GNPS-assisted identification of bioactive constituents from Scoparia dulcis and their anti-inflammatory mechanisms via network pharmacology and experimental validation.

Scoparia dulcis L. (SD) is an edible medicinal herb traditionally consumed as a cooling beverage in tropical Asia for the management of metabolic and inflammatory disorders; however, its dietary bioactive constituents and anti-inflammatory mechanisms have not yet been fully elucidated. In the present study, the metabolite profile of the aerial parts of SD (ASD) was characterized, and their anti-inflammatory potential was evaluated through an integrated approach incorporating LC-HRMS/MS-based molecular networking, network pharmacology, molecular docking, molecular dynamics simulations, and in vitro validation in macrophages. Untargeted LC-HRMS/MS combined with GNPS feature-based molecular networking enabled the annotation of 184 metabolites in the ASD extract, mainly comprising shikimates and phenylpropanoids (44.0%) and terpenoids (39.7%), with flavonoids constituting the most abundant superclass and including known food bioactives such as β-sitosterol, pentamethoxyflavone, naringin, and catechin. ADMET-based screening prioritized 31 absorbable constituents, while network pharmacology identified 179 inflammation-related targets, with TNF-α, IL6, STAT3, AKT1, and JAK2 emerging as key hubs. Enrichment analysis highlighted the TNF, PI3K-AKT, JAK-STAT, and AGE-RAGE pathways as major signaling routes associated with low-grade diet-related inflammation. Molecular docking and 300 ns molecular dynamics simulations further identified zeorinone as a stable dual binder of TNF-α and IL6, exhibiting the most favorable MM-PBSA binding free energies among the compounds selected for molecular dynamics analysis. Consistent with these computational findings, the ASD extract showed no cytotoxicity in RAW264.7 macrophages at 10-100 µg mL-1 and significantly reduced LPS-induced TNF-α and IL6 secretion at 50-100 µg mL-1, with effects comparable to dexamethasone. Overall, these findings establish SD as a promising source of dietary anti-inflammatory bioactives and provide a chemical and mechanistic basis for its potential development as a functional food ingredient or herbal beverage targeting chronic diet-related inflammation.