ARECA CATECHU L. EXTRACT 359 pigmentation is thought to be largely due to its antioxidative property that can reduce oxidative stress and inhibit intermediate autoxidation rather than tyrosinase. The safety of CC-516 was evaluated by cytotoxicity on human fibroblasts and skin irritation testing. In cytotoxicity testing on human fibroblasts, the LD5o of CC-516 was about 50 mg/ml, with no change in cell proliferation at a concentration of 50 mg/ml. CC-516 inhibited cellular pigmentation by reducing oxidative stress and inhibiting tyrosinase and autoxidation, and it is expected to have a whitening effect on human skin. Also, CC-516 showed antioxidative activity in vitro as well as topical anti-inflammatory activity in vivo. REFERENCES (1) L. W. D. Wattenberg and L. K. T. Lart, Inhibition of Chemical Carcinogenesis by Phenols, Coumarins, Aromatic Isothiocyanates, Flavons, and Indoles (Plenum Press, New York, 1981). (2) D. E. Pratt, "Natural Antioxidants From Plant Material," in Phenolic Compounds and Their Efj•cts on Health II (ACS Symposium Series 547, American Chemical Society, Washington D.C., 1994). (3) M. Mizuno and T. Tanaka, Chemistry of Photo-Ingredients--Recent Advances of Crude Drugs Re- search," in The Science of Plants in Cosmetics (Fragrance Press, Tokyo, 1986), p. 2. (4) J. M. Pawelek, A.M. Chakraborty, and J. L. Bolognia, Cosmet. Toiletr., 107, 61-68 (1992). (5) G. Imokawa, FragranceJ., 14, 38•48 (1995). (6) M. Masuda, T. Tejima, and G. Imokawa, Cosmet. Toiletr., 111, 65-77 (1996). (7) K. K. Lee, J. H. Kim, J.J. Cho, and J. D. Choi, Int. J. Cosmet. Sci., 20, 1-12 (1998). (8) Y. Y. Shiau and M. Shear, Oral Surg., 47, 453 (1979). (9) I. Dockrat and M. Shear, Fourth Proceedings of the International Academy of Oral Pathology (Gordon and Breach Science Publishers, New York, 1970), p. 57. (10) V. Raghavan and H. K. Baruah, History, chemistry and utilization, Econ. Bat., 12, 315-325 (1958). (11) K. K. Wary and R. N. Sharan, 22nd Annual Meeting of ESRB, Brussels, September 11-17, 1989. (12) H. Ohkawa, N. Ohishi, and K. Yagi, Anal. Biochem., 95, 351 (1979). (13) Y. Fugita, I. Uehara, Y. Morimoto, and T. Okuda, Yakugak Zasshi, 108, 129-135 (1988). (14) G. L. Reissig and L. F. Leloir, J. Biol. Chem., 217, 959-963 (1955). (15) G. Tonnel, L. Thiabalt, and I. Ringlet, Endocrinol., 77, 625 (1965). (16) J.P. Tarayre, M. Barbara, M. Aliaga, and J. Tisne-Versailler, Arzneim Forsch/Drug Res., 40,(II) 10, 1125 (1990). (17) A. Vanni, D. Gastaldi, and G. Giunata, Amnal. di Chimica, 80, 35-60 (1990). (18) T. Maeda and M. Fukuda, J. Soc. Cosmet. Chem., 42, 361-368 (1991). (19) T.J. Mosmann, Immunol Methods, 63, 55 (1983). (20) B. N. Ames, Science, 221, 1256 (1983). (21) M. G. Simic and S. V. Jovanovic, "Inactivation of Oxygen Radicals by Dietary Phenolic Compounds in Anticarcinogenesis," in Food Phytochemicals for Cancer Prevention II (ACS Symposium Series 547, American Chemical Society, Washington D.C., 1994). (22) K. T. A. Davies, Oxidative Damage and Repair (Perganon Press, New York, 1991). (23) M. G. Simic and D. S. Bergtold, Mutation Res., 250, 17 (1991). (24) P. E. Hartman and D. M. Shankel, Environ. A--Mol Mutagen, 15, 145 (1990). (25) L. W. Wattenberg, Cancer Res., 52, 2015 (1992). (26) H. Masaki, FragranceJ., 8, 64 (1995). (27) C. K. Wang and W. H. Lee, J. Agri. Food Chem., 44(8), 2014 (1996). (28) M. Bouclier, D. Cavey, N. Kail, and C. Hensby, Pharmacol. Rev., 42, 127 (1990). (29) S.J. Lee, H.J. Baek, and H. P. Kim, Arch. Pharm. Res., 17, 31 (1994). (30) Y. Mishima, S. Hatta, and M. Inazu, Pigment Cell Res., 1, 367-374 (1988). (31) O. H. Lowry, N.J. Rosebrough, A. K. Fart, and R. T. Randall, J. Biol. Chem., 193, 256 (1951). (32) M. Dobois, K. A. Glles, and F. Smith, Analyt. Chem., 28, 350 (1956). (33) D. G. Roux, J. Soc. Leather Trades Chemists., 35, 322 (1951).

