Methyl salicylate potentiates antimicrobial properties of berberine against Aggregatibacter actinomycetemcomitans

Berberine (BBR), a plant alkaloid, has been documented for its antimicrobial and anti-inflammatory properties. We have previously reported the in vitro synergism between BBR and antimicrobials against selected oral pathogens. However, limited research is available regarding the effectiveness of BBR when combined with natural antimicrobials. Plant essential oils (EOs) have been shown to exert their antimicrobial activity through disruption of cell membrane functions. Objectives: To investigate the effects of BBR in combination with EOs against periodontal pathogens. Methods: The minimal inhibitory concentrations (MICs) of BBR and four EOs (menthol, thymol, eucalyptol, and methyl salicylate) alone or in combination against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were determined. The combination demonstrating synergism was further investigated for its effects on biofilm formation, bacterial viability, BBR uptake, cellular leakage and bacterial lysis. Result: BBR and the test EOs inhibited growth of P. gingivalis, F. nucleatum and A. actinomycetemcomitans at MICs ranging from 15.6 to 125 µg/ml for BBR, 62.5 to 1000 µg/ml for menthol and thymol, and 0.12 to 2% for methyl salicylate (MS) and eucalyptol. The BBR/MS (15.6 µg/ml/ 0.03 %) combination exerted synergistic growth inhibition against A. actinomycetemcomitans (FICI = 0.375) while no synergism was noted against P. gingivalis or F. nucleatum. Among the 4 EOs tested, MS (0.03%) was the only one that demonstrated synergism when combined with BBR (15.6 µg/ml) in inhibition of growth, biofilm formation, bactericidal activity. While this combination enhanced BBR uptake, it did not cause leakage or cell lysis in A. actinomycetemcomitans. Conclusion: Methyl salicylate may enhance the antimicrobial properties of berberine by facilitating its uptake into A. actinomycetemcomitans. The combination of BBR with MS may represent a novel strategy to preferentially control A. actinomycetemcomitans associated diseases. This study was supported by Department of Pediatric Dentistry, University of Illinois at Chicago.