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

Abstract

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.