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1 J. Ginseng Res. Vol. 30, No., 8-87 (006) y r w z yᜠÁÁ½Á *ÁÁ # w t gw tw ( , ) Comparison of Phenolic Compounds Contents between White and Red ginseng and Their Inhibitory Effect on Melanin Biosynthesis Eun Young Hwang, Yeon Hee Kong, Young Chul Lee, Young Chan Kim, Kyung Mi Yoo*, Youn Ock Jo and Sang Yoon Choi # Korea Food Research Institute, Songnam, Korea *Department of Food Science and Technology, NYSAES, Cornell University, Geneva, NY, USA (Received June 7, 006; Accepted June 0, 006) Abstract : Quantitative difference in five phenolic acids between white and red ginsengs was measured in this study. As the results, white ginseng has higher contents of cinnamic acid, quercetin and p-coumaric acid than red ginseng. Maltol was mainly included in red ginseng. These five compounds were recently reported to have tyrosinase inhibitory effects. These reports led us to investigate the de-pigmenting effect of ginseng products. In our examination of effect on tyrosinase activity, UV-protection and melanin production in melan-a cells, ethylacetate fraction of white ginseng extract and cinnamic acid showed potent de-pigmenting properties. The results indicated that white ginseng might be useful as skin whitening material and cinnamic acid proved to be one of active ingredient. Key words : ginseng, phenolic compound, melanin, pigment š w Panax ginseng C. A. Meyer ew 4~6 w w. 1) ƒœ ew y» z w w. ) š w, šx,», wy š y s protopanaxdiol, protopanaxtriol oleanolic acid ginsenosideƒ yw ƒ x. ù s y w w s w ƒ y w # w (y) ; (q) ( ) sychoi@kfri.re.kr š. s wy ù polyacetylene w z x w z, wy zƒ ù phenolic compounds. 3-7) p, phenolic compound w w w šƒ. 8) r y z y l salicylic acid, vanillic acid, p-coumaric acidƒ l ferulic acid, caffeic acid, gentisic acid, p-hydroxybenzoic acidƒ šš, 9,10) y y w y l ƒ zl maltol. y r cinnamic acid, protocatechuic acid, syringic acid, esculetin, 11,1) esculetin, cinnamic acid, p-coumaric acid, quercetin, maltol w w z tyrosinase y š ) y w phenolic 8

2 Vol. 30, No. (006) y r w z 83 compound cinnamic acid, p-coumaric acid, esculetin, maltol, quercetin w w w š, m y w ethylacetate z y w šw. x 005 x v w wš y KT&G w y w w. z w z 80% k/ yw w 1 3 z w q w z w w. ethyl acetate ƒ z H O ƒ z ù ethyl acetate z w r y d w. w 00 nm~500 nm dw. 18) s melan-a s 10% fetal bovine serum 1% penicillin-streptomycin, 00 nm phorbol-1 myristate 13-acetateƒ w RPMI 1640 w 37 o C, 5% CO w. 19) 4 well plate cells/well s wš s w 4 w. Well 990 µl 10 µl 3 (solvent: 50% propylenen glycol, 30% EtOH, 0% H O) r r Jasco Co. (Japan) HPLC w. Bondpack C18 column (4 um, mm) w % acetic acidƒ w water ( A) 0.5% acetic acidƒ w 50% acetonitrile ( B) gradient w. 0.8 ml/ min, column 40 o C w 80 nm dw. 10 mg/ml k z 0.45 µm syringe filter (Millipore) w w. 3 z xw s³e wš t co-injection w peaks e yw. Tyrosinase y 67 mm phosphate buffer (ph 6.8) 8.0 mm L- dopa 10 µl methanol 40 µl 96-well microplate š tyrosinase (15 U/ml) 40 µl ƒw. 37 o C 0 incubationw z dopachrome 49 nm Eilsa Reader w dw. 17) UV-A UV-B d Methanol UV-spectrophoto-meter Fig. 1. HPLC chromatogram of each sample. The peak on 9., 13.6, 19.7, 33.8, 34.9 min were maltol, esculetin, p- coumaric acid, cinnamic acid, quercetin, respectively. Column: bondapak C18, Solvent system: gradient elution of acetonitrile and water, Flow rate: 0.8 ml/min.

