14 JOURNAL OF COSMETIC SCIENCE NaN 3 0.02%, pH 9.6) was added 1:1 (v/v) and incubated overnight at 4 ° C. The supernatants were removed and the coated well was washed with PBS-T three times and blocked with 3% bovine serum albumin (BSA) in PBS-T for two hours at 37°C. After washing three times with PBS-T, 150 µl of 1:2000 diluted primary antibody (anti- tyrosinase) in PBS-T was added to each well and incubated for one hour and 30 minutes. After washing the wells with PBS-T three times, 150 µl of 1:3000 diluted secondary Ab (Goat anti-rabbit IgG conjugated horse-radish peroxidase) in PBS-T was added and incubated for one hour and 30 minutes. After washing with PBS-T five times, 150 µl of 5 mg/ml o-phenylenediamine (OPD) in OPD solution was added and incubated for 40 minutes. The optical density was measured at 490 nm. EFFECT OF A. DAHURICA EXTRACT ON EXPRESSION OF THE TYROSINASE GENE IN Bl6 MELANOMA CELLS Cells (1 x 106) were seeded into a T-75 flask in DMEM supplemented with 5% FCS and then cultured at 37 ° C for 24 hours. The cells were then cultured in DMEM supple- mented with A. dahurica extract for 24 hours. Total cellular RNA was prepared using the Ultraspec II RNA isolation system (Biotecx Lab., Houston, Texas) according to the supplier's instructions. Primers used for RT-PCR analysis in this study were as follows tyrosinase (716 bp), 5'-ACGCCCGAGGGACCTTTACGGCGTAATCCT-3' (5' primer), 5'-TTATAAATGGCTCTGATACAAGCTGTGGTAA-3' (3' primer) �-actin (400 bp), 5'-CGAGCTGCCTGACGGCCAGG-3' (5' primer) 5'-ATTTGCGGTGG- ACGATGGAG-3' (3' primer). These primers were synthesized by Bioneer Corporation (Korea). The extracted RNA was reverse-transcribed and amplified using an Access R T-PCR system kit (Promega, USA) on a thermal cycler (PCR system PCS0 1, ASTEC, Japan). The PCR cycle conditions were: melting for 30 seconds at 94°C, annealing for 30 seconds at 65°C, and extension for 90 seconds at 68 ° C for 28 cycles. PCR products were resolved on 2% agarose gel and visualized by ethidium bromide (EtBr) staining, photographed, and analyzed with a Fluoro S multi-image analyzer (Bio-Rad, USA). ISOLATION AND IDENTIFICATION OF ACTIVE COMPOUNDS FROM ANGELICA DAHURICA Active compounds from A. dahurica were roughly separated using silica gel column chromatography. The dried roots (100 g) of A. dahurica Benth et Hook (Umbelliferae) were refluxed with 70% aqueous ethanol, and the extract was evaporated to afford 30 g of the residue. The residue was dissolved in MeOH and then divided into a MeOH soluble fraction (14 g) and a MeOH unsoluble fraction (9.5 g), respectively. The MeOH soluble fraction (14 g) was chromatographed on a silica gel column (250 g, 230-400 mesh, 15 x 50 cm) using stepwise gradient elution with the solvents CH 2 Cl 2 and MeOH (50:1, 1:1, v/v) to divide the fraction into eleven subfractions (Fr. 1 - Fr. 11). The inhibitory effect of each elution on melanogenesis was examined to select the elution containing active compounds. Further, the fraction that possessed the greater effect, subfraction 3, was rechromatographed on a silica gel column (70 g, 230-400 mesh, 2 x 50 cm) using stepwise gradient elution with the solvents hexane and EtOAc (5:1, 3:1, v/v) to divide the fraction into four subfractions (Fr. I - Fr. IV). The inhibitory effects of four fractions on melanogenesis were examined, and then compound 1 (114 mg) and

SKIN WHITENING BY A. DAHURICA EXTRACTS 15 compound 2 (94 mg) were obtained by recrystallization in MeOH from subfractions 2 and 4, respectively. COMPOUND 1 (ISOIMPERATORIN) Amorphous white powder mp: 101-103°C UVm ax : 220, 249, 309 nm IR max (KBr) cm- 1 : 2950 (C-H), 1720 (C = O) EIMS rn/z: 270[M+} 1 H-NMR (500 MHz, CDC1 3 ) o H : 8.15 (lH, d,J = 9.76 Hz, H-4), 7.60 (lH, d,J = 2.45 Hz, H-2'), 7.16 (lH, s, H-8), 6.96 (lH, d,J = 2.45 Hz, H-3'), 6.27 (lH, d,J = 9.76 Hz, H-3), 5.54 (lH, m, H-2"), 4.92 (2H, d,J = 6.99 Hz, H-1"), 1.81 (3H, s, H-5"), 1.71 (3H, s, H-4")) 1 3 C-NMR (75 MHz, CDC1 3 ) o c (Table I). COMPOUND 2 (IMPERATORIN) Amorphous white powder mp : 95-97°C UVmax : 217, 247, 298 nm IR m ax (KBr) cm - i : 2950 (C-H), 1720 (C = 0) EIMS rnlz : 270[M+} 1 H-NMR (500 MHz, CDC1 3 ) o H : 7.76 (lH, d,J = 9.60 Hz, H-4), 7.69 (lH, d,J = 2.21 Hz, H-2'), 7.36 (lH, s, H-5), 6.81 (lH, d,J = 2.21 Hz, H-3'), 6.36 (lH, d,J = 9.60 Hz, H-3), 5.62 (lH, m, H-2"), 5.01 (2H, d, J = 7.16 Hz, H-1"), 1.75 (3H, s, H-5"), 1.72 (3H, s, H-4") 1 3 C-NMR (75 MHz, CDC1 3 ) o c(Table I). STATISTICAL ANALYSIS The results were expressed as the averages ± S.D. of three independent experiments. The Student's t-test was used to evaluate the differences of the means between the control and the samples. P values of 0.05 were taken to be significant. Table 1 13 C-NMR Data of Compounds 1 and 2 Isolated from Angelica dahurica* 13 C (0) in CDG'l No. 2 2 161.26 160.50 3 112.55 114.68 4 139.55 144.31 5 148.95 113.12 6 114.20 125.84 7 158.12 148.61 8 94.21 131.67 9 152.66 143.82 10 107 .52 116.47 2' 144.87 146.60 3' 105.02 106.69 1" 69.74 70.14 2" 119.09 119.76 3" 139.80 139.71 4" 18.20 18.09 5" 25.79 25.79 * TMS was used as internal standard the data for compounds 1 and 2 were obtained at 75 MHz. CDC1 3 was used as the solvent.