J. Cosmet. Sci., 64, 145–158 (March/April 2013) 145 Inhibitory effects of Korean indigenous plants on tyrosinase and melanogenesis KYUNG HUN SON and MOON YOUNG HEO, Cosmetics Evaluation Division, Korea Food and Drug Administration, Chungcheongbuk-do 363-700 (K.H.S.), and College of Pharmacy, Kangwon National University, Chunchon 200-701 (M.Y.H.), Republic of Korea. Accepted for publication August 6, 2012. Synopsis To search for new depigmenting cosmetic ingredients from Korean herbal extracts of traditional Korean medicines (TKMs), we screened about 17 TKM extracts collected in the Republic of Korea. Samples were prepared from the natural plants, including medicinal plants such as Chrysanthemum indicum (fl ower), using methanol, methylene chloride, ethyl acetate (EtOAc), n-butyl alcohol, and water as the extraction and/or the partitioning solvents. We then tested their inhibitory effects on melanogenesis by using in vitro tyrosinase inhibition assay, in vitro L -3,4-dihydroxy-indole-2-carboxylic acid (L-DOPA) auto-oxidation assay, and B16 melanoma cells. In addition, cytotoxicity testing (NR50 and MTT50) was conducted to evaluate safety. From the results of these assays, four fractions with good effi cacy and low toxicity were selected among them, in- cluding EtOAc fraction of Smilax china (rhizome), Paeonia lactifl ora (root), and Polyporus umbellatus (sclero- tium), and BuOH fraction of Evodia offi cinalis (fruit). In the inhibition assay of intracellular tyrosinase activity and melanogenesis in B16 melanoma cell line, the four plant fractions showed dose-dependent inhibitory effects, and the EtOAc fraction of P. lactifl ora showed the highest activity among the four fractions. The EtOAc fraction of P. lactifl ora was found to be the most effective substrate. INTRODUCTION Skin color is determined by the type, amount, and size of melanin formed by melanocytes between the dermis and epidermis (1,2). Therefore, in the development of depigmenting agents, it is important to understand the skin structure, physiological functions, and mechanisms of melanin biosynthesis. Melanin is formed in the melanosome, a unique intracellular structure of the melanocyte. Melanosomes contain enzymes that are involved in melanin biosynthesis, such as tyrosi- nase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2. It has been reported that TRP-1 stabilizes enzymes in the melanosome. Furthermore, melanosomes contain matrix proteins, which are thought to act as the location of enzyme synthesis. Address all correspondence to Moon Young Heo at myheo@kangwon.ac.kr.

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