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

JOURNAL OF COSMETIC SCIENCE 74 comparison to the standard positive antioxidants (BHT, BHA, and α-tocopherol) using the DPPH, TBAR (Table I) and FRAP assays (Table II), which represent different mech- anisms of antioxidant activities. The ability of sesamol to inhibit radical scavenging and lipid peroxidation are presented with the IC50 value. The results showed that sesamol exerted the strongest radical scavenging activity over against the positive controls with an IC50 value 2 μg/ml (14.48 μM). Sesamol was able to inhibit lipid peroxidation more than BHT but not higher than BHA and α-tocopherol. Total antioxidant activity based on reducing power was measured by FRAP. The standard curve created by FeSO4 was plotted and was linear between 15.625 and 250 μM FeSO4 Table I. Effect of Sesamol, BHT, BHA and α-Tocopherol on Radical Scavenging Activity Using the DPPH Assay and Lipid-Peroxidation Using the TBAR Assay Compounds IC50 (μM) DPPH assay TBAR assay Sesamol 14.48a 6.15 ± 0.2b BHT 21.78 ± 0.9b 9.53 ± 1.4c BHA 40.50 ± 2.8c 1.55 ± 0.2a α-Tocopherol 19.73 ± 4.6b 3.02 ± 0.7a Results presented: mean ± S.D. (n = 3). Values with different superscripts in each column are significantly different (one-way ANOVA, p 0.05). Table II. Effect of Sesamol, α-Tocopherol, BHA, and BHT on Reducing Power as Determined by the FRAP Method Compounds Concentration (mM) FRAP value (μM FeSO4) Sesamol 0.0145 44.17 ± 2.22a 0.0282 84.23 ± 4.59b 0.0565 117.71 ± 14.51c 0.1129 189.88 ± 17.56d α-Tocopherol 0.0046 44.66 ± 4.12a 0.0091 75.91 ± 4.35b 0.0181 109.12 ± 9.07c 0.0362 165.75 ± 10.70d BHA 0.0111 10.75 ± 5.57a 0.0216 29.55 ± 6.86a,b 0.0433 45.59 ± 15.30b 0.0866 70.91 ± 14.04c BHT 0.0091 4.32 ± 2.94a 0.0177 10.21 ± 5.47a 0.0354 20.75 ± 3.62b 0.0708 33.79 ± 5.63c a–d Mean within a column of each compounds having the same superscripts are not signifi cantly different (p 0.05).