Kaempferol from Syzygium myrtifolium Leaves: Isolation, Structural Elucidation, and Antibiofilm Activity Against Pathogenic Root Canal Bacteria

Abstract

Persistent root canal infection remains a major challenge in endodontic therapy, largely attributable to bacterial biofilms formed by Enterococcus faecalis and Staphylococcus aureus that resist conventional irrigants such as sodium hypochlorite. Plant-derived flavonoids, including kaempferol, have shown promising antibiofilm and antibacterial properties, yet the isolation of kaempferol specifically from Syzygium myrtifolium Walp. (red shoot) leaves and its inhibitory activity against these endodontic pathogens have not been previously reported. This study aimed to isolate and structurally characterize kaempferol from S. myrtifolium leaves and to evaluate its inhibitory effect on mixed-species biofilm formation by E. faecalis and S. aureus. The dried leaf powder was macerated in 96% ethanol, partitioned sequentially with n-hexane and ethyl acetate, and the ethyl acetate fraction was subjected to gravity column chromatography guided by thin-layer chromatography. A pure isolate was obtained and characterized using UV, IR, HR-ESI-TOF-MS, and one-dimensional NMR spectroscopy (1H, 13C, DEPT-135). Spectroscopic data (molecular formula C15H10O6; m/z 287.0556 [M+H]+) together with comparison to reference NMR data confirmed the isolate as kaempferol (3,4′,5,7-tetrahydroxyflavone). In a crystal violet biofilm assay, kaempferol produced concentration-dependent inhibition of mixed-species E. faecalis–S. aureus biofilm formation, with the 5 µg/mL concentration showing antibiofilm efficacy statistically comparable to 0.2% chlorhexidine at both 24 h and 48 h (p > 0.05). These findings support kaempferol from S. myrtifolium leaves as a promising natural antibiofilm candidate against clinically relevant endodontic pathogens and provide a structural basis for its further development as an adjunctive agent in root canal disinfection