J. Cosmet. Sci., 63, 225–232 ( July/August 2012) 225 Crude ethanol extracts from grape seeds and peels exhibit anti-tyrosinase activity CHENG-KUANG HSU, SU-TZE CHOU, PEI-JANE HUANG, MEI-CHIN MONG, CHIEN-KUO WANG, YU-PIN HSUEH, and JYUN-KAI JHAN, Department of Food Science, National Chiayi University, No.300, Syuefu Rd., Chiayi City 60004, (C.-K.H.) Department of Health Nutrition and Biotechnology, Asia University, No.500, Lioufeng Road, Wufeng Shiang, Taichung 413 (C.-K.H., P.-J.H., M.-C.M.) Department of Biotechnology, Asia University, No. 500, Lioufeng Road, Wufeng Shiang, Taichung 413 (C.-K.W., Y.-P.H.) and Department of Food and Nutrition, Providence University, No. 200, Chungchi Rd., Shalu, Taichung 433 (J.-K.J.), Taiwan, ROC. Accepted for publication November 30, 2011. Synopsis This study aimed to evaluate the anti-tyrosinase activites of ethanol extracts from the peels and the seeds of Kyoho grapes and Red Globe grapes (KG-PEE, KG-SEE, RGG-PEE, and RGG-SEE). The total phenolic content in KG-SEE and RGG-SEE was 400±11 and 339±7 mg gallic acid equivalent/g, respectively, about 22 times and 13 times that in KG-PEE and RGG-PEE, respectively. Both seed extracts showed signifi cantly higher anti-tyrosinase activity than the peel extracts due to their high total phenolic content. The gallic acid content in RGG-SEE was twice that in KG-SEE, and gallic acid showed high anti-tyrosinase activity thus, RGG-SEE had higher anti-tyrosinase activity than KG-SEE. Lineweaver-Burk plots revealed that the in- hibitory mechanism of the ethanol extracts from the grapes was a mix-type inhibition. Grape seed has a greater total phenolic content and has potential as a skin-lighting agent. INTRODUCTION Tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) is a multifunctional copper-containing enzyme and commonly present in microorgan- isms, plants, and animals. This enzyme mainly is involved in the fi rst two steps of the melanin biosynthesis pathway fi rst, L-tyrosine (monophenolase activity) is hydroxylated and the hydroxylation product, L-dopa (diphenolase activity), is further oxidized into the corresponding o-quinone (1). Address all correspondence to Cheng-Kuang Hsu at ckhsu@mail.nycu.edu.tw.

JOURNAL OF COSMETIC SCIENCE 226 Tyrosinase is responsible for enzymatic browning in plants and therefore is considered to produce undesirable changes in color, fl avor, and nutritive values of plant-derived foods (2,3). Therefore, tyroinase inhibitors may prevent the browning reaction caused by ty- roinase and maintain the appearance of plant foods. Furthermore, tyrosinase catalyzes the reaction of melanin biosynthesis in human skin and results in dark skin (4). Recently, safe and effective tyrosinase inhibitors have become im- portant for their potential applications in preventing pigmentation in human beings (1,4,5). Therefore, tyrosinase inhibitors are also important in cosmetic applications for skin-whitening effects, since certain people prefer a lighter skin color (6). Plants are rich sources of bioactive chemicals and mostly free from harmful side effects, and there is an increasing interest in fi nding natural tyrosinase inhibitors from them. Some potent tyrosinase inhibitors, such as cuminaldehyde (7), oxyresveratrol (8), kaemp- ferol (9), quercetin (10), and gallic acid derivatives (11) have been isolated from various plants. Phenolic compounds are rich in many plants and they have been shown to possess anti-tyrosinase activity (10–13). In this study, the effects of ethanol extracts from the peels and the seeds of two grape cultivars, Kyoho grapes and Red Globe grapes, on mushroom tyrosinase activity were investigated. MATERIALS AND METHODS CHEMICALS Mushroom tyrosinase (4,000 units/mg), and 3,4-dihydroxyphenylalanine (L-dopa) were purchased from Sigma Chemicals Co. (St. Louis, MO). HPLC-grade ellagic acid, gallic acid, cinnamic acid, resveratrol, and catechin hydrate were also purchased from Sigma. Chlorogenic acid and kuromanin chloride were purchased from Extrasynthese (Genay, France). Kyoho grapes (Vitis vinifera × Vitis labrusca) and Red Globe grapes (Vitis vinifera) were purchased from a local farmer in Taichung city, Taiwan, ROC. PREPARATION OF ETHANOL EXTRACT FROM THE PEEL AND THE SEED OF GRAPES Grape peel and seed were homogenized with 95% ethanol (1: 10, weight (g)/volume (ml)) for 1 min and then set in a refrigerator at 4°C for 12 h. After centrifuging the mixture at 7000g at 4°C for 20 min, the ethanolic solution was fi ltrated with Whatman No.l paper, and then ethanol was removed in an evaporator at a temperature lower than 40°C. MEASUREMENT OF TOTAL PHENOLIC CONTENT The total phenol content was measured using Folin-Ciocalteu’s reagent method (14). The sample (0.5 ml, 200 mg sample/ml) was mixed with 0.5 ml of Folin-Ciocalteu’s reagent for 3 min and then mixed with 0.05 ml of 10 % Na2Co3. The absorbance of the mixture was measured at 735 nm after 1-hr incubation at room temperature. Gallic acid was used as the