Evaluation of the Antibacterial Potential of Liquid and Vapor Phase Phenolic Essential Oil Compounds against Oral Microorganisms

<div><p>The aim of the present study was to determine the antibacterial activities of the phenolic essential oil (EO) compounds hinokitiol, carvacrol, thymol, and menthol against oral pathogens. <i>Aggregatibacter actinomycetemcomitans</i>, <i>Streptococcus mutans</i>, Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), and <i>Escherichia</i>. <i>coli</i> were used in this study. The minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), bacterial growth curves, temperature and pH stabilities, and synergistic effects of the liquid and vapor EO compounds were tested. The MIC/MBC of the EO compounds, ranging from the strongest to weakest, were hinokitiol (40–60 μg/mL/40-100 μg/mL), thymol (100–200 μg/mL/200-400 μg/mL), carvacrol (200–400 μg/mL/200-600 μg/mL), and menthol (500-more than 2500 μg/mL/1000-more than 2500 μg/mL). The antibacterial activities of the four EO phenolic compound based on the agar diffusion test and bacterial growth curves showed that the four EO phenolic compounds were stable under different temperatures for 24 h, but the thymol activity decreased when the temperature was higher than 80°C. The combination of liquid carvacrol with thymol did not show any synergistic effects. The activities of the vaporous carvacrol and thymol were inhibited by the presence of water. Continual violent shaking during culture enhanced the activity of menthol. Both liquid and vaporous hinokitiol were stable at different temperatures and pH conditions. The combination of vaporous hinokitiol with zinc oxide did not show synergistic effects. These results showed that the liquid and vapor phases of hinokitiol have strong anti-oral bacteria abilities. Hinokitiol has the potential to be applied in oral health care products, dental materials, and infection controls to exert antimicrobial activity.</p></div

The antibacterial activities of the phenolic EO compounds.

<p>(A) MRSA treated with 500 μg phenolic EO compounds, as assessed using direct contact agar diffusion tests. (B) The phenolic EO compounds were all tested at 500 μg. The microorganisms examined were <i>A</i>. <i>actinomycetemcomitans</i> (Aa), <i>S</i>. <i>mutans</i> (Sm), MRSA, and <i>E</i>. <i>coli</i>. Dotted line, the 0.6 cm diameter of the filter disc. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared with <i>A</i>. <i>a</i>. in each compound group; a, b, c, and d were <i>P</i> < 0.05, compare with <i>A</i>. <i>a</i>., <i>S</i>. <i>m</i>., MRSA, and <i>E</i>. <i>coli</i> in the hinokitiol group, respectively; e, <i>P</i> < 0.01 based on a comparison of the carvacrol and thymol groups.</p

The antibacterial activity of vaporous hinokitiol was stable under different pH conditions.

<p>The antibacterial activities of (A) vaporous hinokitiol, (B) vaporous carvacrol, and (C) vaporous thymol were analyzed under different pH conditions.</p

The physical characteristics of hinokitiol, carvacrol, thymol, and menthol.

<p>The physical characteristics of hinokitiol, carvacrol, thymol, and menthol.</p

Synergistic antibacterial effects of the phenolic EO compounds.

<p>(A) The synergistic effects of hinokitiol (H) and zinc oxide (ZnO) against <i>A</i>. <i>actinomycetemcomitans</i> (<i>A</i>. <i>a</i>.) were tested by direct contact and vapor phase agar diffusion tests. (B) The synergism of the anti-MRSA activity of carvacrol (C) and thymol (T) was tested by direct contact agar diffusion tests. a, <i>P</i> < 0.01 compared with the direct contact 250H group.</p

The MIC and MBC of the four phenolic EO compounds against four microorganisms (μg/mL).

<p>The MIC and MBC of the four phenolic EO compounds against four microorganisms (μg/mL).</p

The vaporous phenolic EO compounds display antibacterial activity.

<p>(A) The vapor phase agar diffusion experimental device. (B) MRSA treated with 500 μg phenolic EO compounds was examined by vapor phase agar diffusion tests. (C) The vapors from 500 μg phenolic EO compounds were tested by vapor phase agar diffusion. The liquid and vapor phases of (D) hinokitiol, (E) carvacrol, and (F) thymol showed different antibacterial activities after incubation at different temperatures. *<i>P</i> < 0.05, compared with 4°C in each curve. Dotted line, the 0.6 cm diameter of the filter disc.</p

The phenolic EO compounds were heat stable.

<p>The phenolic EO compounds (500 μg) were pre-incubated at 4–100°C for 1 h before the direct contact agar diffusion test. (A) Hinokitiol; (B) carvacrol; (C) thymol. Dotted line, the 0.6 cm diameter of the filter disc.</p

Phenolic EO compounds delay the microorganism growth curves in a concentration-dependent manner.

<p>Various concentrations of the phenolic EO compounds were used to test their impact on the bacterial growth curves. The bacterial growth curves in the presence of various phenolic EO concentrations (hollow diamond, triangle, square, and circle) were compared to each control (solid circle). Broth-only treatment served as a negative control (solid square). Y axis, OD600; X axis, time (sec).</p

The chemical structures of the phenolic EO compounds.

<p>(A) hinokitiol; (B) carvacrol; (C) thymol; (D) menthol.</p