510 JOURNAL OF COSMETIC SCIENCE fragrance (16) in perfumes, soaps, and creams. The extraction of so many different essential oils from aromatic and medicinal plants and different antioxidant activities have been reported in the literature (17-19). However, it is true that the compositions of commonly and commercially used essential oils are different from the original ex­ traction and are rarely investigated. It is important not only for their antioxidant properties, but also because they are natural, non-synthetic products, that their appre­ ciation by consumers be very favorable. In this study, the antioxidant capacity of forty-five essential oils was investigated using two complimentary in vitro assays: DPPH free-radical scavenging ability and total phenolic content. These essential oils were compared with those of the commercial standard antioxidants and butylated hydroxyanisole (BHA), and the components of some essential oils showing antioxidant activity were further identified by GC-MS. MATERIALS AND METHODS CHEMICALS The chemicals 1, l-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxyanisole (BHA), �-caryophyllene, and eugenol acetate were purchased from TCI. Gallic acid, eugenol, benzyl acetate, p-cresol, linalool, and Folin-Ciocalteau's phenol reagents were purchased from Merck. Sodium carbonate was purchased from SHOWA. Benzyl ben­ zoate, indole, and thymol were purchased from ACROS. Jasmone and a-pinene were purchased from Aldrich. Farnesene was purchased from SAFC. The forty-five essential oils were purchased from Australian Botanical Products (TGA warrant by the Australian government, USDA and ACO certification). All the other chemicals used were of stan­ dard analytical grade, and solvents were of HPLC grade. DPPH FREE-RADICAL SCAVENGING ASSAY The antioxidant activity of the essential oils was measured in terms of hydrogen donat­ ing or radical-scavenging ability using the stable DPPH method (20) as modified by Sanchez-Moreno et al. (21). The reaction mixture containing 0.5 ml of essential oil (10 mg/ml EtOH) and 2.5 ml of DPPH (1.52 x 10- 4 M) was vigorously shaken and incubated in darkness at room temperature. When DPPH reacted with an antioxidant compound in an essential oil that can donate hydrogen (Figure 1), it was reduced and the resulting decrease in absorbance at 517 nm was recorded at 10-min intervals up to 30 min using a UV-vis spectrophotometer (Hitachi UV-vis 2001), and the mean values were obtained from triplicate experiments. The percentage of remaining DPPH was plotted against the sample or standard (BHA) concentration to obtain the amount of antioxidant necessary to decrease the initial concentration of DPPH to 50% (EC50). A lower EC50 value indicates greater antioxidant activity. The scavenging effect of a DPPH free radical was calculated by using the following equation: ( 1 - absorbance of sample at 5 1 7 nm ) Scavenging effect ( % ) = b b f 1 X 100 a sor ance o contra at 5 1 7 nm

DPPH FREE-RADICAL SCAVENGING ABILITY Q N J) I N• 02NhN02 y N02 / HOR Q N J) I NH 02NhN02 y N02 Figure 1. The reaction of DPPH free radical and antioxidant. DETERMINATION OF TOTAL PHENOLIC CONTENT 511 The essential oils were diluted with ethanol to a suitable concentration for analysis. The total phenolic content of essential oils was assessed approximately by using the Folin­ Ciocalteau phenol reagent method (22). To 500 µl of the essential oil were added 1.0 ml of Folin-Ciocalteau reagent and 1 ml of sodium carbonate (7.5% w/v), and the contents were mixed and allowed to stand for 30 min. Absorption at 765 nm was measured in a UV-vis spectrophotometer. The total phenolic amounts were expressed as gallic acid equivalents (GAE) in milligrams per gram of sample, using a standard curve generated with gallic acid. GAS CHROMATOGRAPHY-MASS SPECTROMETRY The analyses of the volatile compounds were run on a Thermo GC-MS system (GC-MS Trace DSQ-Mass Spectrometer, MSD 201351, Thermo, USA). A TR-5MS column was used (5% phenyl polysilphenylene-siloxane, Thermo 30 m·0.25 mm ID., film thickness 0.25 um). The oven temperature was programmed as follows: isothermally at 40°C for 1 min then increased to 250°C, at a rate of 10°C/min and subsequently held isother­ mally for 40 min then increased to 300°C, at a rate of 4 ° C/min. The carrier gas was helium (1 ml/min). The injection port temperature was 220°C and the detector tem­ perature was 280°C. Ionization of the sample components was performed in the EI mode (70 eV). The injected volume was 1 µl. The linear retention indices for all the com­ pounds were determined by co-injection of the samples with a solution containing a homologous series of C8-C22 n-alkanes (23 ). The individual constituents were identified by their identical retention indices referring to the compounds known from the litera­ ture data (24), and also by comparing their mass spectra with spectra of either the known compounds or those stored in the Trace DSQ-MASS spectral database (Thermo, USA). RESULTS AND DISCUSSION DPPH RADICAL SCAVENGING ACTIVITY Information on the forty-five essential oils is listed in Table I. Cinnamon leaf, clove bud, jasmine absolute, and thyme red essential oils have apparently shown DPPH radical scavenging activity in the forty-five kinds of commonly used essential oils. In 10 mg/ml