In vitro antibacterial effects and mechanism of action of six constituents of Litsea cubeba essential oil against Helicobacter pylori.

Litsea cubeba essential oil (EO) has been previously proven to possess a potent anti-Helicobacter pylori (H. pylori) activity. The current study investigated antibacterial activity of six single compounds present in the EO and their mechanisms of action against the stomach pathogen. The results demonstrated that citronellol displayed the most potent anti-H. pylori activity with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.04 and 0.16 mg/mL, followed by citral with MIC and MBC of 0.08 and 0.31 mg/mL, respectively. Notably, all tested compounds exerted bactericidal effects at 24 h, except for limonene and 1,8-cineole at 36 h of treatments. Citral and citronellol effectively inhibited 88.09 and 71.69% of biofilm formation at ¾⋅MIC. Citral and citronellal exhibited excellent inhibitory activity against the bacterial urease with IC50 values of 0.145 and 0.332 mg/mL, respectively. Citral, citronellol, and limonene at MBCs significantly increased membrane permeability of H. pylori with respective crystal violet uptake of 80.10%, 80.80%, and 85.57% but less pronounced than that of the EO (93.25%). Citronellal, citronellol, and limonene at MBC induced remarkable protein leakage in the bacterial cells (13.51, 15.96, and 14.36 µg/mL, respectively). SEM images revealed that citronellol and the EO at MBC had significant effects on the cell shape and disrupted the outer membrane. Especially, the H. pylori treatment with citronellol and the EO also caused extensive DNA fragmentation leading to the cell death. These findings highlighted the role of citral, citronellal, citronellol, and limonene as promising candidates for further development as anti-H. pylori agents.