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.

WHITENING AND ANTIAGING EFFECT OF SESAMOL 73 DETERMINATION OF CYTOTOXICITY SK-MEL2 and Vero cells in complete DMEM medium were added to each well of a 96-well plate (5 × 104 cells per well). After 24 h incubation at 37°C in 5% CO2 incubator, 100 μl of sesamol and the positive control (melphalan) were added to each well and incubated for 48 h. The cells were centrifuged for 5 min at 675 ×g to obtain the cell pellet. Next, 190 μl of medium was removed from each well and 100 μl of freshly prepared NR solution (50 μg/ ml of stock solution) was added to each well including the blanks (media combined with sam ple) and the controls (non-treated cells). The solution mixture was incubated at 37°C in an incubator with 5% CO2 for 2 h. After incubation, the cells were pelleted by cen- trifugation for 5 min at 675 ×g and the 100 μl of NR and medium were discarded. The cells were rinsed with 150 μl PBS (pH 7.4) and centrifuged for 5 min at 675 ×g. Then, 200 μl of 0.33% HCl in isopropanol solution was added to each well—including the controls and blanks—followed by thorough mixing. Finally, the absorption of the solu- tions was measured at 520 nm and the concentration resulting in 50% cytotoxicity vs. the non-treated cells (IC50) was calculated. DETERMINATION OF CELLULAR TYROSINASE INHIBITION ACTIVITY The inhibition of cellular tyrosinase activity was performed according to Sapkota et al. (16) with some modifi cations. The SK-MEL-2 cells were cultured at 9×105 cells/well in six-well plates then incubated at 37°C, 5% CO2 for 24 h. The cells were subsequently treated (48 h exposure) with sesamol or positive controls, kojic acid and β-arbutin. After treatment, the cells were washed with ice-cold PBS and slowly lysed with 1 ml of PBS 0.1 M, pH 6.8, containing 1% Triton X-100, at room temperature for 30 min. Lysed cells were centrifuged at 10,000 ×g for 10 min. The supernatant that contained the tyrosinase en- zyme was checked for protein content using the Lowry method and bovine serum albu- min as the standard protein. Supernatant containing the same amount of tyrosinase enzyme was added to each well of the 96-well plate. Then L-DOPA in 0.1 PBS (pH 6.8) was added to each well to achieve a fi nal concentration of 4.5 mM and incubated at 37°C for 18 h. The absorbance was measured at 475 nm and percentage inhibition calculated. STATISTICAL ANALYSIS The data were expressed as a mean ± S.D. (n = 3). Statistical differences between the treated and untreated groups were tested using a one-way ANOVA with a 95% confi - dence interval. RESULTS EFFECT OF SESAMOL ON RADICAL ANTIOXIDANT ACTIVITY Skin exposure to UV radiation can generate radicals or trigger signaling pathways to in- duce melanin formation. This hyperpigmentation of melanin is a defense mechanism in the skin. Radical generation can, however, be disrupted by using suffi cient effective antioxidant(s). The antioxidant activities of sesamol were therefore investigated in