Molecular Networking-Guided Isolation of a Phenolic Constituent from Prunus mume Seed and Its Antioxidant and Anti-Inflammatory Activities

Abstract

<i>Prunus mume</i> (Maesil) is used in health foods and alternative medicine in Korea. In the present study, the anti-inflammatory and antioxidant effects of phenolics from <i>P. mume</i> seed extracts were examined. First, the biological activities of various <i>P. mume</i> extracts were evaluated, and the profiles of their chemical compounds were investigated by Global Natural Products Social (GNPS)-molecular networking. Among these extracts, fermented Maesil seed extract (FMSE) showed potent anti-inflammatory and antioxidant activity, and demonstrated the presence of phenolic clusters in GNPS-based studies. Thus, the chemical constituents of this extract were further investigated. Subsequently, the chemical composition of the active CH₂Cl₂ fraction of FMSE was explored using an advanced GNPS analysis tool, MolNetEnhancer. In addition, the molecular structure of compound <b>1</b> from the CH₂Cl₂ fraction was similarly predicted with Network Annotation Propagation (NAP). Finally, the anti-inflammatory and antioxidant effects of compound <b>1</b> were confirmed by lipopolysaccharide (LPS)-induced nitric oxide production and DPPH assay. Western blot analysis revealed that compound <b>1</b> downregulated the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. The molecular docking simulation additionally confirmed significant interactions of <b>1</b> with iNOS and COX-2 proteins. Our findings suggested that an integrated GNPS-based approach could prioritize samples in the early fractionation process and improve the accuracy of target compound prediction.