SKIN WHITENING BY A. DAHURICA EXTRACTS 17 100 �- i� 75 Q) 0 "' "' �u 50 �s [� 25 0 Ctrl 10 100 Cone. of A. dahurica(µg/ml) Figure 2. Effects of A. dahurica extract on the activity of tyrosinase. The lysates of Bl6 melanoma ceHs containing tyrosinase were incubated with A. dahurica extract and DOPA for three hours. Tyrosinase activity was measured as described in Materials and Methods. Results are the averages of three independent experiments ± SD. 100 'iii 75 �c:- c: e �§ II) .... ll 0 -� * e -- 50 25 0 Ctr1 50 100 1000 Cone. of A. dahurica (µ9/mL) Figure 3. Inhibitory effects of A. dahurica extract on tyrosinase synthesis in Bl6 melanoma ceHs. Bl6 melanoma cells were cultured in the presence of A. dahurica extract at concentrations of 50, 100, and 1000 µg/ml for 24 hours. The tyrosinase synthesis was examined by ELISA as described in Materials and Methods. Results are the averages of three independent experiments ± SD. *p 0.05 compared to the control. 1.5% of the control value, respectively. These results suggested the possibility that A. dahurica extract inhibited tyrosinase synthesis in B 16 melanoma cells. EFFECT OF A. DAHVRICA EXTRACT ON EXPRESSION OF THE TYROSINASE GENE IN B16 MELANOMA CELLS To elucidate the action mechanism of A. dahurica extract, we investigated the changes in the mRN A level of tyrosinase using the RT -PCR technique. B 16 melanoma cells were treated with 20 and 100 µg/ml of A. dahurica extract for 48 hours, respectively, and then each mRNA level was examined. The results are shown in Figure 4. When normalized with the mRNA level of �-actin, the mRNA level of tyrosinase was decreased by 75% at 100 µg/ml of the untreated control value. These results suggest that A. dahurica extract might act on the common upstream event that controls the transcription of the tyrosinase gene.

18 JOURNAL OF COSMETIC SCIENCE (A) 2 3 Tyrosinase b-actbt (B) 120 100 0 80 0 C: 60 0 :E 40 .!: ?fl. 20 0 Ctrl 20 100 Cone. of A. dahurica (µg/mL) Figure 4. Inhibitory effects of A. dahurica extract on expression of the tyrosinase gene in B 16 melanoma cells. Bl6 mouse melanoma cells were seeded into a T-75 flask at a density of 1 x 106 cells per flask and treated with or without A. dahurica extract for 48 hours. (A) is the result of gel electrophoresis of the RT-PCR products. Lane 1 shows the tyrosinase (716 bp) and J3-actin gene (400 bp) of the control cell. Lane 2 shows those of cells treated with 20 µg/ml of A. dahurica extract. Lane 3 shows those of cells treated with 100 µg/ml of A. dahurica extract. (B) shows the decreased mRNA level of cells treated with A. dahurica extract expressed as % inhibition of the control. Results are the averages of three independent experiments ± SD. *p 0.05 compared to the control. ISOLATION AND IDENTIFICATION OF ACTIVE COMPOUNDS FROM A. DAHURICA The ethanolic extract of A. dahurica decreased significant intracellular melanin content and tyrosinase biosynthesis. Thus, a laboratory investigation was performed on the active ethanolic extract. Activity-guided fractionation led to the isolation of compounds 1 and 2 as active compounds. Compound 1 was obtained as an amorphous white powder, and showed [Mr at mlz 270 in the EIMS spectrum. The UV spectrum exhibited typical bands of the 5-substituted linear furanocoumarin ring at 220, 249, and 309 nm (17 ,18). In the 1 H-NMR spectrum, two doublet signals (lH,J = 9.76 Hz) at o 6.27 and 8.15 were assignable to the ortho coupled protons of the pyrone ring, and another two doublet signals (lH,J = 2.45 Hz) were detected at 6.96 and 7 .60 ppm, attributable to the protons of the furan ring. A peak of the aromatic proton was detected as a singlet signal (lH, H-8) at 7 .16 ppm, and the signal for H-4 was observed at a rather lower field (o 8.15) to indicate the absence of proton at C-5 (17,18). Therefore, a side chain was suggested to substitute at the C-5