REDUCTION OF HYPERPIGMENTATION BY A. INCISUS EXTRACT 3 dried at 50°C by using a hot-air oven. Then the dried chipped heartwood was milled into powder. After that, 500 grams of the powder was extracted with 800 ml of diethyl ether (analytical grade, LabScan Asia, Co. Ltd., Bangkok, Thailand) at room temperature for two days, according to previous studies (5,6) with minor modifi cation. The obtained mixture was fi ltered through a cloth to remove particulates and the diethyl ether was then removed by evaporation with a vacuum evaporator set at 33°C. The resultant powder was stored in a tight amber glass at -20°C for further studies. QUANTIFICATION OF ARTOCARPIN IN THE EXTRACT The quantity of artocarpin, the major component of A. incisus’s heartwood extract, was determined by using isocratic high-performance liquid chromatography (HPLC) accord- ing to our previous study (5) with minor modifi cation. The artocarpin standard was pro- vided by Assist. Prof. Atawit Somsiri of the Faculty of Pharmaceutical Sciences, Naresuan University (11). The HPLC instrument consisted of an SPD-10M10AVP diode array detector and an SCL-10A central unit (Shimadzu Co., Ltd., Kyoto, Japan). An Alltima C18 column (5 μm), 250 × 4.60 mm in diameter (Alltech Associates Inc., Illinois), was applied as stationary phase. The effl uent consisted of a mixture of methanol (HPLC grade, LabScan Asia Co. Ltd):water (80:20). The fl ow rate of the effl uent was 1 ml/min and the injection volume was 20 μ1. The quantifi cation of artocarpin was based on the peak area at 282 nm. Determinations were performed in triplicate and the results were the average of three independent determinations. IC50 VALUE OF MELANOGENESIS INHIBITORY ACTIVITY OF THE EXTRACT IN MOUSE MELANOCYTE CELLS The IC50 value, the equivalent concentration to provide 50% melanogenesis inhibition, was determined by log prohibit analysis using six different fi nal concentrations of the extract. In the present study, the extract concentration used for nanoemulsion formula- tion was based on this value. CELLS AND TREATMENT B16-F1 mouse melanoma cells (Lot No. 300122-43) were purchased from Cell Lines Services, Eppelheim, Germany. First, B16F1 melanoma cells were initially cultured in a 25-cm2 fl ask (3.2 × 106 cells/cm2) in Dulbecco’s Modifi ed Eagle’s Medium (low glucose, Sigma-Aldrich Co., St. Louis, Missouri), supplemented with 10% fetal bovine serum (FBS, GIBCO, California) at 37°C in a humidifi ed 5% CO2 atmosphere. The medium was changed every two days. The passage numbers (the number of times the cell has been replated and allowed to grow back to confl uency) of 5 to 8 were used in this study. Before being tested, the cell suspension was transferred from a 25-cm2 fl ask into a 24-well plate (1 × 105 cells/well) and kept in an incubator (37°C, 5% CO2) overnight for complete adherence of the cells on the culture plate. After 24 h of cultivation, the old medium was replaced with 1.0 ml of new DMEM medium containing various concentrations (10, 15, 25, 40, 80, and 100 μg/ml) of A. incisus extracts dissolved in dimethyl sulfoxide (DMSO,

JOURNAL OF COSMETIC SCIENCE 4 99.5% GC plant cell culture tested, Merck, Darmstadt, Germany). At the fi nal concen- tration, 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 (Sigma-Aldrich) of various concentrations (10, 15, 25, 40, 80, and 100 μg/ml) was used as a positive marker. MELANIN CONTENT ASSAY After treatment for four days, the treated cells were trypsinized with trypsin EDTA (GIBCO, Ontario, Canada) and washed twice with phosphate buffer saline (PBS). Then the collected cells were lysed in 1 N NaOH containing 10% v/v of DMSO and heated at 80°C for 1 h (12). Finally, the amount of melanin was determined from the absorbance at the wavelength of 490 nm by using a microplate spectrophotometer (model Spectra Count®, Perkin Elmer, Connecticut, USA). The percentage of inhibition was calculated from the reduction in the absorbance value of the treated cells compared with that of the control adjusted to 100%. All experiments were performed in triplicate. CELL VIABILITY MEASUREMENT A hemocytometer was used for counting viable cells that were not stained with the blue dye of a trypan blue solution (R&D grade, Sigma-Aldrich). A microscopic technique was also used to investigate the phenotypic appearance of the melanocyte cells before and after treatment with the extract. NANOEMULSION FORMULATION Nanoemulsions containing A. incisus extract were prepared by the phase inversion tem- perature (PTT) method with some modifi cations (13,14). The water phase was composed of ceteareth-10 (Brij 56®, nonionic emulsifi er, Sigma-Aldrich), 0.05% w/w trietha- nolamine (TEA, Riedel-de Haen, RdH Laborchemikalien GmbH & Co. KG, Seelze, Germany), 0.03% w/w carbopol 940 (BASF, Ludwigshafen, Germany), and deionized water. The oil phase contained the self-bodying agent glyceryl monostearate (GMS, co- emulsifi er, Huls AG, Witten, Germany), 0.03% w/w α-tocopherol (Cognis, Düsseldorf, Germany), 41.6% w/w isopropyl myristate (IPM, Cogins), and 0.02% w/w A. incisus extract. The concentration of the extract used was about six times the IC50 value of the melanogenesis-inhibitory activity of the extract, according to the above-mentioned study. For preparation processes, ceteareth-10 was dissolved in the required amount of deionized water and preheated to 75°C before being added to the oil phase. In the oil phase, the A. incisus extract was fi rst dissolved in IPM. The obtained solution was then mixed with other compositions of the oil phase, followed by heating to 70°C. Afterwards, the two phases were homogeneously mixed using an Ultra-Turrax T25 homogenizer (Janke & Kunkel IKA Labortechnik, Staufen, Germany) for 5 min at a rate of 8,000 rpm to form coarse o/w emulsions. The resultant emulsions were heated to a specifi c phase-inversion temperature (90°C), according to our preliminary study, at which point w/o emulsions were formed. Then, the hot emulsions were cooled rapidly in an ice bath without a high input of mechanical energy, resulting in fi nely dispersed o/w emulsions. Carbopol 940