3 84 yᜠÁÁ½Á ÁÁ šwz w z 4 w. s d z PBS washingwš well crystal violet (CV 0.1%, 10% EtOH, ù PBS) 00 µl ƒw. 5 incubationw z washing w. EtOH 1 ml ƒw 10 shaking w z 590 nm UV dw. d z PBS wš well 1N NaOH 1ml ƒw z 400 nm UV d w. š r yw w y wš 80% k w w z ethyl acetate ƒ z H O ƒ z ù w ƒƒ Table 1. y esuletin, cinnamic acid, p-coumaric acid, quercetin, maltol w HPLC w w y chromatogram ql quercetin 0.4% w. Maltol y w yp, 8) y w y p maltol w 3.8% w ù. Tyrosinase y w z Tyrosinase» tyrosine L- dopa y w z. w 5 r tyrosinase y e w d w Fig.. Esculetin 00 ppm 46.5% ƒ tyrosinase wy ùkü quercetin cinnamic acidƒ 00 ppm ƒƒ 35%, 33% wy ùkþ. wr, y ethyl acetate z 00 ppm ƒƒ 13.7% 10.5% wy ù z wy ùkü. s e z s melanosome». 0) l v yw w w», Á,, v y w. mouse s melan-a s w ƒƒ (Fig. 1). t maltol, p-coumaric acid, esculetin, cinnamic acid, quercetin k g ƒƒ 9., 13.6, 19.7, 33.8, y z ethylacetate z w dw Table. cinnamic acidƒ 1.15% ƒ w š, Table 1. Yields of ginseng extracts (%). Samples 80% Methanol extract Ethyl acetate fraction extract White ginseng 6.5 ± ± 0.07 Red ginseng 31. ± ± 0.39 Each value represents the % in drying materials Fig.. Inhibitory effects of each sample on tyrosinase activity. Tyrosinase was incubated with test substances and dopa for 0 min. The absorbance was read at 490 nm. Table. Comparison of contents in ethyl acetate fraction of white and red ginseng (%). Samples esculetin cinnamic acid p-coumaric acid quercetin maltol White ginseng < Red ginseng < 3.8

4 Vol. 30, No. (006) y r w z 85 Fig. 3. Effects of each sample on cell growth and melanin production of melan-a cells. Viability and melanin content of vehicle was set to 100%. The cell viabilities and the melanin contents of melan-a cells were determined after 3 days. The data shown representative the mean±s.e. of three experiments. y s e w dw. phenyl thiourea (PTU) 100 µg/ml 8.1% s ù 74.7% gš, kojic acid s y. ethyl acetate z j s 10 µg/ml g 100 µg/ml 8.6%. y ethyl acetate z s wì s y ù. w tyrosinase y cinnamic acidƒ j s 10 µg/ml 17.0%, 100 µg/ml 30.1% g, quercetin 10 µg/ml s 13.1% z ùkþš esculetin Fig. 4. UV absorption of each sample in UV-A and UV-B. UV-A: nm, UV-B: nm

