12 JOURNAL OF COSMETIC SCIENCE Concerns of changes in skin color are frequently raised for medical or cosmetic reasons. Hyperpigmentation disorders are often treated with hydroquinones, retinoids, arbutin, kojic acid, ascorbic acid, and tyrosinase inhibitors, but results of such treatments are sometimes disappointing (7). Nowadays, tyrosinase inhibitors from natural plants, such Morus alba L. (8), Glycyrrhiza glabra L. (9), and green tea (10), have been studied in consideration of safety. To develop plant materials protecting hyperpigmentation, we have checked the effect of 20 medicinal plant extracts on the inhibition of melanogen- esis, the inhibition of tyrosinase synthesis, and the activity of tyrosinase in B16 mela- noma cells, respectively. From the results of these screening procedures, we found that Angelica dahurica exerted a strong melanogenic inhibitory effect on B16 mouse mela- noma cells. A. dahurica has been used in Korea, Japan, and China as a folk medicine to treat menstrual disorder, abdominal pain, hysteria, bleeding, headache, and excessive leukorrhea (11). We also identified the active compounds in the extract of A. dahurica. To elucidate the action mechanism of the active compounds of A. dahurica, we inves- tigated the changes in the mRNA level of tyrosinase using the reverse transcription- polymerase chain reaction (RT-PCR) technique. EXPERIMENTAL MATERIALS Medicinal plants were purchased from a local market (Kyeong-Dong market, Korea). The plants were extracted in 7 0% aqueous ethanol under reflux for four hours. The extracts were filtered and concentrated in vacuo. For evaluation, the plant extracts were dissolved in dimethylsulfoxide (DMSO). The melting points were taken on a Mel-Temp II (Laboratory Devices, USA) melting- point-determining apparatus and uncorrected. 1H- and 1 3C-NMR spectra were recorded with a Unity Inova 500 (Varian, USA) and a DPX 300 (Bruker, Germany). Chemical shifts were given in O (ppm) from TMS. IR, EIMS, and UV spectra were measured on an FT/IR-5300 Qasco, Japan), a JMS 700 Qeol, Japan), and a Cary 1E (Varian, Austra- lia), respectively. The purity of the compounds isolated was identified with high- performance liquid chromatography (Waters Alliance 2695-996 PDA detector, Waters, USA). Thin-layer chromatography (TLC) was performed on pre-coated Kieselgel 60 F 254 (Art. 1.05554 and 1.13895) and RP-18 F 254 (Art. 1.05559) plates (Merck, Germany). Silica gel 60 was purchased from Merck. The organic solvents and chemicals were obtained from Sigma (USA), Bio Whittaker (USA), and Gibco BRL (USA), and purified by the appropriate methods before use. CELL VIABILITY ASSAY Cell survival was measured by the level of mitochondrial respiration in cells after treating the samples, which was determined by the reduction of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), into a blue formazan precipitate. The cells were plated at a density of approximately 1 x 104 cells/well in a 96-well microplate. After sample treatment for 24 hours, the cells were incubated in MTT solution (0.5 mg/ml) for four hours at 3 7°C. The blue formazan produced was solubilized in 0.4 ml of acid-isopropanol (0.04 N HCl in isopropanol), and

SKIN WHITENING BY A. DAHURICA EXTRACTS 13 the optical density was read at 565 nm. Only cells with functional mitochondria are capable of cleaving MTT to generate the dark purple formazan. The results were ex- pressed in percentages relative to the control (12). EFFECT OF A. DAHURICA EXTRACT ON MELANOGENESIS IN Bl6 MELANOMA CELLS The A. dahurica extract was quantitatively evaluated regarding its inhibitory activity on melanogenesis in B16 melanoma cells. The inhibitory activity on melanogenesis of A. dahurica extract was demonstrated according to the procedure oflmokawa et al. (13) with minor modification. B16 melanoma cells were seeded into a 12-well microplate at a density of 8 x 10 5 cells, and incubated in 37°C in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS). After 24 hours, the medium was exchanged with DMEM supplemented with 2% FCS and the plant extract at various concentrations, and cultured at 3 7°C. The intracellular melanin content and the cell number were measured after 72 hours. The melanin content of B16 melanoma cells was quantified by the following procedure. The cells were washed with phosphate buffer saline (PBS) and then collected by trypsinization and centrifugation. The cells were lysed with phosphate buffer (50 mM, pH 6.8) containing 1 % Triton-X, 2 mM phenyl methyl sulfonyl fluoride (PMSF) and collected by centrifugation. The pellets of the cells were solubilized in lN NaOH containing 10% DMSO to determine the melanin content. The melanin content was quantified with an absorbance at 405 nm. A standard curve for melanin determination was prepared using synthetic melanin (Sigma Chemical Co., St. Louis, MO). The cell number was determined with a Coulter counter. EFFECT OF A. DAHURICA EXTRACT ON THE ACTIVITY OF TYROSINASE IN B16 MELANOMA CELLS Cells (5 x 10 4 ) were seeded into a 96-well microplate in DMEM supplemented with 5% FCS and cultured at 3 7°C for 24 hours. After washing with PBS twice, the cells were lysed for 30 minutes at 3 7°C with 50 µl of phosphate buffer (50 mM, pH 6.8) con- taining 1 % Triton-X, 2 mM PMSF. Then 50 µl of phosphate buffer (50 mM, pH 6.8) containing 0.1 % L-3, 4-dihydroxyphenylanine (DOPA) and 50 µl of A. dahurica extract was added to the lysate and incubated at 3 7°C for three hours. When DOPA was used as a substrate of tyrosinase, melanin was yielded as a final product. Therefore, to evaluate tyrosinase activity, melanin content was determined by the method described in the previous section. A protein in the cells was quantified with the Bio-Rad protein assay kit (Pierce Co., Rockford, Illinois) according to the supplier's instruction. Tyrosinase ac- tivity was expressed as µg melanin/µg protein. EFFECT OF A. DAHURICA EXTRACT ON THE SYNTHESIS OF TYROSINASE IN Bl6 MELANOMA CELLS The synthesis of tyrosinase was assayed by enzyme-linked immunosorbent assay (ELISA) by Fuller et al. ( 14). Cells (5 x 10 4 ) were seeded into a 96-well microplate in DMEM supplemented with 5% FCS and cultured at 37°C for 24 hours. The cells were cultured in DMEM supplemented with A. dahurica extract for 24 hours. After washing with PBS, the cells were lysed at 37°C for 30 minutes with 75 µl of phosphate buffer (50 mM, pH 6.8) containing 1 % Triton-X and 2 mM PMSF. Then the supernatants were transferred into a 96-well microplate and the coating buffer (Na2CO3 0.159%, NaHCO 3 0.293%,