INHIBITORY EFFECT OF C. OFFICINALIS ON MELANOGENESIS 513 0.8 0. 7 .-. E 0.6 C LO 0. 5 0 V 0.4 ._, 0 0.3 0 0.2 0. 1 0.0 DOPA + + + + + + + CO extract 0 0 5 10 50 5 10 50 0 0 a 0 (mg/ml) cornuside 0 0 0 0 0 0 0 a 10 50 10 50 (µM) Gas N2 02 02 02 02 02 02 02 02 02 02 02 ambient condition Figure 2. The inhibitory effects of C. o/ficinalis extract in auto-oxidation of 1-Dopa solution. 1-Dopa oxidative substrate of the whole experiment was measured at OD 405 nm. Each value is the mean+/-SEM of eight determinations. Values are significantly different from the control (C. officinalis extract, 0 mg/ml, 02 ambient condition). ***p 0.01 by Dunnett's multiple comparison. and related compounds in the presence of aldehyde and hydrogen peroxide (H 2 O 2 ) or hydroxyl radical, generated by the Fenton reaction, and determined the relationship between the chemical structures of the flavonoids and their CL intensities and/or radical­ scavenging activities (16). In their study, it was suggested that the intensity of chemi­ luminescence was positively correlated. It will visually reveal more about the area where the radical is located. Therefore, we used bioactive components of C. originalis extract in this visual evaluation system to measure the radical-scavenging activity. The intensities of photons emitted (CL) from caffeic acid, cornuside, and EGCG in this system are shown in Figure 4. The CL intensities of caffeic acid and cornuside were much very stronger than that of EGCG, indicating they would efficiently exchange their excited state energy to photon energy. In this experiment, we examined by measurement of chemiluminescence the inhibitory effect of oxidation by C. officinalis. The mechanism of CL for the purified compounds in C. officinalis extract is unknown in the presence of H 2 O 2 and KHCO3' However, it may be closely related to common CL occurring with singlet oxygen (17). Many compounds emit photons when excited with singlet oxygen. There are several methods to detect reactive oxygen species (ROS), such as DPPH radical-scavenging activity and superoxide scavenging activity. Using those methods,

a) 0.8 0.6 E *** C: 0 0.4 V Q 0 0.2 0 C: e ti ,, .!: G) G) (I) (!) ·u C: "'C - "'C (.) ::J - � "in as "in C: as as (!) .c 0 x u C e (I) ::J L... u w as u (I) � .Q (I) C: ::? 0 3 (I) 0 � rn u E (.) """"" ....I ·crn E e L.. :::t 0 LO E N 0.1 mM b) 0.5 0.4 E C: 0 0.3 Q 0.2 0 0.1 0 C: 0 ti "'C C: (I) � (I) (!) L... ·u C "'C ""O (.) ::J - as "in as "in C: L.. cu as - (!) -e 0 x u C e (I) ::J 0 0 u C: w as (I) � (I) ffl L... ::? Q 3 (I) 0 E (.) B rn ""O 0 """"" ....I ·c(/) 0 E e L... :::t 0 LO E N 0.1 mM Figure 3. The inhibitory effects of C. officinalis extract and its components on melanogenesis in B16 melanoma cells. (a) The vertical axis is melanin content (420 nm). (b) Cell numbers (570 nm) in B16 melanoma cells. Each value is the mean+/-SEM of three determinations. Values are significantly different from the control. **p 0.05, ***p 0.01 by Dunnett's multiple comparison.