Phenolic constituents from Raphiocarpus sesquifolius with neuroprotective and cognition-enhancing activities

Raphiocarpus sesquifolius (C. B. Clarke) Burtt is a unique medicinal plant from Sichuan Province, China. Seven phenolic compounds were isolated and identified from the whole herb for the first time: 6,4'-dihydroxy-7-methoxyflavone-5-O-β-D-(6'-acetoxy) glucoside (1), 6,4'-dihydroxy-7-methoxyflavonoid-5-O-β-D-glucoside (2), 4,9-dihydroxy-3-methyl-3,4-dihydroanthracen-1(2H)-one (3), 10-hydroxy-2-methylanthracene-1,4-dione (4), 4',5,6-trihydroxy-7-methoxyflavone (5), isoverbascoside (6), and caffeoyl ester of apionic acid (7). Compounds 1-4 are undescribed, with their structures elucidated mainly by HRESIMS, 2D NMR, ECD, and acid hydrolysis. Neuroprotective effects were evaluated in BV2 microglial cells, while acetylcholinesterase inhibition assays were performed to assess potential cognitive benefits. Compound 2 markedly suppressed nitric oxide production and exhibited anti-acetylcholinesterase activity. Transcriptomic analysis further revealed that compound 2 modulated multiple pathways associated with neuroinflammation and neuroprotection-including Toll-like receptor, TNF-α, MAPK, and NF-κB signaling-and upregulated genes related to cholinergic synapses. Collectively, our data demonstrate that compound 2 possesses dual functionality-conferring neuroprotection and enhancing cognition-thereby laying the mechanistic groundwork for its future development as a therapeutic agent for Alzheimer's disease and related neurodegenerative conditions.