Synergistic formulation of terpinen-4-ol and 1,8-cineole: A chitosan-based strategy for sustained antibacterial activity

Artificial formulation has higher antibacterial activity than tea tree oil (TTO). • The formation and physical properties of nanoemulsions is studied systematically. • Nanoemulsions exhibit superior antibacterial efficacy than reported nano-TTO. • Chitosan and the essential oil components exhibit a synergistic antibacterial effect. The volatility and instability of essential oil components often limit their practical application in antibacterial materials. In this study, a chitosan-based nanoemulsion (NE) loaded with an artificially designed synergistic formulation of two key components from tea tree oil (TTO) was developed to achieve enhanced and sustained antibacterial activity. The optimal 9:1 (v/v) ratio of terpinen-4-ol to 1,8-cineole, produced inhibition zones of 25.8 mm against S. aureus and 22.8 mm against E. coli , thereby outperforming commercial TTO (14.5 mm and 12.5 mm). Guided by pseudo-ternary phase diagrams, the formation of NE was determined. A 3:1 mass ratio of surfactant Tween 80 to co-surfactant ethanol yielded the largest nanoemulsifying region (38.33%), whereas 0.1% chitosan aqueous solution provided optimal antibacterial efficacy, uniform particle size (Z-Ave: 16.89 nm; PDI: 0.26), and good stability. Conductivity and viscosity changes were conducted to analysis the critical transition points of NEs, and NEs transitioned to the oil in water (O/W) phase when chitosan aqueous content exceeded 60% by mass. Interestingly, unlike the essential oil, the nanoemulsion exhibited higher antibacterial activity against E. coli than S. aureus , with minimum inhibition concentration of 3.91 μL·mL -1 and 7.81 μL·mL -1 , respectively. Chitosan rendered the nanoemulsion positively charged, facilitating easier interaction with the negatively charged cell walls of gram-negative bacteria. SEM revealed that NEs disrupted bacterial cell wall and membrane structures, leading to cell death and antibacterial activity. Additionally, the NEs demonstrated notable long-lasting effects. Overall, these eco-friendly NEs offer a promising alternative to antibiotics, especially for gram-negative bacteria inhibition. We designed chitosan-based nanoemulsion (NE) loaded with an artificially designed synergistic formulation of two key components from tea tree oil (TTO), achieving enhanced and sustained antibacterial activity. Guided by pseudo-ternary phase diagrams, the formulation of NE was determined. Moreover, conductivity and viscosity changes were conducted to analysis the critical transition points of NEs. Notably, the nanoemulsion exhibited minimum inhibition concentration of 3.91 μL·mL -1 and 7.81 μL·mL -1 against Escherichia coli and Staphylococcus aureus , respectively, which outperformed reported nano-TTO of 48.8 μL·mL -1 . Additionally, the NEs demonstrated notable long-lasting effects. Overall, these eco-friendly NEs offer a promising alternative to antibiotics. Download: Download high-res image (263KB) Download: Download full-size image