15 CHARACTERIZATION AND ACTIVITY OF ESSENTIAL OILS Acne lesions have been treated by topical and systemic (oral) antibiotics for more than 40 years. Macrolides such as erythromycin and clindamycin are frequently found in topical formulations of liquid, gel, lotion, and ointment (7). Current treatments are used to reduce sebum production and inflammation, inhibit bacterial growth, and normalize keratinization. Oral therapy is mostly successful but shows some side effects. Isotretinoin is known to be a powerful anti-acne agent but causes serious ailments such as headaches, muscle pain, and suicidal tendencies. Topical therapy like benzoyl peroxide, retinoids, antibiotics, and salicylic acid that reduce the effects of acne pathogens are preferred but cause skin irritation (8–13). Inappropriate and excessive use of antibiotics causes resistant bacteria (14). Complementary and alternative medicine treatments have less toxicity and side effects and are frequently preferred, especially in dermatology (15,16). Essential oils (EOs) are the secondary metabolites of plants, showing antioxidant, antifungal, and antibacterial activities due to their different phytochemical constituents such as flavonoids, alkaloids, phenol and phenolic compounds, phenylpropanoid glycosides, and bisnaphthicione derivatives (17,18). Plant-derived antioxidants accelerate the wound healing process (19). EOs are often used in combination to increase their therapeutic efficacy (20). In this study, we aimed to determine antibacterial properties of different EOs both alone and in combination with S epidermidis ATCC 12228 and C acnes ATCC 6919 microbial strains, and to investigate the chemical components, antioxidant potential, total phenolic content (TPC) of effective EOs, and their cytotoxicity against NIH 3T3 cell line by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MATERIALS AND METHODS ESSENTIAL OILS EOs extracted from oregano leaves (Origanum vulgare, lot 1110), garlic bulb (Allium sativum, lot 2512), cinnamon trunk bark (Cinnamomum verum, lot 00018), orange peels (Citrus aurantium, lot 1708), laurel leaves (Laurus nobilis, lot 03112022), lavender flowers and leaves (Lavandula officinalis, lot 1510), rosemary leaves (Rosmarinus officinalis, lot 10112022), tea tree leaves and twigs (Melaleuca alternifolia, lot 1510), sage leaves and twigs (Salvia officinalis, lot 02022021), eucalyptus leaves (Eucalyptus globulus, lot 2810), clove oil flower buds (Eugenia caryophyllus, lot 2510), lemon peels (Citrus limon, lot 0402), and coconut seeds (Cocos nucifera, lot 211010318) were acquired commercially produced by Naturoil, Turkey. Ginger rhizome (Zingiber officinale, lot 19680) and chamomile flowers (Matricaria recutita, lot 20549) were purchased from Arifoglu (Turkey), and sandalwood trunk bark (Santalum album, lot SM48A) was purchased from Misbahce, Turkey. All EOs were obtained by steam distillation method and stored in the dark at room temperature before the test. Each EO was dissolved in 0.5% Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) and sterilized using a syringe filter. GAS CHROMATOGRAPHY/MASS SPECTROMETRY ANALYSIS—CHEMICAL COMPOSITION Separation and characterization of chemical constituents of EOs were performed via Agilent 6890N GC system coupled with a 5973 inert mass selective detector and equipped

16 JOURNAL OF COSMETIC SCIENCE with a fused-silica HP-Innowax polyethylene glycol column (60m × 250µm × 0.25µm) (Agilent Technologies, Santa Clara, CA, USA). The GC oven was programmed as follows: The initial temperature was 60°C for 10 minutes and the temperature increased to 150°C at a rate of 5°C/min for 20 minutes and then increased to 250°C held for 30 minutes. Helium was used as a carrier gas. The flow rate was 1.7 ml/min. The split ratio was 30:1. The temperature of the injector was set at 250°C. Electron ionization was set at 70eV. After injection of each EO, the phytocomponents were determined by comparison of their mass spectrometry data with the National Institute of Standards and Technology (NIST 08) and Wiley library database. BACTERIAL STRAINS AND CULTURAL METHODS S epidermidis ATCC 12228 and C acnes ATCC 6919 standard bacterial strains causing acne were selected as test microorganisms and purchased from the collection of the Microbiologics (Kiwk-Stik™, MediMark Europe, France). S epidermidis strain was stored at −20°C in brain heart infusion broth (BHI) (Biokar, Allonne, France) and added with glycerol (20%) until its use. The strain was cultured on brain heart agar (BHA) (Biokar) and incubated at 37°C for 24 hours, aerobically. C acnes strain was stored at −20°C in sterile beaded tubes containing skimmed milk broth with 10% glycerol. The strain was cultured on Brucella Blood Agar (BBA) supplemented with haemin and vitamin K1 (Becton Dickinson, Heidelberg, Germany) at 37°C for 72 hours in an anaerobic environment using an H 2 /CO 2 gas-generating kit (Becton Dickinson GasPak® EZ Container System). In the antimicrobial activity assays, the bacterial suspensions were adjusted to 0.5 McFarland standard with BHI. ANTIBACTERIAL ACTIVITY OF ESSENTIAL OILS IN VITRO Disc diffusion assay. The test was used to detect the antimicrobial activity of 16 EOs against 2 bacteria according to the Clinical and Laboratory Standards Institute (CLSI) (21). An amount of 0.1 ml bacterial suspension (1.5 × 108 CFU/ml) was spread on BHA (for S epidermidis) and BBA supplemented with haemin and vitamin K1 (for C acnes) plates. An amount of 20 µl of EO-Tween 80 mixture was pipetted onto sterile blank paper discs and aseptically placed on the inoculated agar surface (one blank disc per plate). Then, the plates were incubated for 20 minutes at room temperature. After incubation at 37°C, the inhibition zone sizes were measured in mm. Erythromycin (15 μg/disc, Bioanalyse, Ankara, Turkey) and clindamycin (2 μg/disc, Bioanalyse) were used as positive controls, and Tween 80 was used as a negative control. All experiments were done independently four times. The growth inhibition zone diameters (IZDs) were interpreted by the Singh et al. algorithm, i.e., extremely sensitive: 20 mm very sensitive: 15–19 mm sensitive: 9–14 mm and exhibiting no antimicrobial activity: 8 mm (22). Minimum inhibitory concentration and minimum bactericidal concentration assays. EOs with inhibition diameter ≥12 mm were analyzed for their antibacterial activity against strains by microdilution broth method as described by CLSI with some modifications (23). Two-fold serial dilutions of EOs containing 0.5% Tween 80 were prepared in culture medium (BHI) ranging from 0.03125% to 64% (v/v) using sterile 96-well microtiter plates. Then, 20 µl of