Molecular complex formation with 1,8-Cineole protects clavulanic acid from acidic degradation and preserves its antibacterial activity: experimental, and computational studies

Clavulanic acid is well known for its chemical instability under various conditions, such as temperature, pH, humidity, and storage duration. This instability reduces its direct antibacterial efficacy both in vitro and in vivo . The main objective of this study was to evaluate the stability of clavulanic acid by assessing its antibacterial activity and to enhance this stability by adding 1,8-cineole, while investigating the molecular interactions between these two molecules. For this purpose, the antibacterial activity of clavulanic acid alone was investigated at different pH levels. Subsequently, the antibacterial activity of clavulanic acid combined with 1,8-cineole was examined at pH 2 using disc diffusion and time-kill assays against Escherichia coli ATCC 25922 ( E. coli ) and Klebsiella pneumoniae ATCC 13883 ( K. pneumoniae ). This study reveals that 1,8-cineole significantly stabilizes clavulanic acid activity under acidic conditions. Density Functional Theory (DFT) analysis using B3LYP/6-31G(d) and RDG (Reduced Density Gradient)-NCI (Non-Covalent Interaction) analysis revealed significant molecular interactions between clavulanic acid and 1,8-cineole primarily mediated by hydrogen bonding. Molecular docking analysis performed on the Penicillin-binding protein 2 (PBP2) and β-lactamase targets further showed that the resulting molecular complex exhibits a higher binding affinity toward the targets (−8.2 kcal/mol and −7.2 kcal/mol, respectively) than clavulanic acid alone (−6.6 kcal/mol and −6.3 kcal/mol, respectively), indicating a more stable and favorable interaction. Overall, the results suggest that 1,8-cineole is a promising molecule for stabilizing clavulanic acid in acidic aqueous solution.