JOURNAL OF COSMETIC SCIENCE 146 The factors involved in melanin biosynthesis inside the melanosome include intracel- lular regulating factors that are involved in the synthesis and transfer of enzymes such as tyrosinase, dopachrome tautomerase, peroxidase, catalase, and glutathione reductase metal ions such as copper, zinc, and iron and interferon (IFN), prostaglan- din (PG), and histamine. In this study, 17 plant species were selected among the traditional Korean medicines (TKMs) mentioned in traditional herbal medicine books. Until now, reports have been published on tyrosinase inhibition and L -3,4-dihydroxy-indole-2-carboxylic acid (L-DOPA) auto-oxidation inhibition by methanol extracts of Chrysanthemum indicum, Evodia offi cinalis, Prunus mume, and Poncirus trifoliata (3) tyrosinase inhibition by E. offi cinalis, Gentiana scabra, Forsythia viridissima, Smilax china, Lonicera japonica, Paeonia lactifl ora Pall, P. trifoliata, Vitex rotundifolia, and P. mume (4) and DOPA auto-oxidation inhibition by C. indicum, E. offi cinalis, G. scabra, Houttuynia cordata, L. japonica, F. viridissima, Euonymus alatus, P. lactifl ora, Perilla frutescens, P. trifoliata, and V. rotundifolia (5). However, they only described investigation at the screening stage, and later investigation results have not been found yet. A total of 17 kinds of plants were extracted and fractionated using fi ve different solvents. Eighty-fi ve extracts and fractions were screened to elucidate their depigmenting effects using a tyrosinase inhibition assay and an L -DOPA auto-oxidation inhibition assay, re- sulting in the identifi cation of 10 kinds of candidate depigmenting fraction. The result- ing 10 fractions were tested again at various concentrations for tyrosinase inhibition and L -DOPA auto-oxidation inhibition to determine 50% inhibitory concentration (IC50). Finally, four fractions with good effi cacy were selected through the in vivo melanin syn- thesis inhibition and cytotoxicity tests. From these activities, ethyl acetate (EtOAc) frac- tion of P. lactifl ora was found to be the most effective constituent and was fi nally selected to develop a skin-whitening agent. MATERIALS AND METHODS MATERIALS Seventeen plants were obtained from the Oriental Medicinal Market in Seoul, South Korea, and were identifi ed with help from the Korea Pharmaceutical Traders Associa- tion (Table I). The B16 melanoma cell line and transformed mouse fi broblast L929 were obtained from the Korean Cell Line Bank (Seoul, Korea). EXTRACTION AND ISOLATION OF PLANTS The scheme of the fractionation of plant extracts is shown in Figure 1. About 500 g of each sample was soaked twice in 2000 ml of 80% methanol at room temperature for 24 h twice and fi ltered with a Whatman No. 5 fi lter paper to remove the solid material. The fi ltrates were evaporated in vacua to dryness (methanolic extract). To isolate the pure compounds, the methanolic extract was mixed with distilled water and partitioned with methylene chloride (MC), EtOAc, and n-butanol (n-BuOH). About 10–50 g

INHIBITORY EFFECTS OF KOREAN INDIGENOUS PLANTS 147 of the methanol extract was resuspended in 200 ml of water and was then extracted three times with the same volume of MC. The resulting aqueous layer was sequentially extracted with EtOAc and then n-BuOH using the same procedure as that used for MC extraction. The MC, EtOAc, n-BuOH, and aqueous layer were evaporated in vacua (VV2000, Heidolph, Schwabach, Germany), and approximately 1–20 g of powder or gel was obtained for testing (6,7). Table I List of Plants and Their TKM Names in Korea Scientifi c name TKM name Part used Chrysanthemum indicum Gamguk Flower Lonicera japonica Geumeunhwa Flower bud Vitex rotundifolia Manhyeongja Fruit Forsythia viridissima Yeongyo Fruit Evodia offi cinalis Osuyu Fruit Gentiana scabra Yongdam Root and rhizome Perilla frutescens (leaf and sprig) Jasoyeop Leaf and sprig P. frutescens (fruit) Jasoja Fruit Polyporus umbellatus Jeoryeong Sclerotium Houttuynia cordata Jeupchae Whole plant Celosia cristata Cheongsangja Seed Smilax china Tobokryeong Rhizome Prunus mume Omae Steamed and dried fruit Poncirus trifoliata Jisil Fruit Euonymus alatus Gwijeonu Alated sprigs Thuja orientalis Baekjain Seed Paeonia lactifl ora Jakyak Root Figure 1. The isolation of active fractions from plant samples.