j. Cosmet. Sci., 49, 361-367 (November/December 1998) An in vitro method for screening skin-whitening products GOPA MAJMUDAR, GEORGE JACOB, YOLANDA LABOY, and LOUIS FISHER, Mary Kay Holding Corporation, Dallas, TX 75247. Accepted for publication October 15, 1998. Synopsis Melanoderm (Mat-Tek) is an in vitro model of the human epidermis consisting of well-differentiated, cultured human keratinocytes and melanocytes. We utilized this model to evaluate the efficacy, stability, and cytotoxicity of whitening agents. Magnesium ascorbyl phosphate (MAP), kojic acid, and lactic acid in aqueous or anhydrous base were applied to Melanoderm. Following incubation, tyrosinase activity was measured using L-dihydroxyphenylalanine (L-DOPA). Melanocyte staining was observed under the micro- scope. Melanoderm treated with either MAP, kojic acid, or lactic acid showed 33%, 48%, and 46% reduction, respectively, of tyrosinase activity. Microscopic examination of treated Melanoderm clearly showed the dendritic nature of melanocytes, and normal morphology of keratinocytes and MTT assay suggested that the test materials were not cytotoxic. The kojic acid effect declined with the age of the preparation, and subsequent analysis via high performance liquid chromatography (HPLC) showed kojic acid to be unstable in the aqueous base. Clinical tests using a chromameter to evaluate skin color indicated that kojic acid in an anhydrous base can induce more skin lightening than in the aqueous base. We obtained a good correlation between the Melanoderm, HPLC, and clinical tests. The data show that Melanoderm is a suitable tool for screening whitening agents and developing whitening products. A combination of two in vitro tests, such as the Melanoderm and HPLC methods, is useful to evaluate the relative activity, stability, and cytotoxicity of whitening ingredients and products before testing on humans. INTRODUCTION Melanin pigments in skin play a key role in determining skin color and are synthesized by large dendritic cells known as melanocytes, which are located at the epidermal- dermal junction (1). Tyrosinase in melanocytes is a key enzyme in the synthesis of melanin pigments (2-4). Melanocytes transfer melanin pigments to neighboring cells such as keratinocytes. Melanin production and transport of melanin is increased by factors such as UV rays, hormones, and chemicals, resulting in darkening of the skin and development of age spots, freckles, melasma, and other disorders of hyperpigmentation (5-8). In Asia, skin-whitening products are very popular and are used to lighten the skin and to treat freckles and skin hyperpigmentation (9-11). The development of successful whitening skin care products depends on the use of effective whitening or depigmenting ingredients that inhibit melanin formation in melanocytes. A number of in vitro screen- Yolanda Laboy's present address is BASF Corporation, Washington, NJ 07882. 361