JOURNAL OF COSMETIC SCIENCE 2 INTRODUCTION Melasma is a common acquired hyperpigmentary disorder characterized by dark patches or macules located on the cheeks, forehead, upper lip, chin, and neck. Pigmentation is pre- dominantly found in females, accounting for 90% of all cases (1,2). It appears in all racial types, but it occurs more frequently in persons with darker complexions (Fitzpatrick’s skin type IV) (3,4), especially those living in high-ultraviolet-radiation areas, including Thailand. Although melasma is a disfi guring skin condition that worsens with sunlight, it is considered to be a cosmetic problem, as there is no pain or other associated symptoms. Nowadays, safe and effective components extracted from natural sources have been re- ported for their potential application in improving hyperpigmentation disorders. These extracts are mostly free from harmful side effects. For this reason, there is an increasing interest in fi nding natural extracts for application in personal care products and cosmetics. Our previous study revealed that the extract from heartwood of Artocarpus incisus (bread- fruit or Sa-kae in Thai) provided tyrosinase inhibitory and anti-oxidation activities (5). Additionally, it was found that artocarpin, the major component of A. incisus extract, exhibited skin lightening effects on the UVB-induced hyperpigmented dorsal skin of brown- ish guinea pigs (6,7). These fi ndings indicated the extract from A. incisus heartwood to be a potential source of depigmenting agents and that its site of action is in the substratum, in particular within the epidermis and hair follicles. Therefore, to increase the effi cacy of the extract, the tools for increasing penetration of the extract through skin are needed. As we know, delivery systems play an important role in the fi elds of cosmetics and dermophar- maceuticals. The key aspects of the delivery systems are increasing and/or improving penetra- tion and effi cacy, controlling delivery, separating incompatible actives, prolonging shelf life, and/or decreasing the degradation of active compounds. Nanoemulsions have increasingly become one of the most popular delivery systems. Nanoemulsions can be defi ned as oil- in-water (o/w) emulsions, with mean droplet diameters ranging from 50 to 1,000 nm (8). They can be “transparent” (mean droplet size 200 nm) or “milky” (mean droplet size ≈ 500 nm) (9,10). Nanoemulsions would infl uence the transport properties of the active ingredients since their compositions such as emulsifi ers and co-emulsifi ers can enhance the skin penetra- tion of active ingredients and increase their concentrations in the skin. Additionally, due to smaller particle sizes, they offer higher stability against creaming or sedimentation as com- pared to macroemulsions (10). Furthermore, nanoemulsions are more suitable for the trans- portation of lipophilic compounds, according to the lipophilic interior of nanoemulsions. Therefore, in the present study, the natural extract from heartwood of A. incisus was for- mulated into various nanoemulsion formulations. An in vivo study of UVB-induced hyper- pigmentation in C57BL/6 mice was performed to investigate visible results of the formulated nanoemulsion in application for the purpose of depigmenting. The results from this study showed that a nanoemulsion containing heartwood extract from A. incisus could remarkably improve the hyperpigmented lesion on the dorsal skin of C57BL/6 mice. MATERIALS AND METHODS PREPARATION OF HEARTWOOD EXTRACT OF A. INCISUS The heartwood of A. incisus was collected in July 2007 from Phitsanulok Province, Thai- land. To prepare the extract, the heartwood portion was chipped, exposed to the sun, and

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,