LC–MS/MS Chemical Profiling and HPLC/PDA Quantification of Hypericum perforatum Extract and Its Anti-Inflammatory Properties

Background/Objectives: Hypericum perforatum L. is a medicinal plant containing diverse specialized metabolites, including flavonoids, naphthodianthrones, phloroglucinols, and phenolic acids. However, the relationship between its quantitative flavonoid composition and extract-level anti-inflammatory activity remains insufficiently characterized. This study aimed to profile the chemical constituents of H. perforatum extract (HPE), quantify selected flavonoid markers, and relate these data to nitric oxide (NO) inhibitory activity in LPS-stimulated RAW 264.7 macrophages. Methods: HPE was chemically characterized by LC–MS/MS, and selected flavonoid constituents were quantified using HPLC/PDA analysis. Cell viability and NO production were evaluated in RAW 264.7 macrophages using MTT and Griess assays, respectively. Results: LC–MS/MS profiling tentatively identified 36 compounds in HPE, with flavonoid-related metabolites representing the largest portion of the annotation list. Multiple quercetin-derived glycosides and conjugates were detected, indicating a flavonoid-rich phenolic profile. HPLC/PDA analysis showed that hyperoside (2) was the most abundant quantified marker at 12.44 mg/g extract, followed by rutin (1, 9.18 mg/g), quercetin (4, 6.64 mg/g), and avicularin (3, 5.55 mg/g), whereas amentoflavone (5) was detected only at trace level. At non-cytotoxic concentrations, HPE significantly suppressed LPS-induced NO production, iNOS protein expression, and the mRNA expression of the pro-inflammatory cytokines Tnf and Il6 in RAW 264.7 macrophages. Compounds 1–4 also reduced NO production and iNOS protein expression, although their effects on cytokine mRNA expression differed among compounds. Conclusions: HPE exhibited anti-inflammatory potential in LPS-stimulated macrophages, as shown by inhibition of NO production, downregulation of iNOS protein expression, and reduced Tnf/Il6 mRNA expression. However, extract-equivalent comparison indicated that the extract-level response could not be directly assigned to any single quantified flavonoid marker. The broader flavonoid-rich phenolic matrix may provide a plausible context for this response, but additive or synergistic interactions among constituents were not directly demonstrated and require further validation.