SKIN WHITENING BY A. DAHURICA EXT RACT S 19 position. A doublet signal (2H,j = 6.99 Hz, H-1") and a multiplet signal (lH, m, H-2") were detected at 4.92 and 5.54 ppm, respectively, and protons of methyl groups were detected at 1.71 and 1.81 ppm as singlet signals. In the 13 C-NMR spectrum, carbonyl carbon was shown at 161.26 ppm, along with twelve sp2 carbons (o 94.12 to 158.12) and three sp3 carbons (o 18.20 to 69.74). From these data, compound 1 was postulated to be isoimperatorin, and the structure was verified by the reported NMR data (18,19). Compound 2 was obtained as an amorphous white powder, and [Mr at rnlz 270 in the EIMS spectrum. In the 1 H- and 13C- NMR spectrum, most of the spectral aspect of compound 2 was similar to that of compound 1, but the proton signal for H-4 was detected at a higher field (o 7.76) than that of compound 1, and two carbon signals for C-5 and 8 were changed. From these data, compound 2 was assumed to be the position isomer of compound 1, and identified as imperatorin. This was finally confirmed by comparing its NMR data with those in the reported references (18,19). The structures of compounds 1 and 2 are presented in Figure 5 and 13C-NMR data are listed in Table I. We investigated the inhibitory effects of these compounds on melanogenesis in B16 melanoma cells. Compounds 1 and 2 at a concentration of 80 µMand 100 µM reduced the melanin content by 50% compared with non-treated control cells, respectively. These compounds inhibited melanogenesis of B16 melanoma cells in a dose-dependent manner (data not shown). We further studied by ELI SA the inhibitory effects of these compounds on tyrosinase expression in B16 melanoma cells. The treatment of B16 melanoma cells with these compounds significantly suppressed tyrosinase production at the protein levels in a dose-dependent manner: by an average of 20± 5% (p 0.05) at 10 µM 40± 4.5% (p 0.05) at 50 µM and 60± 5.6% (p 0.05) at 100 µM of compound 1 35± 5% (p 0.05) at 50 µM 50± 3.4% (p 0.05) at 100 µM and 75± 6.5% (p 0.05) at 200 µM of lsoimperatorin lmperatorin J" 4 � -o 5" H H _r('" -o 5" Figure 5. Chemical structure of compounds 1 and 2 isolated from the root of Angelica dahurica.

20 JOURNAL OF COSMETIC SCIENCE compound 2, compared with non-treated control cells, respectively (data not shown). Therefore, in this study, we investigated in B16 melanoma cells whether these com- pounds modulate the expression of tyrosinase steady-state mRNA levels. These com- pounds significantly reduced tyrosinase production at the mRNA level (data not shown). These results suggest that these compounds suppress the tyrosinse production at the protein and mRNA levels. CONCLUSIONS We found that A. dahurica extract had a strong inhibitory activity against melanogenesis and exerted its melanogenic inhibitory effect through the modulation of mRNA levels of tyrosinase. Also, we isolated isoimperatorin and imperatorin, new cosmetic agents for skin whitening, from Angelica dahurica. These results suggest that the active compounds, in their action mechanism, are novel whitening agents different from other those being used in the cosmetic industry. REFERENCES (1) H. Y. Park, J. M. Perez, R. Laursen, M. Hara, and B. A. Gilchrest, Protein kinase C-13 activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain, ]. Biol. Chern., 274(23), 16470-16478 (1999). (2) A. M. Korner and J. Pawelek, Dopachrome conversion: A possible control point in melanin biosyn- thesis, J. Invest. Derrnatol., 75, 192-195 (1980). (3) V. J. Hearing and M. Jimenez, Analysis of mammalian pigmentation at the molecular level, Pigment Cell Res., 2, 75-85 (1989). (4) T. Kobayashi, K. Urabe, A. Winder, C. Jimenez-Cervantes, G. lmokawa T. Brewington, F. Solano, J. C. Garcia-Borron, and V. J. Hearing, Tyrosinase related protein 1 (TRPl) functions as a DHICA oxidase in melanin biosynthesis. EMBO ]., 13, 5818-5825 (1994). (5) K. Yokoyama, H. Suzuki, K. Yasumoto, Y. Tomita, and S. Shibahara, Molecular cloning and func- tional analysis of a cDNA coding for human DOPAchrome tautomerase/tyrosinase-related protein-2, Biochim. Biophys. Acta., 1217, 317-321 (1994). (6) V. J. Hearing and K. Tsukamoto, Enzymatic control of pigmentation in mammals, FASEB ]., 5, 2902-2909 (1991). (7) S. M. Hacker, Common disorders of pigmentation: When are more than cosmetic cover-ups required?, Postgrad. Med.]., 99(6), 177-186 (1996). (8) S. H. Lee, S. Y. Choi, H. Kim, J. S. Hwang, B. G. Lee, J. J. Gao, and S. Y. Kim, Mulverroside F isolated from the leaves of Marus alba inhibits melanin biosynthesis, Biol. Pharm. Bull., 25(8), 1045- 1048 (2002). (9) G. D. Jung, J. Y. Yang, E. S. Song, and J. W. Par, Stimulation of melanogenesis by glycyrrhizin in Bl6 melanoma cells, Exp. Mo!. Med., 33(2), 131-135 (2001). (10) J. K. No, D. Y. Soung, Y. J. Kim, K. H. Shim, Y. S. Jun, S. H. Rhee, T. Yokozawa, and H. Y. Chung, Inhibition of tyrosinase by green tea components, Life Sci., 65(21), PL241-246 (1999). (l l) T. Kimura, P. P. H. But, J. X. Guo, and C. K. Sung, International Collation of Traditional and Folk Medicine, Part 1 (World Scientific, Singapore, 1996), pp. 117-118. (12) T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays,]. Immunol. Methods, 65(1-2), 55-63 (1983). (13) G. lmokawa and Y. Mishima, Loss of melanogenic properties in tyrosinases induced by glycosylation inhibitors within malignant melanoma cells, Cancer Res., 42, 1994-2002 (1982). (14) B. B. Fuller, D. S. Iman, and J. B. Lunsford, Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme-linked immunoadsorbent assay and by immunotitration analysis,]. Cell. Physiol., 134(1), 149-154 (1988). (15) S. Akiu, Y. Suzuki, T. Asahara, Y. Fujinuma, and M. Fukuda, Inhibitory effect of arbutin on melano-