JOURNAL OF COSMETIC SCIENCE 180 To examine the tyrosinase x-ray crystal structure as a receptor, fi ve tyrosinase structures of Bacillus megaterium and Streptomyces castaneoglobisporus (protein data bank [PDB] ID: 3NM8, 3NQ1, 1WXC, 2ZWD, 3AWS) were downloaded from the RCSB PDB, and the protein se- quences were compared. A homology modeling analysis was performed using the modeler (University of California, San Francisco, CA) focusing on the locations with similar protein sequences. Docking was designed considering the structure of human tyrosinase, which was created by homology modeling using AutoDock Tools (The Scripps Research Institute, La Jolla, CA). Through this program, docking of ligand and target protein was simulated using 3D grid box. The structures of the whitening derivative molecules were generated using the software Cornica (Molecular Networks GmbH, Erlangen, Germany). The process of docking was simulated using the following steps (the same procedure was performed for studying ar- butin, another tyrosinase inhibitor): • Converting 2D structures of whitening derivatives and arbutin into 3D structures. • Calculating polarities and eliminating water molecules. • Attaching hydrogen molecules to polar molecules and identifying the location where the ligand of the protein attaches. • Proceeding with the simulation by using the AutoDock tools. RESULTS INHIBITORY EFFECTS OF CYCLOHEXANE DIESTER DERIVATIVES (1A–1O) AND BENZENE DIESTER DERIVATIVES (2A–2O) ON MUSHROOM TYROSINASE ACTIVITY The mushroom tyrosinase inhibitory activities of cyclohexane diester and benzene diester de- rivatives were determined using L-DOPA as a substrate. These diester compounds were as- sayed at a variety of concentrations. However, all compounds showed no inhibitory activities (data not shown). STRUCTURE–CYTOTOXICITY RELATIONSHIPS OF CYCLOHEXANE DIESTER DERIVATIVES (1A–1O) AND BENZENE DIESTER DERIVATIVES (2A–2O) ON B16F10 CELLS In an endeavor to identify the structure–cytotoxicity relationships between cyclohexane dies- ter derivatives and benzene diester derivatives in the B16F10 cells, the levels of cytotoxicity were observed in various diester locations (i.e., 1,2-, 1,3-, 1,4-) and by the changing chain length of the acyl group. The cytotoxicity test results in Table II suggest that, at the concentration of 500 μM, the cy- totoxicity of 1,2-cyclohexane diester derivatives and 1,3-cyclohexane diester derivatives in- creased as the carbon number of the side chain went up. On the other hand, the cytotoxicity of 1,4-cyclohexane diester derivatives tended to decline with the carbon number of the side chain on the rise. In the case of 1,2-benzene diester derivatives, their cytotoxicity decreased with the carbon number of the side chain going up, but then it started increasing from the time when the carbon number of the side chain reached 10 onward. For 1,3-benzene diester derivatives and 1,4-benzene diester derivatives, their cytotoxicity grew lower with an increase in the carbon number of the side chain.

