INHIBITORY EFFECTS OF R. MORI EXTRACTS 139 The flow-chart of Ramulus mori extracts Ramulusmori 10 Kg I 70% EtOH 60 L, after 5 days filter Ethylacetate extraction water fraction IC50=1001• g/ml Ethylacetate Extracts 92.3 g IC5o=10.0 g g/ml I Prep-TLC I ...... Rf=0.23, IC5o=1.35 gg/ml •" Recrystalization with CHC13 dark-brown 45.4 gl IC5o=4.53 la g/ml Silicagel Column Chromatography Fraction 10.2 g IC5o=1.35 l•g/ml Prep-LC 2'-Oxyresveratrol, 1.67g IC5o=0.23 [tg/ml Figure 4. Flow chart of purification of single compound from R. mori extracts. changes of body weight in the test animals or abnormalities of organs from gross findings of necropsy (data not shown). Skin irritation test. The primary skin irritation from R. mori extracts was investigated in six rabbits. We examined skin irritation and clinical signs for 24-72 hours after a seven-day treatment using Draiz's P.I.I. (primary irritation index). We did not observe side effects such as erythema or edema. Eye irritation test. The potential toxicity of the compound was determined according to AFNOR (Association Francaise de Normalization) guidelines in an eye irritation test on nine rabbits. We examined both eyes at one hour, and one, two, three, four, and seven days after treatment. We did not see any clinical signs.

140 JOURNAL OF COSMETIC SCIENCE A •H NMR(500 MHz, acetone-de): 6 6.05(s, 1H), 6.22(d, J=8.4 Hz, 1H), 6.31(d, J=2.3 Hz, 1H), 6.33(s, 2H), 6.76(d, J=16.5 Hz, 1H), 7.14(d,J=16.5 Hz, 1H), 7.33(d, J=8.5 Hz, 1H) •3C NMR (125 MHz, acetone-de): 6 102.19, 103.39, 104.82, 108.06, 116.09, 124.00, 125.44, 127.99, 140.87, 156.87, 158.97, 159.21, 159.35 Mass(tel intensity): 245(M+, 15.8), 244(100), 243(12.6), 227(15.2), 226(19.4), 198(16.3), 197(10.5), 69(8.0) Figure 5. The spectrum data (A) and structure (B) of 2-oxyresveratrol(2,3',4,5'-tetrahydroxystilbene). 0.25 0.20 ._ "• 0.15 0 • 0.10 0.05 -0.002 -0.001 0.000 0.001 0.002 0.003 0.004 1/[L-tyrosine] Figure 6. Kinetics of mushroom tyrosinase by 2-oxyresveratrol. Lineweaver-Burk plot of enzyme activity. Circle: 1.5 lag/mi. Triangle: 1.0 lag/mi. Rectangle: 0.5 lag/mi. Diamond: 0 lag/mi. Skin sensitization test. The sensitization potential of the compound was assessed in 33 guinea pigs by the methods of the Magmusson and Kligman maximization test. We did not observe any skin responses in the test animals. Acute oral toxicity. The acute oral toxicity of the compound was assessed in 60 rats and 24 rabbits by the "Toxicity Guideline of Drug" proposed by KFDA (Korea Food & Drug Administration). We did not observe any oral toxicity in the test animals.