THREE Holistic Research Center Announces Research Results on "Anti-Biofilm Activity" of Spice-Based Essential Oil Blends and "Oxidative Stress Protection" of Rosemary Hydrosol

ACRO Co., Ltd. (Headquarters: Shinagawa-ku, Tokyo; President: Toshiaki Miyazaki), part of the Pola Orbis Group, announced that its THREE Holistic Research Center has clarified that blending specific spice-based essential oils exhibits inhibitory and bactericidal effects against "biofilms" formed by Staphylococcus aureus.

In addition, through joint research with Josai University (Headquarters: Chiyoda-ku, Tokyo; Chairman: Hirohisa Mizuta), it was discovered that rosemary hydrosol has the function of protecting the skin from oxidative stress and suppressing inflammation.

### **Discovery of "Anti-Biofilm Activity" in a Blended Essential Oil of Cinnamon, Clove, and Oregano**

**Background of this initiative**

Biofilm refers to a structure formed by the aggregation of microorganisms and substances they produce (mainly extracellular polysaccharides, proteins, DNA, etc.). Biofilms are highly adhesive and function as a barrier to protect bacteria from external factors such as antibacterial agents and immune cells, making them difficult to wash away and resistant to penetration by antibacterial and bactericidal agents.

Staphylococcus aureus, known as a skin resident bacterium, is known to form biofilms and is considered one of the factors that inhibit wound healing. It has also been suggested to be a factor in worsening skin inflammation, so anti-biofilm effects are considered to play an important role in skin wound healing.

While there have been reports on the antibacterial properties of single essential oils, we explored the effects of blended essential oils combining multiple essential oils.

**Research Results**

We verified the effects on Staphylococcus aureus using a blended essential oil containing cinnamon, clove, and oregano essential oils.

To evaluate the biofilm formation inhibitory ability, when the blended essential oil was added during Staphylococcus aureus culture, a significant decrease in biofilm formation was confirmed (Figure 1).

Figure 1. **Inhibition of Biofilm Formation by Blended Essential Oil**

Test method: After adding the blended essential oil to a suspension of Staphylococcus aureus, it was cultured with shaking at 37°C. The amount of biofilm formed was calculated and compared with and without the addition of the blended essential oil.

Source: Survey by THREE Holistic Research Center

Furthermore, observation using a fluorescence microscope to determine the viability of bacteria within the biofilm revealed that even against already formed strong biofilms, adding the blended essential oil had a significant bactericidal effect on the bacteria inside.