A Multi-Layered Analytical Pipeline Combining Informatics, UHPLC–MS/MS, Network Pharmacology, and Bioassays for Elucidating the Skin Anti-Aging Activity of Melampyrum roseum

Oxidative stress, UV exposure, inflammation, and extracellular matrix degradation collectively drive skin aging, underscoring the need for safe, multi-target therapeutic options. We developed and applied an integrated analytical pipeline combining UHPLC-MS/MS metabolomics, computational analyses (network pharmacology, molecular docking, and molecular dynamics simulation), and experimental bioassays to efficiently identify and characterize novel natural products with anti-aging potential. This workflow was applied to <i>Melampyrum roseum</i> Maxim., a previously unassessed hemiparasitic plant of the Orobanchaceae family, to elucidate its bioactive potential against skin aging. UHPLC-MS/MS profiling annotated 13 secondary metabolites, predominantly flavone aglycones, iridoid glycosides, and phenylpropanoid derivatives. Network pharmacology analysis linked these metabolites to 172 potential skin-aging-associated targets, mainly within inflammatory, ECM, and oxidative-stress pathways. Molecular docking and 100-ns molecular dynamics simulations confirmed stable ligand-target interactions with favorable binding energies, particularly with AKT1, EGFR, PTGS2 and XDH. Validating these predictions, the <i>M. roseum</i> extract demonstrated significant antioxidant activity and effectively suppressed key inflammatory mediators (IL-6, TNF-α, COX-2) and MMP-1 levels in UVB-exposed fibroblasts, notably without significant cytotoxicity. Collectively, these findings demonstrate that <i>M. roseum</i> harbors multifunctional metabolites that modulate key inflammatory and matrix-regulatory pathways, providing preliminary mechanistic evidence for its potential as a promising candidate for natural anti-aging applications.