Antibacterial Activity of Essential Oils from Clove and Elsholtzia Plants Against Common Pathogens of Bovine Mastitis, Antioxidant Activity, and Toxicity to Bovine Mammary Epithelial Cells.

In light of concerns regarding milk quality deterioration and the rising threat of bacterial resistance, developing alternative treatments to antibiotics for mastitis has become imperative. Clove oil (CEO), a well-established natural antimicrobial, has not been extensively investigated for its synergistic potential when combined with Elsholtzia EO. Essential oils (EOs) from the plants were extracted using steam distillation. Gas chromatography-mass spectrometry (GC-MS) results showed that the main component of CEO was eugenol (76.59%), the main component of Elsholtzia densa EO was limonene (22.05%), the main component of Elsholtzia ciliata EO was carvone (31.63%), the main component of Elsholtzia fruticosa EO (EfEO) was eucalyptol (40.1%), and the main component of Elsholtzia stauntonii EO was cuminol (27.60%). Antimicrobial assays demonstrated CEO's superior efficacy against bovine mastitis pathogens (MIC: 1.875-7.5 mg/mL), while CEO/EfEO combination exhibited marked synergy (FICI ≤ 0.75). Further studies on the antimicrobial mechanisms of CEO, EfEO, and the CEO/EfEO combination showed that they were able to affect the integrity of the bacterial cell wall and increase membrane permeability. Such effect led to the leakage of biomolecules, including proteins and nucleic acids, accompanied by a concurrent decline in intracellular ATPase activity. The results of antioxidant activity assay showed that CEO exhibited stronger antioxidant capacity than EfEO. Cytotoxicity assessments conducted in bovine mammary epithelial cells delineated safe therapeutic windows, with EC values ranging from 0.120 to 0.148 mg/mL for CEO and 0.261-0.286 mg/mL for EfEO. Furthermore, the combined application of CEO and EfEO resulted in a reduction of the EC value. These findings indicate that CEO and EfEO represent a promising phytotherapeutic alternative, offering a potential solution to the growing challenge of antibiotic resistance in dairy production systems.