5 86 yᜠÁÁ½Á ÁÁ šwz š 100 µg/ml z ù s g. p-coumaric acid maltol z ùkü (Fig. 3). ww û s y ethyl acetate z ƒ y cinnamic acid ww melan-a s y cinnamic acidƒ w w w q. d v j e w. UV-A (350~370 nm) x e j UV-B (70~90 nm) ƒ Ÿy j x e k. ƒ nm dw ethyl acetate z y ethyl acetate z UV-B p ùkþù ethyl acetate z ƒ. w y cinnamic acidƒ UV-B ƒ p ùk üš, y p maltol UV-B w(fig. 4). ethyl acetate z v s UV-B z»w q y cinnamic acid. y quercetin, cinnamic acid, p-coumaric acid, maltol, esculetin w dw cinnamic acidƒ ƒ w š, y p maltol ƒ w ùkþ. ethyl acetate z melan-a s s y d, UV- d, tyrosinase y d w y ùkü, y cinnamic acidƒ w w w q. wr, maltol ww y ethyl acetate z UV- y ù y ùkü. ethyl acetate z v» ƒ q. w t»š w. x 1. Ha, D.C., Ryu, G.H. : Chemical components of red, white and extruded root ginseng. J. Korea Soc. Food Sci. Nutr. 34(), (005).. Park, S.Y., Jung, I., Jung, T.L. and Park, M.K. : Difference between steaming and decocting ginseng. J. Gingseng Res. 5(1), (001). 3. Park, C.W. : The studies of pharmacology of ginseng. Biochemistry News. 4, (1984). 4. Jin, H.K., Kim, S.H. and Lee, J.K. : Studies of the physiological activity of Korean ginseng. Korean J. Appl. Microbiol. Bioeng. 10, (198). 5. Choi, C., Yoon, S.H., Bae, M.J. and An, B.J. : Protein and amino acid composition of Korean ginseng classified by years. Korean J. Food Sci. 17, 1-4 (1985). 6. Choi, H.J., Zhang, Y.B., An, B.J. and Choi, C. : Identification of biologically active compounds from Panax ginseng C.A. Meyer. Korean J. Food Sci. Technol. 34(3), (00). 7. Kwak, Y.S., Park, J.D. and Yang, J.W. : Present and prospect of red ginseng efficacy research. Food Industry and Nutrition 8(), (003). 8. Yoo, B.S., Lee, H.J. and Byun, S.Y. : Differences in phenolic compounds between Korean ginseng and mountain ginseng. Korean J. Biotechnol. Bioeng. 15(), (000). 9. Han, B.H., Park, M.H., Woo, L.K., Woo, W.S. and Han, Y.N. : Studies on the antioxidant components of Korean ginseng. Korean Biochem. J. 1(1), 33 (1979). 10. Han, B.H., Park, M.H. and Han, Y.N. : Studies on the antioxidant components of Korean ginseng(). Identification of phenolic acid. Arch. Pharm. Res. 4, (1981). 11. Wee, J.J., Park, J.D., Kim, M.W. and Lee, H.J. : Identification of phenolic antioxidant components isolated from Panax ginseng. J. Korean Agric. Chem. Soc. 3(1), 50 (1989). 1. Wee, J.J., Park, J.D., Kim, M.W. and Lee, H.J. : Isolation of phenolic antioxidant components from Panax ginseng. J. Korean Agric. Chem. Soc. 3(1), 44 (1989). 13. Masamoto, Y., Murata, Y., Baba, K., Shimoishi, Y., Taba, M. and Takahata, K. : Inhibitory Effects of Esculetin on Melanin Biosynthesis. Biol. Pharm. Bull. 7(3), 4-45 (004). 14. Kahn, V. and Benshalom, N. : Effects of Maltol on the Oxidation of DL-Dopa, Dopamin, N-Acetyldopamine (NADA), and Norepinephrine by Mushroom Tyrosinase. Pigment Cell

6 Vol. 30, No. (006) y r w z 87 Res. 10, (1997). 15. Lim, J.Y., Ishiguro, K. and Kubo, I. : Tyrosinase Inhibitory p- Coumaric acid from Ginseng Leaves. Phytotherapy Res. 13, (1999). 16. Chun, H.J., Choi, W.H., Baek, S.H. and Woo, W.H. : Effect of Quercetin on Melanogenesis in Melan-a Melanocyte Cells. Kor. J. Pharmacogn. 33(3), (00). 17. Shin, N.H., Ryu, S.Y., Choi, E.J., Kang, S.H., Chang, I.M., Min, K.R. and Kim, Y.S. : Oxyresveratrol as Potent Inhibitor on Dopa Oxidase Activity of Mushroom Tyrosinase. Biochem. Biophys. Res. Commun. 43, (1998). 18. Matsuda, H., Higashio, M., Nakai, Y., Iinuma, M., Kubo, M. and Frank, L. : Studies of Cuticle Drugs from Natural Sources. IV. Inhibitory Effects of Some Arctostaphylos Plants on Melanin Biosynthesis. Biol. Pharm. Bull. 19(1), (1996). 19. Choi, S.Y., Kim, S., Kim, H., Suk, K., Hwang, J.S., Lee, B.G., Kim, A.G. and Kim, S.Y. : (4-Methoxybenzylidene)-(3-methoxyphenyl) amine, a Nitrogen Analog of Stilbene as a Potent Inhibitor of Melanin Production. Chem. Pharm. Bull. 50(4), (00). 0. Bennett, D., Cooper, P. and Hart, I. : A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumor promotor for growth. Int. J. Cancer 39, (1987).