1-42 and D-galactose-induced toxicity in neuronal and microglial cell lines. A C. elegans AD model (CL4176 strain) was employed to assess the impact on Aβ-induced neurodegeneration (paralysis). Based on this comprehensive screening, the ethyl acetate extract (AMEA) emerged as the most promising fraction. Its mechanism of action was investigated using network pharmacology and validated in vivo using APP/PS1 transgenic mice. Finally, key compounds within AMEA were isolated and characterized.

RESULTS: Compared to other extracts, AMEA showed stronger antioxidant, anti-inflammatory, and neuroprotective effects. In AD model mice, AMEA treatment protected the hippocampal neuronal structure, reduced damage to the pyramidal cell layer, and prevented dendritic spine loss. AMEA also significantly lowered the levels of key pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and reduced markers of microglial (IBA-1) and astrocyte (GFAP) activation, indicating reduced neuroinflammation. AMEA inhibited Aβ aggregation and improved cognitive function by reducing Aβ deposition in APP/PS1 transgenic mice. Network pharmacology analysis showed that AMEA is associated with neurodegenerative disease pathways and can regulate inflammatory mediators. UPLC-OE/MS analysis tentatively identified thirty-three phytochemicals in AMEA, including sesquiterpenoids, phenylpropanoids, coumarins, and flavonoids. Molecular docking revealed high binding affinity of compounds 3, 9, and 11 towards TNF-α, IL-6, and IL-1β, and ELISA results confirmed that compounds 3 and 11 markedly downregulated the expression of key neuroinflammatory factors. Structural isolation and bioactivity analysis of AMEA revealed that it contains bioactive components capable of protecting nerve cells from Aβ toxicity.

CONCLUSIONS: This research concludes that the AMEA fraction of AM demonstrates significantly superior neuroprotective effects against the primary pathological features of AD compared to other solvent fractions. AMEA exhibited greater efficacy in inhibiting Aβ aggregation, neutralizing free radicals, protecting nerve cells, and reducing brain inflammation across various models, including C. elegans and AD mice. Furthermore, chemical component isolation and structural identification of the AMEA fraction yielded compounds such as atractylenolide and atractylenolactam, along with their dimers, many of which possess neuroprotective properties. In conclusion, this study establishes a basis for the future identification and development of neuroprotective compounds from AM.