Chemicals
Pure standard substances of α-pinene and γ-terpinene were purchased from Sigma-Aldrich (Sigma, St. Louis, MO) and limonene, β-pinene, α-terpinene were from Tokyo Chemical Industry Co., Ltd. (TCI, Tokyo, Japan). Ascorbic acid was obtained from Shinyo chemical Co. Ltd. Butylated Hydroxytoluene (BHT), butylated Hydroxyanisole (BHA), 2,20-diphenylpicrylhydrazyl (DPPH), 2,2'-azino-bis 3-ethyl benzothiazoline-6-sulfonic acid (ABTS), Earle’s basal salt solution (EBSS), trypsin solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), trolox were obtained from Sigma Chemical Co. (St Louis, MO). Fetal bovine serum (FBS) and Dulbecco’s modified eagle medium (DMEM) were from Gibco BRL (NY).
Essential oil
Commercial silver fir essential oil which extracted by steam distillation from leaf and twig of Abies alba (Pinaceae) was obtained from Skinmate (Puchon, Gyeonggi-do, Korea). It was stored in glass vials with Teflon sealed caps at 4°C in the absence of light.
GC-MS analysis
GC-MS spectrometry analysis of the oils was performed on a GC/MSD Polaris Q (Thermo Finnigan, TX) instrument. Briefly, HP5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.32 µm) was used with the following temperature program: initial oven temperature, 40°C for 5 min; then gradient of 4°C/min to 200°C, held 5 min; followed by a gradient of 20°C/min to 280°C, held 10 min. Injector line temperatures was set at 260°C, and a split ratio of 10:1 was applied. Helium was used as the carrier gas at a flow rate of 1.0 ml/min. The interface temperature was 280°C. For GC-MS detection, electron ionization system with ionization system energy 70 eV was used. Total ion current (TIC) chromatograms were recorded in a mass range of 30–350 amu. The components were identified by comparison of their relative retention times with authentic substances and mass spectra with NIST, WILLY library data of the GC-MS system and literature data.
Cell culture and treatment
Skin CCD-986SK human fibroblast cells were obtained from Korean cell line bank (Seoul, Korea). Cells were cultured at 37°C in the presence of 5% CO2 in DMEM supplemented with 10% FBS. For the treatment, the oil was diluted with a culture medium. In every experiment, cells in negative control group were treated with the same concentration of dimethyl sulfoxide (DMSO) (0.1%) as contained in the silver fir oil-treated group.
Determination of cell viability
Cell viability that was assessed by the MTT staining assay [
12] based on the reduction of a MTT into formazan dye by active mitochondria reflects the cytotoxicity of silver fir oil. CCD-986SK cells were seeded at a density of 10,000 cells/well in 96-well microplate. The next day, cells were treated with the oil at 37°C for 48 h. At the end of culture, 100 µl of MTT (5 mg/ml) were added to each well, and plates were incubated at 37°C for 4 h. One hundred microliters DMSO were added to each well to dissolve the cells. The plates were kept at room temperature for 5 min and the absorbance was measured at 550 nm using a multiwell spectrophotometer (Molecular Devices, Sunnyvale, CA).
Bacterial cultures
The S. aureus were grown in tryptic soy agar broth (Becton Dickinson, MD), and the S. mutant and L. monocytogenes were grown in brain heart infusion broth (Becton Dickinson, MD). Other bacteria strains were grown in nutrient broth (Becton Dickinson, MD) with aeration at 37°C.
Antibacterial screening
The agar diffusion method was employed to determine the antibacterial activity of the essential oil [
13]. Briefly, sterile 8-mm paper discs were individually impregnated with 25 µl of the essential oil and placed on the solid media plates, which had been surface spread with the tested bacteria (2 × 10
8 CFU/ml). Plates were incubated at 37°C for 24 h and the inhibition zones were measured against bacterial strains tested.
DPPH radical scavenging activity
Radical scavenging activity was determined using DPPH as a free radical by the method described in the literature [
14] with some modifications. This spectroscopic assay uses the stable radical DPPH as a reagent. Briefly, 200 µL of various concentrations of sample was added to 50 µL of DPPH solution (0.15 mM in methanol) in a 96-well microtiter plate. The sample was dissolved in methanol and used. After 30 min incubation in the dark at room temperature, the decrease in absorbance was measured at 517 nm. Methanol was used as blank solution, and DPPH solution without the essential oil served as control. BHT, BHA, and ascorbic acid were used as synthetic references. Reduction of DPPH radical in percent (
R%) was calculated in following way:
R% = (Ablank – Asample / Ablank) × 100,
where Ablank is the absorbance of the control reaction (containing all reagents except the test sample), and Asample is the absorbance of the test sample. Tests were carried out in triplicate. For the final results, RC50 values (the concentrations required for 50% reduction of DPPH (0.15 mM) at 30 min after starting the reaction) were calculated from the absorbance diminished by 50%. The experiment was performed in triplicate.
ABTS radical scavenging activity
The antiradical activity using ABTS was measured by ABTS cation decolorisation assay method, as described by Re
et al. [
15]. Briefly, ABTS radical cation was freshly prepared by mixing 14 mM ABTS with an equal volume of 4.95 mM potassium persulfate and kept for 24 h in dark at room temperature. This ABTS radical cation solution was used for the assay after dilution in phosphate buffer saline (PBS) appropriately. To 50 µl of various concentration of sample, 150 µl of ABTS radical solution was added. After 1 min incubation at room temperature, the absorbance was measured at 732 nm. Methanol was used as blank solution, and ABTS solution without the essential oil served as control. Trolox was used as reference synthetic antioxidant compound. The experiment was performed triplicate. Reduction of ABTS radical in percent (
R%) and RC
50 value were calculated the same as described in DPPH radical assay.
Statistical analysis
All the experiments were performed three to five times. Data are expressed as the mean ± standard error of the mean (SEM) or SD. A significant difference from the respective controls for each experimental test condition was assessed using Student’s t test for each paired experiment. A p-value of <0.05 was regarded as indicating a statistical significance.
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