Investigation of Free and Bound Phenolic Profiles in Walnut Male Flowers during Ripening and Their Contribution to Antibacterial and Antioxidant Activities

This study examined the dynamic changes in free and bound phenolic compounds in young and mature walnut male flowers (WMF, <i>Juglans regia L</i>.) during ripening alongside the total phenolic content, antioxidant potential, and antibacterial activity. Phenolic profiles were identified using LC-MS/MS. Distinct variations were observed between maturation and phenolic forms. Free phenolics were significantly dominant in young WMF; their levels decreased during maturation, with bound phenolics becoming predominant in the mature fraction. Bound fractions showed higher antioxidant activity (DPPH IC₅₀: 34.33 μg/mL; ABTS IC₅₀: 4.51 μg/mL), consistent with their higher total phenolic content. The bound fraction of mature flowers exhibited the strongest antibacterial activity against <i>Bacillus cereus</i> and <i>Escherichia coli</i> (MIC: 256 μg/mL). FTIR analysis demonstrated characteristic absorption bands of phenolic functional groups, while correlation analysis revealed associations between individual phenolics and antioxidant activity. The findings highlight the influence of phenolic form and maturity on the bioactive potential of WMF.