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

17 CHARACTERIZATION AND ACTIVITY OF ESSENTIAL OILS inoculum suspension (standardized 0.5 McFarland) was added to each well. Growth control and sterility control were used. After incubation, 20 µl of 0.2 mg/ml iodonitrotetrazolium chloride (Sigma-Aldrich) solution was added to each well to determine minimum inhibitory concentration (MIC). To identify minimum bactericidal concentration (MBC) values, 10 µL of inoculum was taken from wells without visible growth and transferred onto BHA. Checkerboard assay. The checkerboard microtiter plate method was used to evaluate the effects of binary combinations of EOs against strains as shown by Gutierrez et al. with modifications (24). Two-fold dilutions (ranging from 64% to 0.03125% [v/v]) of each EO were prepared. Decreasing concentrations of EOs were dispensed into the 96-well plates horizontally and vertically according to the MIC values. The 12th-H well included growth control. The interaction between EOs was determined by the fractional inhibitory concentration index (FICI) using the following formula: FIC MICEO1combined and FICEO2 MICEO2combined EO1 :/MICEO1alone :MICEO2alone /FICI FIC FICEO2 EO1 =+ANTIOXIDANT ACTIVITY Antioxidant activity of EOs (oregano, cinnamon, tea tree, lavender, lemon, sandalwood, eucalyptus, and laurel) was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and Folin–Ciocalteu methods. Antioxidant assay. The antioxidant potential of the EOs was determined by the DPPH free radical scavenging assay as described by Shimamura et al. (2014) (25) using DPPH antioxidant assay kit (Dojindo, Kumamoto, Japan). The DPPH and Trolox standards were dissolved in ethanol (99.5%) (Isolab, Eschau, Germany) using sonication before use. DPPH reagent and Trolox standard solutions (100, 80, 60, 40, and 0 µg/ml) were prepared. Sample, Trolox (standard), and blanks were put into wells. The total volume was 200 µl. Incubation conditions of the plates were set at 25°C for 30 minutes in the dark. Then their absorbance was measured at 517 nm using a microplate reader (Thermo Fisher Scientific. Watham, MA, USA). The experiment was done in duplicate. The inhibition ratio was calculated as follows: DPPHscavengingactivity(%) A A ()Control Sample:EO Control =-/A × 1 100 IC 50 values of the Trolox and EOs were calculated, and the antioxidant capacity of each EO was expressed as the Trolox equivalent antioxidant capacity (TEAC). TEAC: IC 50 Trolox/IC 50 Sample Folin–Ciocalteu assay. Phenolic compounds of EOs were determined by the Folin–Ciocalteu method as explained by Slinkard and Singleton (1977) with little modifications (26). Briefly, 1.36 ml of distilled water, 0.04 ml of each EO, and 0.8 ml of 0.5 N phenol reagent (Merck, Darmstadt, Germany) were added into a tube and mixed for 3 minutes, then 0.8 ml of 10% Na 2 CO 3 (Isolab) solution was put into the tube. The reaction mixture vortexed and incubated at 25°C for 30 minutes and blue color was observed. The absorbance was measured at 760 nm by a spectrophotometer (Shimadzu Spectrophotometer UV-1800,