INHIBITORY EFFECTS OF R. MORI EXTRACTS 137 100,. • 80 • 60 • 40 • , 2o i 0 Control 50 1 O0 R. mori extracts(ug/ml) Figure 1. Effect of R. mori extracts on tyrosinase synthesis (band intensity) Figure 2. Effects of R. mori extracts on tyrosinase gene expression (RT-PCR). Lane 1' Tyr-•control. Lane 2: Tyr--R. mori 50 pg/rnl. Lane 3: Tyr--R. mori 100 pg/rnl. Lane 4: Actin--control. Lane 5: Actin--R. mori 50 pg/rnl. Lane 6: Actin--R. mori 100 pg/rnl. INHIBITORY EFFECT OF R. MORI EXTRACTS ON UVB-INT)UCED PIGMENTATION OF BROWNISH GUINEA PIGS R. mori extracts inhibited the pigmentation induced by UVB in brownish guinea pigs (Figure 3). In the histological comparisons, the melanin content produced by UV radiation in the basal layer of the epidermis (control) was increased as compared to the skin treated with R. mori extracts. These results showed that R. mori extracts had an inhibitory activity on UV-induced pigmentation in the brownish guinea pig model. R. mori extract induced no irritant signs such as redness in this study. IDENTIFICATION OF AN ACTIVE COMPOUND IN R. MORI EXTRACTS AND EVALUATION OF A SINGLE COMPOUND ON MELANOGENESIS We purified a single compound from R. mori extracts using column chromatography and Prep LC. (Figtire 4). It was found that four compounds showed high inhibitory effects on tyrosinase activity (data not shown). From these compounds we isolated a single compound by recrystallization and identified its structure using mass chromatography, IR spectroscopy, and NMR analysis. The NMR data is shown in Figtire 5. The com- pound was identified as 2,3',4,5'-tetrahydroxystilbene(2-oxyresveratrol). INHIBITION MECHANISM OF 2-OXYRESVERATROL ON TYROSINASE ACTIVITY We checked the inhibitory effect of 2-oxyresveratrol on tyrosinase activity. It showed very high activity in tyrosinase inhibition (IC5o = 0.23 pg/ml). Significant inhibition in enzyme activity was shown by this compound in concentrations of more than 0.1 pg/ml. When L-tyrosine was used as a substrate, 2-oxyresveratrol decreased the Km value of tyrosinase but did not change the Vmax, and thus was a competitive inhibitor with a Ki value of 5 x 10 -6 M (Figure 6).

138 JOURNAL OF COSMETIC SCIENCE Figure 3. Effects of R. mort extracts on the inhibition of UV-induced pigmentation. (a) Placebo. (b) 1% (v/v) R. mori-extracts-treated skin. (c) 5% (v/v)R. mori-extracts-treated skin. SAFETY TESTS OF R. MORI EXTRACTS Acute toxicity test. We investigated the potential toxicity of the R. mori extracts. Accord- ing to the CTFA guidelines, we assessed acute oral toxicity and acute derreal toxicity of the compound in 60 rats and 24 rabbits, respectively. We examined acute toxicity for 14 days after treatment. No death occurred, and abnormality was not detected at clinical findings in rats and rabbits administered orally with the compound. We did not observe