BREADFRUIT EXTRACT AS SKIN LIGHTENER 45 the Spectra Count® microplate reader. The radical scavenging activity was calculated as a percentage of DPPH decoloration using the following equation: %Radical scavenging activity= [1-(A5/AB)] X 100 where A5 = an absorbence of DPPH with the tested sample and AB = an absorbence of DPPH without the tested sample. EC50 , the equivalent concentration to give the 50% effect, was determined by log-probit analysis using six to ten different final concentrations of the samples. DETERMINATION OF MELANOGENESIS-INHIBITORY ACTIVITY B16-Fl (ATCC No. CRL-6323) mouse melanoma cells were purchased from the Ameri­ can Type Culture Collection (Lot No. 2634963, Virginia). This cell line was isolated from the melanocyte of mouse strain C57BL/6J B16Fl melanoma cells were initially cultured in a 25-cm2 flask (3.2 x 106 cells/ml) with DMEM (analytical grade, Lot No. 054K8302, Sigma-Aldrich, Missouri) supplemented with 10% fetal bovine serum (FBS, analytical grade, Lot No. 40f3634K, Gibco, Paisley, UK) in air containing 5% CO2 and at a temperature of 37°C. The medium was changed every two days. The passage numbers of 5 to 8 were used in this study. Before being tested, the cell suspension was transferred from the 25-cm2 flask into a 24-well plate (1 x 105 cells/well) and kept in an incubator (5% CO 2 and temperature of 3 7°C) overnight for complete adherence of the cells on the plate. After 24 hr of cultivation, the old medium was replaced with 1.0 ml of new DMEM medium con­ taining the various concentrations of A. incisus extract or artocarpin dissolved in DMSO. At the final concentration, the amount of DMSO used was not more than 0.1 % (v/v). The control cells were treated with 0.1 % (v/v) DMSO. Kojic acid and hydroquinone were used as positive controls in this study. A melanin content assay was performed by using a modified method (9) with triplicate run. After treatment for three days, the treated cells were harvested by using trypsin­ ization (Tyrpsin-EDTA, analytical grade, Lot No. 1212385, Gibco Canada Ltd., On­ tario, Canada) and washed twice with phosphate buffer saline. The samples were air­ dried and dissolved in 200 µl of 1 N NaOH containing 10% of DMSO (99.5% GC plant cell culture tested, CAS No. 67685, Merck, Shuchardt, Germany). The obtained solu­ tions were heated at 80 ° C for 1 hr and then cooled at room temperature. The absorbence of melanin was measured at a wavelength of 490 nm by using the Spectra Count® microplate reader. The melanin content per cell was calculated by comparing it to the absorbence of the control adjusted to 100%. DETERMINATION OF CELL VIABILITY Before being tested, B16Fl melanoma cells were initially cultured and treated as in the above procedure. After treatment for three days, the cell proliferation was measured by directly counting the number of cells treated with trypan blue. A hemocytometer was used for counting viable cells that were not stained with blue dye (trypan blue solution, R&D grade, Lot No. 55K2342, Sigma Chemical Co, Ltd.). The microscopic technique

46 JOURNAL OF COSMETIC SCIENCE Figure 1. The appearance of A. incisus ether (A) and methanol (B) extract. was used to investigate the phenotypic appearance of the melanocyre cells at, before, and after treatment with the rested samples. STATISTICAL ANALYSIS All experimental data were analyzed using analysis of variance (ANOV A), and the significant difference of the mean from triplicate analysis at p 0.05 was determined by Duncan's multiple range test using SPSS 12.0 for Windows (SPSS Inc., Illinois). RESULTS AND DISCUSSION APPEARANCE AND AR TOCARPIN CONTENT OF THE EXTRACTS The appearance, moisture content and percent yield of the diethyl ether or methanol extract are shown in Figure 1 and Table I. Extraction of diethyl ether provided a yellow-powder solid, whereas that of methanol provided a deep brown paste. The HPLC chromatogram of artocarpin contained in both kinds of extract is shown in Figure 2. The amount of artocarpin contained in the ether and methanol extract was 45.19 ± 0.45 and 19.61 ± 0.05 % w/w, respectively. Because of the higher content of the major component, the ether extract of A. incisus was selected for further studies to clarify its action on melanogenesis-inhibitory and antioxidation activity. Table I Appearance, Percentage of Yield, and Moisture Content of Extracts from the Heartwood of A. incisus Characteristics % Yield % Moisture content Appearance A. incisus ether extract 0.82 7.19 ± 0.36 Yellow powder Type of extraction A. incisus methanol extract 1.10 11.36 ± 0.78 Deep brown paste