INHIBITORY EFFECTS OF KOREAN INDIGENOUS PLANTS 149 INTRACELLULAR TYROSINASE INHIBITION TEST From tyrosinase inhibition and cell cytotoxicity tests, four samples with a good inhibition effect and no cytotoxicity were selected to conduct intracellular tyrosinase inhibition and melanin synthesis tests. A six-well plate containing DMEM media (Gibco, Carlsbad, CA) with 10% fetal bovine serum (FBS Gibco) (2 × 104 cells/well) was inoculated with 2 ml of murine melanoma (B16) cells and incubated at 5% CO2 and 37°C (CO2 air-jacketed incubator NuAire, Plymouth, MN) for 24 h. The medium was then replaced with fresh DMEM containing 10% FBS, 2 μM α-melanocyte-stimulating hormone (MSH), and 2 mM theophylline. Selected plant extracts and fractions were diluted with fresh medium and incubated with cells at 5% CO2 and 37°C until more than approximately 80%. After incubation, the medium was removed, cells were washed with phosphate-buffered saline (PBS), and trypsin was applied for the recovery of cells. The cell pellet was trans- ferred to an eppendorf tube and centrifuged at 10,000 rpm for 10 min to remove the supernatant. The cell pellet was washed two or three more times with PBS and centrifu- gation was repeated. Then, 100 μl of 0.1% Triton X-100 was added to dissolve cells and obtain intracellular tyrosinase extract, and 220 μl of 0.1 M phosphate buffer (pH 6.5), 40 μl of 1.5 mM tyrosine solution, and 20 μl of tyrosinase extract were added sequentially (10). After reacting at 37°C for 60 min, the absorbance was measured at 490 nm with an ELISA reader. For a blank test, only B16 melanoma cells were incubated without sample and the recovered tyrosinase extract was added. INTRACELLULAR MELANIN SYNTHESIS INHIBITION TEST A six-well plate containing DMEM with 10% FBS (2 × 104 cells/well) was inocu- lated with 2 ml of murine melanoma (B16) and incubated at 5% CO2 and 37°C for 24 h. The medium was then replaced with fresh DMEM containing 10% FBS, 2 μM α-MSH, and 2 mM theophylline. Murine melanoma B16 cells were grown, incu- bated, and processed until a pellet was formed as described earlier. The cell pellet was dried at 60°C. Then, 100 μl of 1 N NaOH was added to the pellet to dissolve intracellular melanin. After dilution of melanin with PBS, the absorbance was measured at 490 nm with an ELISA reader to determine the melanin synthesis inhibition rate. For a blank test, only B16 melanoma cells were incubated without sample and the recovered tyrosinase extract was added. IN VIVO DEPIGMENTING EFFICACY TEST Based on the results of the in vitro tyrosinase inhibition test, in vitro L -DOPA auto- oxidation inhibition test, intracellular tyrosinase inhibition test, intracellular melanin synthesis inhibition test, and cell cytotoxicity test, the EtOAC fraction of P. lactifora and BuOH fraction of E. offi cinalis were proven to have good depigmenting effects and no toxicity, and were then subjected to an in vivo effi cacy study using guinea pigs to estimate the depigmenting effect in human body. Four female brown guinea pigs were used for an in vivo depigmentation study. To in- duce pigmentation, the backs of the animals were completely shaved with an electric

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.