508 Loganin (2) Fr.6 JOURNAL OF COSMETIC SCIENCE Camus officinalis I 50% EtOH extract I HP-20 (MeOH/H2O) Fr.1-5 I ODS (MeOH/H2O) 1. ODS 1. ODS (CH3CN/H2O) (CHJCN/H2O) 2. SiO2 2. ODS (CH3CI/MeOH) (MeOH/H2O) 3.ODS 1. acetylation (CH3CN/H2O) 2. HPLC SiO2 4. HPLC ODS (hexane/EtOAc) (CH3CN/H2O) Morroniside acetate ( 4) 11 Cornuside (5) Scheme 2 1. SiO2 (CH3CN/H2O) 2. ODS (MeOH/H2O) 3. SiO2 (CH3CI/MeOH/H2O) Morroniside acetate was obtained as a colorless crystal (18). The FABMS of morroniside acetate proved the [M+Hr ion at m/z 407, which coincided with the molecular formula C17H 2 6 O 11 . The 1 H-NMR spectrum of morroniside acetate (in CD 3 OD): ol.27 (2H, d, j = 7 .0 Hz), 1.53 (lH, dt, J = 13.5, 3.9 Hz), 1.66 (3H, s), 1.78 (lH, m), 1.19 (lH, m), 3.07 (lH, m), 3.71 (3H, s), 4.14 (lH, dd, J = 2.4, 12.3 Hz), 4.34 (lH, m), 5.00 (lH, m), 5.10 (lH, t,J = 9.7 Hz), 5.23 (lH, t,J = 9.2 Hz), 5.67 (lH, d,J = 8.77 Hz), 6.14 (lH, d, j = 3.2 Hz), 7.43 (lH, s). 13C NMR (CDC13 ): 018.7, 20.5, 20.6, 21.2, 25.9, 30.9, 31.3, 39.1, 51.4, 61.6, 67 .2, 68.4, 71.0, 72.1, 91.2, 94.3, 96.7, 111.1, 152.5, 166.6, 169.2, 169.4, 169.6, 170.1, 170.6. It was thought that this compound without acetylation was morroniside, because it was identified when the isolated compound was morroniside-5-acetate. Caffeic acid (12), loganin (13), sweroside (14,15), and cornuside (11) were identified by comparison with 1 H-, 13C-NMR spectral data. (See Scheme 3 for the chemical structures of these components.) DPPH RADICAL-SCAVENGING ASSAY For this experiment, 0.05 ml of a solution of the compound isolated from the C. officinalis extract was added to 1.95 ml of DPPH (Sigma-Aldrich Co) MeOH solution, 6.0 x 10- 5 M. After the mixture was incubated at room temperature for 30 min, the absorbance at

INHIBITORY EFFECT OF C. OFFICINALIS ON MELANOGENESIS 509 0 OH OH caffeic acid ( 1 ) OH HO 0 OGlu loganin (2) OH morroniside acetate ( 4) HO HO 0 OGlu sweroside (3) cornuside (5) Scheme 3 515 nm was monitored. In a similar way, 0.05 ml of solvent (MeOH or water) was added instead of a test sample as a control. The blank was 1.95 ml of MeOH plus 0.05 ml of solvent (MeOH or water). DPPH radical scavenging activity was measured by compari­ son with the control value. DPPH scavenging rate(%)= [{ABSc -(ABS 5 - ABS B )}/ ABSc] x 100 where ABSc = the absorbance value of the control, ABS5 = the absorbance value of the test sample, and ABSB = the absorbance value of the blank. MEASUREMENT OF CHEMILUMINESCENCE Weak chemiluminescence was observed with compounds from C. officinalis that showed DPPH scavenge activity and EGCG. A half milliliter of cornuside (or caffeic acid) isolated from C. officinalis was mixed with 0.5 ml of 5% (w/w) H2O2 and 0.5 ml of 100 mM KHCO 3 . Using the low-level light detection unit C8801 (Hamamatsu Photonics K.K.), photons from test samples (final cone. 0.05 mM) were measured and integrated for 20 min at room temperature.