Abstract
BACKGROUND AND AIM: Bovine mastitis is one of the most economically significant diseases affecting dairy cattle, leading to substantial losses due to decreased milk yield, higher treatment costs, and lower milk quality. Iodine-based teat disinfectants are commonly used to prevent mastitis but have several limitations, such as milk residue contamination, potential skin irritation, environmental persistence, and concerns about antimicrobial resistance. Plant-derived antimicrobials have thus gained interest as potential alternatives. Clove leaf oil from Syzygium aromaticum (L.) Merr. & L.M. Perry contains eugenol and other bioactive compounds with strong antimicrobial activity. However, its use is limited by poor water solubility and physicochemical instability. This study aimed to develop and optimize a clove leaf oil nanoemulsion (CLON) using a self-nanoemulsifying drug delivery system (SNEDDS) and to evaluate its physicochemical properties and in vitro antimicrobial activity against pathogens associated with bovine mastitis. MATERIALS AND METHODS: This study followed a three-phase sequential workflow. Phase I involved screening and optimizing the formulation of CLON using SNEDDS technology. Nine ratios of clove leaf oil, Polysorbate 80 (Tween 80), and Polyethylene glycol 400 (PEG 400) were initially tested, followed by optimization with an I-optimal mixture design. Phase II included physicochemical characterization and assessing the thermodynamic stability of the optimized nanoemulsion, such as measurements of transmittance, pH, emulsification time, droplet size, polydispersity index, zeta potential, and transmission electron microscopy morphology. Phase III examined the in vitro antimicrobial activity of CLON against Staphylococcus aureus, Escherichia coli, and Candida albicans through agar diffusion assays, determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and killing-time kinetics. Inhibition zone data were analyzed using analysis of variance, followed by Duncan's multiple range test. RESULTS: The optimized CLON formulation contained 14.29% clove leaf oil, 68.56% Tween 80, and 17.15% PEG 400. The nanoemulsion showed high optical clarity (93.36 ± 0.25%), physiological pH (7.13 ± 0.01), rapid emulsification (35.46 ± 0.08 s), nanoscale droplet size (25.19 ± 2.31 nm), low polydispersity index (0.30 ± 0.04), and strong negative zeta potential (-45.07 ± 8.15 mV), indicating excellent stability. CLON demonstrated significant concentration-dependent antimicrobial activity (p < 0.01) against all tested microorganisms. The MIC and MBC values were 12.5% and 50% (v/v), respectively, indicating bactericidal activity. In the killing-time assay, 25% CLON achieved >3 log(10) colony-forming units/mL reduction within 60 min. CONCLUSION: The optimized CLON displayed favorable physicochemical properties, strong thermodynamic stability, and broad-spectrum in vitro antimicrobial activity against key bovine mastitis pathogens. These results indicate that CLON has potential as a plant-based alternative teat disinfectant to prevent mastitis. However, further in vivo studies and field trials are needed to verify its safety and practical use in dairy production systems.