Dried fruit of M. fragrans and the barks of C. verum were grounded, and the powdered materials were hydrodistilled into steam distillation apparatus, as mentioned above. Isolated volatile oil extracts

collected from each distillation process were added to each other and dried over anhydrous sodium sulphate and stored in dark glass bottles in a fridge at 4°C until use. Macrophage Infection Healthy human macrophage cells were collected and cultured in RPMI. medium supplemented with 10% heat-inactivated fetal calf serum. For macrophage growth assays, click here 96-well microtiter Inhibitors,research,lifescience,medical plates were seeded with 2×105 macrophages/well and infected with B. abortus 544 at a ratio of 1:100 bacteria/macrophage. Cells were incubated for one h at 37°C in 5% CO2. Extracellular bacteria were removed by three washes with PBS, Inhibitors,research,lifescience,medical followed by treatment with 25 µg/ml of gentamicin for 30 min. Then, the cells were maintained by the addition of medium containing

5 µg/ml of gentamicin. To evaluate the effect of plants volatile oil extracts on the ability of Brucella to invade human macrophage, 1% concentration of the five studied volatile oil extracts, or 0.1% of C. verum plus 1% of the other four volatile oil extracts, were added after 2, 4, 24, 48, 72, 96, 120 and 144 h of infection, the cells were Inhibitors,research,lifescience,medical washed three times by PBS, and lysed with 0.1% Triton. Five minutes after the incubation at room temperature, the lysates were plated on 2YT agar and incubated at 37°C for 48 h; in order to determine the intracellular bacterial count. All experiments were performed in triplicate. Macrophages infected with B. abortus 544 at a ratio of one bacteria/100 macrophage without adding any oil extract as a control. Statistical Methods Inhibitors,research,lifescience,medical Antibacterial properties of oil extracts were analyzed with one-way repeated Inhibitors,research,lifescience,medical measures analysis of variance (ANOVA) fallowed by Tukey’s multiple comparison test to compare the difference between each pair of means. Data were transformed into log10 CFU prior to analysis to homogenize the variance. All analyses

were conducted by using GraphPad Prism Statistical Software V5.03. Differences were considered statistically significant at P<0.05. Results Brucella abortus 544 log10 counts in human macrophages were significantly suppressed (F5,35=22.7; P<0.0001) by volatile oil extracts treatments compared with the (-)-p-Bromotetramisole Oxalate untreated control. For example, the inhibitory effect of C. verum at a concentration of 1% was started 24 h and continued till 144 h after the infection, and the log10 counts increased only from 3.11 to 4.9. The repeated measures ANOVA followed by Tukey’s test of multiple comparisons revealed that C. verum volatile oil possessed the strongest antibacterial effect compared to all the other essential oil extracts (figure 1). It is worth pointing out that no significant difference occurred between the antibacterial activity of lemon, peppermint, sweet marjoram and nutmeg volatile oil extracts.