Targeted isolation and antipyretic mechanisms of 5,7,4'-trimethoxyflavone and p-hydroxybenzoic acid from Mimosa pudica root via cell membrane-coated magnetic carbon spheres integrated with thermal shift assay.

This study introduces a cell membrane-coated magnetic carbon sphere-integrated thermal shift assay (CMMCS-TSA) platform, combined with metabolomics and gut microbiota analysis, to identify bioactive compounds in Mimosa pudica root and elucidate their antipyretic mechanisms. Using yeast-induced febrile rats and LPS-stimulated RAW 264.7 macrophages, the ethyl acetate fraction (EA) of M. pudica root exhibited potent antipyretic effects by reducing fever, inflammatory cytokines (IL-6, TNF-α, IL-1β), thermoregulatory factors (PGE2, cAMP, 5-HT), and suppressing TLR4/NF-κB pathway proteins (NF-κB p65, COX-2, TLR4). CMMCS-TSA enabled targeted isolation of two bioactive compounds - 5,7,4'-trimethoxyflavone (TF) and p-hydroxybenzoic acid (HA) - confirmed via NMR and LC-MS. Molecular docking revealed strong binding affinities of HA and TF to TLR4 and COX-2 catalytic domains. In vitro, both compounds inhibited LPS-induced NO production via TLR4 suppression, validated by TAK antagonist experiments. In vivo, HA and TF alleviated fever, restored amino acid/sphingolipid metabolism, and rebalanced gut microbiota. Mechanistically, their antipyretic effects involved coordinated modulation of the TLR4/NF-κB pathway, gut-brain axis signaling, and metabolic reprogramming. This work establishes CMMCS-TSA as a transformative platform for precision isolation of bioactive phytochemicals and provides the first evidence of M. pudica root's dual-compound antipyretic mechanism through multi-omics regulation. The findings highlight the potential of nanomaterial-integrated approaches in natural product research and advance the development of plant-derived antipyretics with multi-target mechanisms.