EFFECT OF CYCLOHEXANE AND BENZENE DIESTER ON MELANOGENESIS 181 The results of cytotoxicity tests on octanoyl and 2-ethylhexanoyl—conducted to identify the impact of the structural isomer of the side chain on cytotoxicity—indicate that the levels of cytotoxicity were lower in the branch type than in the linear type across all derivatives except the 1,2-cyclohexane diester derivatives. When the side chain was the same yet the location of diesters (i.e., 1,2-, 1,3-, 1,4-) varied, the 1,3-diester derivatives demonstrated a lower level of cytotoxicity than the 1,2-diester and 1,4-diester derivatives. Table II Viabilitya of Cyclohexane Diester Derivatives (1a–1o) and Benzene Diester Derivatives (2a–2o) on B16F10 Cells Compounds Viability (%) 25 μM 50 μM 125 μM 250 μM 500 μM 1a 100.0 ± 1.4 96.9 ± 1.9 89.3 ± 1.5 84.2 ± 1.8 70.8 ± 2.1 1b 94.8 ± 1.5 93.0 ± 2.5 86.5 ± 1.8 73.1 ± 1.9 65.0 ± 2.3 1c 100.0 ± 1.8 95.3 ± 1.4 79.6 ± 1.9 66.8 ± 2.5 54.7 ± 1.8 1d 100.0 ± 2.6 94.3 ± 1.6 77.9 ± 0.6 54.5 ± 2.3 40.7 ± 1.8 1e 98.9 ± 1.7 90.7 ± 1.8 73.8 ± 2.6 58.4 ± 2.3 40.1 ± 2.8 1f 100.0 ± 0.4 100.0 ± 3.1 100.0 ± 3.3 94.1 ± 2.2 68.1 ± 3.2 1g 100.0 ± 2.7 100.0 ± 2.4 100.0 ± 1.3 96.9 ± 1.0 74.1 ± 2.7 1h 100.0 ± 2.5 98.1 ± 2.4 91.8 ± 1.4 72.7 ± 3.1 41.0 ± 2.3 1i 100.0 ± 2.1 100.0 ± 2.6 99.3 ± 2.9 99.2 ± 1.6 98.5 ± 1.3 1j 100.0 ± 2.2 100.0 ± 1.1 100.0 ± 1.9 96.1 ± 2.4 96.0 ± 1.8 1k 75.2 ± 1.5 62.1 ± 2.7 57.9 ± 1.6 50.7 ± 1.6 40.2 ± 1.4 1l 91.9 ± 2.1 89.7 ± 0.2 88.8 ± 0.8 85.7 ± 0.9 61.8 ± 1.3 1m 100.0 ± 2.0 100.0 ± 3.1 89.5 ± 2.7 89.5 ± 2.3 79.1 ± 2.4 1n 99.3 ± 3.5 96.8 ± 2.1 95.0 ± 2.2 89.9 ± 3.7 82.4 ± 2.2 1o 93.0 ± 1.2 90.6 ± 0.4 90.0 ± 1.3 87.4 ± 1.7 84.9 ± 1.9 2a 75.6 ± 1.9 64.9 ± 2.4 59.6 ± 3.4 44.3 ± 2.6 7.6 ± 1.5 2b 75.8 ± 0.5 73.1 ± 2.5 59.3 ± 2.8 46.6 ± 2.6 40.2 ± 2.8 2c 93.1 ± 3.0 77.8 ± 3.0 72.3 ± 2.3 61.2 ± 1.8 57.9 ± 2.2 2d 61.3 ± 1.6 58.6 ± 1.3 56.0 ± 2.3 53.0 ± 0.7 48.0 ± 2.7 2e 100.0 ± 2.1 100.0 ± 2.2 100.0 ± 2.5 100.0 ± 2.3 74.3 ± 1.6 2f 92.7 ± 1.0 88.3 ± 1.6 76.8 ± 2.2 65.9 ± 1.8 59.8 ± 2.5 2g 100.0 ± 0.4 98.5 ± 0.6 86.5 ± 1.0 80.6 ± 2.3 62.5 ± 2.3 2h 100.0 ± 2.7 97.4 ± 1.2 94.1 ± 2.1 78.4 ± 2.7 66.9 ± 0.4 2i 100.0 ± 1.4 100.0 ± 2.0 100.0 ± 1.8 99.7 ± 2.4 98.9 ± 2.9 2j 100.0 ± 2.0 100.0 ± 2.1 100.0 ± 2.6 97.7 ± 2.3 94.2 ± 1.2 2k 66.3 ± 1.4 57.5 ± 1.8 34.4 ± 2.3 3.3 ± 0.3 3.2 ± 0.1 2l 49.4 ± 2.6 47.1 ± 2.9 36.2 ± 1.4 21.6 ± 1.5 7.5 ± 0.1 2m 41.5 ± 0.9 33.9 ± 2.1 23.4 ± 0.8 19.8 ± 1.1 18.4 ± 1.1 2n 80.5 ±1.3 78.4 ± 1.4 77.1 ± 0.5 76.4 ± 1.0 74.2 ± 1.5 2o 69.2 ± 1.5 59.3 ± 3.6 58.6 ± 0.5 56.6 ± 1.9 52.9 ± 0.9 a Values are means of three experiments.