BREADFRUIT EXTRACT AS SKIN LIGHTENER 43 antioxidant activity of the extracts. The obtained results indicate the potential of the new source for skin-lightening application. MATERIALS AND METHODS PLANT MATERIALS The heartwood of A. incisus was collected from May to June of 2005 from Phitsanulok Province, Thailand. The heartwood was chipped in the size of 1 x 1 x 10 cm and exposed to the sun for two days. Then the chipped heartwoods were dried at 50°C for two days by using a hot-air oven and were ground into a powder by using a mill. The obtained powder was kept in a tight container at room temperature before being used. EXTRACTION PROCESS Two solvent systems, diethyl ether (analytical grade, Batch No. 04090204, LabScan Asia Co. Ltd., Bangkok, Thailand) or methanol (HPLC grade, Batch No. 03040021, LabScan Asia Co. Ltd.) were used for preparation of A. incisus extract with a modified method (3). According to our preliminary study, the different ratios between the amount of A. incisus powder and each solvent were selected due to the different capacity of each solvent to provide the highest percent yield of artocarpin in the extracts (8.25 g/1 kg for ether extract, 10.95 g/1 kg for methanol extract). Five-hundred grams of the A. incisus powder was placed in a percolator and then soaked with 800 ml of diethyl ether at room temperature for two days. The solution of A. incisus ether extract was filtered through a woven cloth filter and evaporated to a concentrate under reduced pressure with a vacuum evaporator set at 30°C. For preparation of the methanol extract, one kilogram of A. incisus powder was placed in the glass container and then soaked with 2 1 of methanol. The tight container containing the extracts was shaken at room temperature for one week. The solution of A. incisus extract was filtered and evaporated to a concentrate under reduced pressure with a vacuum evaporator at 50°C. After that, both kinds of extract were dried in a dessiccator. The dried extract was stored at -20°C in a tight amber glass before being used. DETERMINATION OF ARTOCARPIN CONTENT Isocratic high-performance liquid chromatography (HPLC) was used to determine the amount of artocarpin, a major component contained in the A. Incisus's heartwood extract. Artocarpin standard (purified artocarpin) was kindly provided by Dr. Kuniyoshi Shimizu, Faculty of Agriculture, Kyushu University, Japan. The HPLC instrument consisted of an SPD-l0MlOAVP diode array detector and an SCL-lOA central unit (Shimadzu Co., Ltd., Kyoto, Japan). A 5-µm Alltima Cl8 column of 250 x 4.60 mm diameter (Alltech Associates Inc., Illinois) was applied. The effluent consisted of a mixture of methanol:water (85:15). The flow rate of the effluent was 1 ml/min, and the volume of injection was 50 µl. Identification and quantification of the artocarpin was based on a peak area at 282 nm. All the experiments were performed in triplicate.

44 JOURNAL OF COSMETIC SCIENCE DETERMINATION OF TYROSINASE-INHIBITORY ACTIVITY The tyrosinase-inhibitory activity of the ether crude extract was determined by spec­ trophotometry with a modified method (5 ,6). The sample solutions of A. incisus extract or a positive control, kojic acid (analytical grade, Lot No. 08325 34, Sigma-Aldrich, Steinheim, Germany), were prepared by dissolving the crude extract or kojic acid in dimethylsulfoxide (DMSO, analytical grade, Lot No. 0320064, Sigma Chemical Co. Ltd., Missouri). For each concentration of the sample solution, fours well were desig­ nated as A, B, C, and D. Each contained a reaction mixture (180 µl) as follows: (A) 20 µl of 426 units/ml of mushroom tyrosinase (analytical grade, Lot No. 023K7024, Sigma-Aldrich), 140 µl of 20 mM phosphate buffer (pH 6.8), and 20 µl of DMSO (B) 160 µl of 20 mM phosphate buffer (pH 6.8) and 20 µl of DMSO (C) 20 µl of 426 units/ml of mushroom tyrosinase solution, 140 µl of 20 mM phosphate buffer (pH 6.8), and 20 µl of the sample solution (D) 160 µl of 20 mM phosphate buffer (pH 6.8) and 20 µl of the sample solution The mixed solution was incubated at room temperature for 10 min, and then 20 µl of 0.85 mM 3,4-dihydroxyphenylalanine (L-DOPA, analytical grade, Lot No. 023K7024, Sigma-Aldrich) was added to each well. After incubation at 25°C for 20 min, an amount of DOP Achrome produced in each well was measured with an absorbence at 490 nm by using a Spectra Count® microplate reader (Perkin Elmer, Inc., Massachusetts). Tyrosinase-inhibitory activity was calculated by using the following equation: %Tyrosinase inhibition= [(A-B) - (C-D)/(A-B)] x 100 where A = an absorbance of the mixture well (A), B = an absorbence of the mixture well (B), C = an absorbence of the mixture well (C), and D = an absorbence of the mixture well (D). IC 50 , the 50% inhibition of tyrosinase activity was calculated as the concentration of test samples that inhibit 50% of tyrosinase activity under experimental conditions. This study was performed in triplicate. DETERMINATION OF ANTIOXIDANT ACTIVITY We used 2,2-diphenyl-l-picrylhydrazyl (DPPH, analytical grade, Lot No. 083K0830, Sigma-Aldrich) to measure the free-radical scavenging activity of the extract, comparing it to positive controls including butylhydroxytoluene (BHT, analytical grade, Lot No. 78H0689, Merck, Danstadt, Germany) and L-ascorbic acid (ACS reagent, Lot No. 60780, Riedel-deHaen, Seelze, Germany). The degree of DPPH decoloration indicated the scavenging efficiency of the added sample solution. This DPPH assay was performed in triplicate under a modified method (7 ,8). The sample solutions of the tested samples including A. incisus ether extract, BHT, and L-ascorbic acid were prepared by dissolving each of them with DMSO, methanol, and deionized water, respectively. The reaction mixture consisted of 150 µl of DPPH (0.2 mM, stored at -20°C until use) and 75 µl of the sample solution. This sample solution was replaced with methanol, DMSO, or deionized water for use as a blank solution. The mixture was vortex-mixed for 15 sec and left to stand for 30 min. After incubation, the absorbence of the remaining DPPH was measured at a wavelength of 515 nm by using