Dual-Action Antimicrobials: Prenylated Flavonoids from Paper Mulberry Fruit Targeting Staphylococcus aureus Fatty Acid Synthesis

• Two novel compounds and sixteen known compounds were identified from paper mulberry fruit. • These compounds demonstrated potent antibacterial activity against S. aureus . • Broussoflavonol B, the most potent compound, disrupted the bacterial cell membrane. • These compounds targeted both the FASII pathway and its bypass, mainly inhibiting PlsC. • Prenylated flavonoids maintained their antibacterial activity even in the presence of exogenous fatty acids. Paper mulberry ( Broussonetia papyrifera ) fruit, known for its flavor and health benefits, has previously been reported to exhibit antimicrobial activity, but its potential antibacterial effect against Staphylococcus aureus remains underexplored. This study investigated the anti- Staphylococcal activity of prenylated phenolics isolated from paper mulberry, including two novel compounds. Among these compounds, broussoflavonol B exhibited the strongest antibacterial activity against S. aureus CMCC 26003, with a minimum inhibitory concentration (MIC) of 6.9 µM. Mechanistic studies revealed that broussoflavonol B disrupted the cell structure and significantly reduced the levels of unsaturated fatty acids in bacterial cells. Additionally, it suppressed the expression of genes involved in fatty acid biosynthesis, maintaining its antibacterial effect even in the presence of exogenous fatty acids. Interestingly, all prenylated flavonoids from paper mulberry retained their antimicrobial activity in the presence of exogenous fatty acids, whereas prenylated phenopyran (papyriphenol) did not. This suggests a special mode of action for prenylated flavonoids, possibly involving the inhibition of phosphotransacetylase C (PlsC), as supported by molecular docking studies. In summary, the dual inhibitory effects of prenylated flavonoids from paper mulberry fruit provide a novel approach to combat S. aureus . By targeting multiple pathways, these compounds may offer a potential solution to the growing problem of antimicrobial resistance.