WHITENING AND ANTIAGING EFFECT OF SESAMOL 71 MATERIALS AND METHODS CHEMICALS Sesamol was purchased from Spectrum Chemical (Gardena, CA) α-Tocopherol from Fluka Biochemika (Buchs, Switzerland) butylated hydroxy anisole (BHA) and butylated hydroxy toluene (BHT) from Fluka AG (Buchs, Switzerland) 2,2-Diphenyl-1-picrylhydrazyl hydrate (DPPH•) from Fluka (Buchs, Switzerland) 2,4,6-Tri(2-pyridyl)-1,3,5-Triazine (TPTZ) was from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan) thiobarbituric acid from Sigma-Aldrich chemie GmbH (Buchs, Switzerland) linoleic acid from Sigma-Aldrich chemie GmbH (Steinheim, Germany) dimethyl sulfoxide (DMSO) from Sigma (St Quentin Fallavier, France) ferric chloride (FeCl3.6H2O) and ferrous sulfate (FeSO4.7H2O) from Asia Pacifi c Specialty Chemical Limited (Seven Hills, Australia) mushroom tyrosinase, β-arbutin, and neutral red (NR) from Sigma–Aldrich Chemical Co. (St. Louis, MO) kojic acid from TCI (Tokyo, Japan) L-3,4-dihydroxyphenylalanine (L-DOPA) from Acros Organic Geel (Geel, Belgium) and, DMEM medium, fetal bovine serum (FBS), and penicillin/streptomycin from GIBCO (Grand Island, NY). The UV spectra were recorded on UV–Vis spectrophotometry from Shimadzu, UV-1700 PharmaSpec (Kyoto, Japan), while the microplate reader was from Anthos 2010 (Anthos Labtec Instruments, GmbH, Salzburg, Austria). DETERMINATION OF DPPH· RADIC AL SCAVENGING ACTIVITY The effect of sesamol on radical scavenging by DPPH was determined in comparison to three standard compounds—viz., BHA, BHT, and α-tocopherol. Various concentrations of test compounds in methanol were added to a methanolic solution of the DPPH radical. The fi nal concentration of DPPH was 0.02 mM. The mixture was shaken thoroughly and kept in the dark at room temperature for 30 min. The absorbance of the resulting solu- tion was measured by UV–Vis spectrophotometry at 520 nm (16). DETERMINATION OF FERRIC REDUCING ANTIOXIDANT POWER (FRAP) The FRAP assay was assessed according to Benzie and Strain (17). The method was based on the reduction of the Fe3+-TPTZ complex to the ferrous form (Fe2+) at low pH. This reduction was monitored by measuring the change of absorbance at 600 nm, which was related to the combined or “total” reducing power of the existence of electron-donating antioxidants in the reaction mixture. Briefl y, 50 μl of working FRAP reagent prepared daily was mixed with 200 μl of diluted test compounds. The stock solutions of the test compounds were dissolved in the DMSO. The absorbance at 600 nm was recorded after 8 min incubation at 37°C. FRAP values were obtained from the difference of absorptions in the reaction mixture with those from increasing concentrations of Fe3+ and were expressed as μmol of Fe2+. The standard curve was linear between 15.625 and 250 μM FeSO4 with R2 = 0.9603. DETERMINATION OF INHIBITION OF LIPID PEROXIDATION (LPO) USING THE THIOBARBITURIC ACID REACTIVE SUBSTANCES (TBARS) ASSAY LPO was measured in terms of TBARS according to the reaction with malondialdehyde equivalents formed from the peroxidation of lipids as described by Bae and Lee (18).

JOURNAL OF COSMETIC SCIENCE 72 Sesamol, BHA, BHT, and α-tocopherol were dissolved in DMSO and diluted with 0.1 M PBS, pH 7.0. A volume of 50 μl test compound was added to each well of a 96-well plate. Lipid oxidation was initiated by adding 85 μl of linoleic acid solution in DMSO and shaken at 100 rpm, 40°C for 24 h. To the mixture, 100 μl of thiobarbituric acid (0.67%w/v in phosphate buffer) was added and incubated at 80°C for 1 h. After cooling down, 45 μl of chloroform was added to the mixture, which was then shaken at 20×g for 5 min. The clear solution was taken and read at 520 nm. The plot between the different concentra- tions (μg/ml) of the compounds and percentage inhibition of LPO were used to calculate the IC50 value, i.e., the concentration of compound needed to achieve a 50% inhibition of LPO. DETERMINATION OF TYROSINASE INHIBITION ACTIVITY Tyrosinase is the key enzyme in melanin biosynthesis initiation of the reaction is by con- version of the amino acid tyrosine to other intermediates resulting in the melanin pig- ment. The inhibition of tyrosinase enzyme activity will lead to skin whitening. The inhibition of mushroom tyrosinase activity in vitro was performed as per Momtaz et al. (19), with minor modifi cations. Mushroom tyrosinase enzyme was added to each well of a 96-well plate to achieve a fi nal concentration of 27 units/ml. The test compounds and positive control, kojic acid and β-arbutin (prepared in aqueous solution) were added into each well. The prepared substrate L-DOPA solution in the 0.1 M PBS (pH 6.8) was added to the reaction mixture yielding a fi nal concentration of 4.5 mM. All of the reaction mixtures were incubated at room temperature (28°C) for 20 min and the ab- sorbance was measured at 492 nm using a microplate reader. The concentration pos- sessing a 50% tyrosinase inhibition compared to the control (in an absence of inhibitor) or IC50 value was calculated. Percent inhibition of tyrosinase activity was calculated as the following: ¯ ¢ ± % = × - - - - Tyrosinase inhibition A B C D 100 A B Note: A = absorbance of blank solution with enzyme B = absorbance of blank solution without enzyme C = absorbance of sample solution with enzyme D = absorbance of sample solution without enzyme CELL CULTURE The African green monkey kidney cell line (Vero) was maintained at the Centre for Re- search and Development of Medical Diagnostic Laboratories, Khon Kaen University, while SK-MEL2 was purchased from CLS-Cell Lines Service, Eppelheim, Germany. Both cell lines were maintained in Dulbecco’s modifi ed Eagle medium (DMEM) supplemented with 10% FBS, 1% penicillin/streptomycin and were cultured at 37°C in a humidifi ed atmosphere at 5% CO2.