INHIBITORY EFFECT OF C. OFFICINALIS ON MELANOGENESIS 511 RESULTS AND DISCUSSION Two C. officinalis extracts were prepared, one with hot water and the other with 50% ethanol (EtOH). The antioxdative activities of extracts were determined by the DPPH radical-scavenging method. The extracts were subjected to HP-20, silica gel, and ODS column chromatography, and then to ODS HPLC to purify cornuside (11), together with caffeic acid (12), loganin (13), sweroside (14,15), and morroniside acetate. Caffeic acid and cornuside, strong anti-oxidants, were isolated, respectively, from the hot water extract and the 50% EtOH extract. DPPH radical scavenging activities of cornuside, caffeic acid, loganin, sweroside, and morroniside acetate are shown in Table I. The activities of caffeic acid and cornuside are quite different from those of the other compounds. Major components, loganin, swero­ side, and morroniside, in C. officinalis do not show the potent radical scavenging of DPPH as does caffeic acid and cornuside. We have confirmed that among these ingre­ dients, caffeic acid and cornuside are the main components as far as DPPH radical­ scavenging activity is concerned. Skin forms melanin in order to protect its tissues from UV rays, and excessive exposure to UV rays causes freckles. UV-induced pigmentation influences melanogenesis by melanocyte-stimulating factors, such as iNOS (9). We evaluated the inhibitory effect of DVB-induced skin pigmentation in C. officinalis extract through its radical-scavenging activity. In addition, the melanin synthesis pathway in melanocytosis is an oxidative reaction, and this polymerization reaction continues automatically in the presence of free radicals. Therefore, it is understood that improvement of DVB-induced skin pigmen­ tation by C. officinalis extract is due to its potent free-radical-scavenging activity. We used arbutin as a positive control to evaluate its influence on UV-induced skin pig­ mentation. We conducted the following experiment to evaluate the inhibitory effect of C. officinalis extract on DVB-induced skin pigmentation. The dorsal skin of guinea pigs (N = 5) was irradiated with a constant level· of UVB and C. officinalis extract applied during two weeks. Dorsal skin lightness was compared before and after this experiment. The DVB-induced pigmentation in brownish guinea pigs treated with C. officinalis extract Table I DPPH Radical Scavenging Activity of Components in C. officinalis Extract and component Hot water extract EtOH extract Caffeic acid Loganin Sweroside Morroniside acetate Cornuside Ascorbic acid Each value is the mean of three determinations. 0.1 mg/ml 21.3 28.4 21.0 16.6 13.2 1.1 12.0 79.2 Inhibition rate (%) 1.0 mg/ml 83.7 97.5 97.0 30.7 10.1 1.8 96.4 97.0

512 JOURNAL OF COSMETIC SCIENCE (100 mg/ml) decreased significantly in comparison with the control, and the degree of pigmentation was equivalent to that of skin treated with 30 mg/ml of arbutin (Figure 1). It is well known that the two major melanin types, eumelanin and pheomelanin, are synthesized from tyrosine by a continued oxidative process (10). Therefore, we evaluated the free-radical-scavenging activity in C. officinalis extract and its components, since there was a significant improvement in pigmentation in an in vivo test. In general, to generate dopaquinone in vitro from 1-Dopa takes about one week. We used an optical density (OD) value at 405 nm as an indicator of melanogenesis inhibition in vitro. The inhibitory effect of C. officinalis extract on auto-oxidation of 1-Dopa in aqueous solution is shown in Figure 2. As expected, the OD value for 1-Dopa aqueous solution was higher in an ambient atmosphere than that in an N2 atmosphere. Addition of C. officinalis extract or cornuside tended to decrease the OD in a dose-dependent manner. To evaluate the effect of C. officinalis extract and its components on melanocytes, we used cultured B16 melanoma cells. The inhibitory effect of C. officinalis extract (25 µg/ml) on mela­ nogenesis judged by the melanin content was greater than that of arbutin (0.1 mM). The melanin content in B 16 melanoma cells treated with caffeic acid (0.1 mM), loganin (0.1 mM), or cornuside (0.1 mM) diminished significantly in comparison with the control, and the inhibitory effects of loganin (0.1 mM p 0.01) and cornuside (0.1 mM p 0.01) were found to be greater than that of arbutin as shown in Figure 3a). C. officinalis extract and its components did not show cytotoxicity or affect cell proliferation (Figure 36). The values obtained for EGCG with galloyl groups did not show significant differences from the control. We compared the radical-scavenging activity of (-)-epigallocatechingallate (EGCG) with other radical-scavenging activity of the purified components caffeic acid and cor­ nuside in C. officinalis. Yoshiki et al. measured the chemiluminescence (CL) of flavonoids 12 10 Q) :::, 8 6 l 4 2 0 a C: a 0 0 (_) *** C: :::, -e C'O Figure 1. The inhibitory effects of C. officinalis extract on DVB-induced pigmentation in brownish guinea pig (N = 5). Each delta L* value is the mean+/-SEM of ten determinations after UVB irradiation for two weeks. C. officinalis extract: (100 mg/ml) and arbutin: (30 mg/ml) were applied twice a day for two weeks.