Antioxidant and Anti-Inflammatory Activities of Silene dichotoma Ehrh. subsp. dichotoma: Phytochemical Characterization and Metabolomic Insights into Oxidative Stress-Related Responses in HaCaT Cells

Background/Objectives: Silene dichotoma Ehrh. subsp. dichotoma is a relatively understudied member of a genus known for diverse secondary metabolites. This study integrated phytochemical characterization, biological activity assessment, and LC–MS-based cellular metabolomics to investigate its antioxidant, NO-inhibitory, and cytoprotective potential. Methods: An 80% ethanol extract of the aerial parts was partitioned into n-hexane, ethyl acetate, n-butanol, and aqueous fractions and evaluated for DPPH and superoxide radical-scavenging activities and total phenolic and flavonoid contents. The most active fraction was subjected to bioactivity-guided isolation, and fractions were assessed for cytotoxicity, LPS-induced nitric oxide inhibition, H2O2-induced cytoprotection, and untargeted LC–MS metabolomic profiling. Results: The ethyl acetate fraction exhibited the highest phenolic (51.84 mg GAE/g) and flavonoid (176.56 mg QE/g) contents and the strongest antioxidant activity, yielding isorhamnetin, isorhamnetin-3-O-β-D-galactopyranoside, and quercetin, reported for the first time from this species. None of the extract or fractions showed marked cytotoxicity toward RAW 264.7 macrophages or HaCaT keratinocytes. The ethyl acetate fraction most strongly inhibited LPS-induced nitric oxide production (IC50 = 118.34 µg/mL). In H2O2-challenged HaCaT cells, the n-hexane fraction modestly increased viability to 67.77% at 200 µg/mL; however, no statistically significant overall cytoprotective effect was demonstrated. Untargeted LC–MS profiling yielded 99 putatively annotated metabolites. After averaging analytical duplicates within biological replicates, 38 of 75 metabolites eligible for the overall seven-group analysis showed an FDR-adjusted difference. Exploratory pairwise comparisons suggested treatment-associated changes in energy-related, redox-associated, nucleotide, amino-acid, and lipid-associated features. Conclusions: These findings identify this species as a source of flavonoids with antioxidant and nitric oxide-inhibitory potential while demonstrating the value of metabolomics in characterizing cellular responses beyond viability endpoints.