JOURNAL OF COSMETIC SCIENCE 150 shaver and marked for ultraviolet (UV) irradiation (1.5 × 1.5 cm2, six points). They were irradiated with 500 mJ/cm2 UV rays using Waldmann UV 800 (Herbert Waldmann, Villinogen-Schwenningen, Germany, GmbH&E, Philis TL/12 lamp emitting 280–305 nm). This irradiation process was repeated three times at 1-week intervals (total 1500 mJ/cm2) (11). The plant extract and fraction samples were dissolved in vehicle (propylene glycol : ethanol : water at 5:3:2) to 2% and were applied on the backs of the guinea pigs after the UV induction pigmentation procedure was completed. Application of the sample was continued twice a day for 6 weeks. Hydroquinone was dissolved in the same vehicle. Hydroquinone and vehicle were used as positive and negative blanks, respectively. The degree of pigmentation on treated areas was measured according to melanin index with a mexameter (Courage-Khazaka Electronic, Koln, Germany) at 0, 1, 2, 3, 4, 5, and 6 weeks, and potential adverse effects were assessed. RESULTS AND DISCUSSION SCREENING OF EFFECTIVE DEPIGMENTING PLANT EXTRACT In vitro tyrosinase inhibition. Methanol, MC, EtOAc, n-BuOH, and water fraction of 17 natural plants, including C. indicum (Gamguk), were diluted to 0.1% test solutions with ethanol. These were used for in vitro tyrosinase (mushroom tyrosinase) inhibition tests (data not shown). From this test, the n-BuOH fraction of E. offi cinalis, EtOAc fraction of P. frutescens, n-BuOH fraction of P. frutescens, EtOAc fraction of P. umbellatus, methanol extract of S. china, EtOAc fraction of S. china, n-BuOH fraction of S. china, EtOAc fraction of P. mume, EtOAc fraction of P. trifoliata, n-BuOH fraction of P. trifoliata, methanol extract of P. lactifl ora, MC fraction of P. lactifl ora, EtOAc fraction of P. lactifl ora, and n-BuOH fraction of P. lactifl ora showed good in vitro tyrosinase inhibition results (more than 90%). The n-BuOH fraction of L. japonica, n-BuOH fraction of P. frutescens, methanol extract of E. alatus, EtOAc fraction of E. alatus, and n-BuOH fraction of E. alatus also showed good tyrosinase inhibition results (80%–90%). However, G. scabra, H. cordata, and Celosia cristata showed relatively low activity (less than 70%). In vitro L -DOPA auto-oxidation inhibition. Samples showing good results in the in vitro tyrosinase inhibition tests were selected for in vitro L -DOPA auto-oxidation inhibition tests (data not shown). The n-BuOH fraction of E. offi cinalis, EtOAc fraction of P. frutescens (leaf and sprig), n-BuOH fraction of P. frutescens (leaf and sprig), EtOAc fraction of P. umbellatus, methanol extract of S. china, EtOAc fraction of S. china, and EtOAc fraction of P. lactifl ora showed good auto-oxidation inhibition effect (more than 60%). Meanwhile, the EtOAc fraction of P. frutescens (leaf and sprig), n-BuOH fraction of P. frutescens (leaf and sprig), EtOAc fraction of P. umbellatus, and EtOAc fraction of P. mume, which had good in vitro tyrosinase inhibition results, did not show desirable antioxidation inhibition effects (less than 10%). From these results, it was determined that the two tests have to be conducted in parallel to appropriately evaluate new candi- date substances.

INHIBITORY EFFECTS OF KOREAN INDIGENOUS PLANTS 151 IN VITRO TYROSINASE INHIBITION RATE AND L-DOPA AUTO-OXIDATION INHIBITION RATE For this test, EtOAc fractions of S. china, P. lactifl ora, P. frutescens, E. alatus, P. mume, and P. trifoliata MC fraction of P. lactifl ora and n-BuOH fractions of E. offi cinalis and H. cordata were selected. Samples were dissolved in ethanol and the in vitro tyrosinase inhibition test was conducted using mushroom tyrosinase with arbutin, a depigmenting substance widely used in Korea, as a control. The results are shown in Figure 2. Figure 2. Inhibitory effects of S. china EtOAc fraction, P. lactifl ora EtOAc fraction, P. lactifl ora MC fraction, E. offi cinalis BuOH fraction, P. umbellatus EtOAc fraction, E. alatus EtOAc fraction, P. frutescens EtOAc fraction, H. cardata BuOH fraction, P. mume EtOAc fraction, P. trifoliata EtOAc fraction, and arbutin on (A) tyrosinase activity, (B) L -DOPA auto-oxidation, and (C) melanin synthesis. Results are shown as the aver- ages ± SD of three independent experiments.