Integrated Metabolomics and Multilevel Validation of the Anti-Inflammatory and Skin-Soothing Effects of Bioactive...

Prinsepia utilis Royle seeds are traditionally used in Northwestern Yunnan to relieve skin inflammation and irritation. However, its active constituents and potential molecular mechanisms remain unclear. To investigate the bioactive constituents and anti-inflammatory and skin-soothing effects of P. utilis seed-derived extracts, we characterized their chemical profiles and investigated the potential molecular mechanisms through phytochemical, computational, and complementary biological approaches, as well as in vitro and in vivo tests in this study. The results of this study show that the 75% ethanol eluate (QC04) of P. utilis seeds exhibited the strongest inhibitory effect on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages, with an IC₅₀ value of 239.21 ± 6.70 μg/mL. Subsequently, untargeted metabolomics identified 1039 metabolites, and 20 representative compounds were selected for downstream analysis. Furthermore, network pharmacology analysis identified AKT1, MAPK1, MAPK8, and MAPK14 as core targets, which are mainly involved in the TNF and MAPK signaling pathways. Molecular docking confirmed favorable binding interactions between representative metabolites and these core proteins; meanwhile, a 100 ns molecular dynamics simulation verified the conformational stability of the euscaphic acid-AKT1 complex. Moreover, in the UVB-induced 3D epidermal model (EpiKutis^®), QC04 treatment reduced IL-6 and PGE₂ secretion and downregulated TNF-α and NF-κB p65 expression. In vivo zebrafish assays further demonstrated that QC04 inhibited copper sulfate-induced neutrophil recruitment and histamine-induced vasodilation. Overall, these findings link the metabolites identified in the activity-enriched QC04 fraction with predicted molecular targets and anti-inflammatory and skin-soothing effects, providing experimental evidence for the biological potential of P. utilis seed-derived preparations and supporting further investigation of QC04 as a potential skin-soothing active fraction.