ANTI-TYROSINASE ACTIVITY OF ETHANOL FROM GRAPES 227 standard for the calibration curve, and the total phenolic content was expressed as gallic acid equivalents (mg/g dry material). CHARACTERIZATION OF PHENOLIC COMPOUNDS IN EXTRACTS Phenolic compounds in the tested extract were analyzed based on the method described by Li et al. (15), with a slight modifi cation. Dried extract (10 mg) was dissolved in l ml of 0.1% phosphoric acid, fi ltered through a 0.45-μm fi lter, and analyzed by HPLC. HPLC analysis was performed using a Hewlett- Packard HPLC system (HP 1100 series, Waldron, Germany), consisting of a quaternary pump and a variable wavelength detector (VWD) at 270 nm and equipped with a Li-Chrospher RP-18 cartridge column (Merck, 250 mm × 4.6 mm, 5 μm). The mobile phase was a stepwise gradient of water (0.1% v/v phosphoric acid)–acetonitrile (0.01 min, 100:0 50 min, 20:80), and the injection volume was 30 μl. The identifi cation of each compound was based on a combination of retention time and spectral matching by comparison with those of known standards. ENZYMATIC ASSAY OF TYROSINASE The tyrosinase activity using L-dopa as substrate was measured according to the method of Kubo and Kinst-Hori (7), with slight modifi cations. First, 0.29 ml of 4.5 mM L-dopa solu- tion (the substrate for tyrosinase) was mixed with 0.3 ml of 25 mM phosphate buffer (pH 6.8) and incubated at 25°C for 10 min. Then, 0.3 ml of tested samples of different concen- trations (1, 5, 10, and 15 mg/ml) was added to the mixture followed by the addition of 0.01 ml of 4000 units/ml mushroom tyrosinase. The formation of dopachrome was immediately monitored by measuring the linear increase in optical density at 475 nm. Triplicate measurements were recorded. The increased absorbance at 475 nm was recorded during 10 min at room temperature. Deionized water was used instead of the extract for the blank. One unit (U) of enzymatic activity was defi ned as the amount of enzyme needed for increasing 0.001 absorbance per min at 475 nm under the experimental conditions. DETERMINATION OF KINETIC PARAMETERS Mushroom tyrosinase (0.03 ml, 4000 units/ml) was incubated with various concentra- tions of 0.27-ml enzyme substrates (L-dopa, 0.6–0.66 mg/ml) in 0.3 ml of 25-mM phos- phate buffer (pH 6.8) at room temperature, and tested samples (0.3 ml) were added to the reaction mixture simultaneously. The kinetic parameters, Km and Vmax, of the tyrosinase activity were calculated by linear regression from Lineweaver-Burk plots. STATISTICAL ANALYSIS For each measurement, three replicates were conducted. The data were presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was conducted using a package (SAS Institute Inc., Cary, NC). Duncan’s multiple ranges test was used to de- termine the signifi cant difference between different treatments.

JOURNAL OF COSMETIC SCIENCE 228 RESULTS AND DISCUSSION ANTI-TYROSINASE ACTIVITY OF GRAPE EXTRACTS L-dopa is an endogenous phenolic substrate for polyphenol oxidase in apple and potato sources (16). It is an intermediate product during oxidation of L-tyrosine and is used commonly as an enzyme substrate of tyrosinase, a polyphenol oxidase (1,9–11,17). In this study, the inhibitory effects of ethanol extracts from peels and seeds of Kyoho grapes and Red Globe grapes (KG-PEE, KG-SEE, RGG-PEE, and RGG-SEE, respectively) toward mushroom tyrosinase activity were evaluated. Therefore, L-dopa was used as the substrate of mushroom tyrosinase to screen the inhibitory activities of ethanol extracts from the grapes. The inhibitory effect of KG-PEE on mushroom tyrosinase activity depended on the concentrations (Figure 1). As the concentrations of KG-PEE increased from 0 mg/ml to 5 mg/ml, the absorbance of OD475nm, an index of dopachrome formation, decreased accordingly, indicating a decrease in tyrosinase activity. All the ethanol extracts showed anti-tyrosinase activity, with the following order: RGG-SEE KG-SEE KG-PEE RGG-PEE. It was noted that the seed extracts from both the Kyoho grapes and Red Globe grapes had higher anti-tyrosinase activities than the peel extracts. To evaluate the inhibitory mechanism of the ethanol extracts, mushroom tyrosinase activ- ity was determined under various L-dopa concentrations. It was found that the reaction rate of tyrosinase decreased as the concentrations of KG-SEE increased from 0.33 mg/ml to 5 mg/ml (Figure 2). KG-PEE, RGG-PEE, and RGG-SEE also reduced the tyrosinase reaction rate in different magnitudes (data not shown). The Lineweaver-Burk plots re- vealed that these extracts served as mix-type inhibitors, and a represented plot for KG- SEE is shown in Figure 3. Figure 1. Relative inhibitory effect of grape extracts on mushroom tyrosinase activity using L-dopa (0.33 mg/ml) as the substrate. Mushroom tyrosinase (44 units/ml) reacted at room temperature for 10 min. Symbols: - - (KG-PEE) - - (KG-SEE) -▲- (RGG-PEE) -×- (RGG-SEE).