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지선 운
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* 1 Division of Beauty Design & Institute for Better Living, Wonkwang University, Iksan, Korea 2 Huvet Co. Ltd, Iksan, Korea 3 Korea DNA Valley Co.

1 Lab. Anim. Res. 21: 26(2), Skin Whitening Effects of Sanguisorba officinalis and Stichopus japonicus Min-Ok Lee 1, Hong-Geun Oh 2, Sang-Ho Park 3, Hyun-A Lee 4, Jeong-Dug Sul 5, Jeho Song 1 * and Okjin Kim 4 * 1 Division of Beauty Design & Institute for Better Living, Wonkwang University, Iksan, Korea 2 Huvet Co. Ltd, Iksan, Korea 3 Korea DNA Valley Co. Ltd, Iksan, Korea 4 Center for Animal Resources Development, Wonkwang University, Iksan, Korea 5 College of Sport Science, Chung-Ang University, Anseong, Korea This study is aimed to evaluate skin whitening effects of Sanguisorba officinalis and Stichopus japonicus for cosmeceutical ingredients. The extract of Stichopus japonicus showed 61.78% inhibition of tyrosinase activity, and the mixture of Sanguisorba officinalis extract and Stichopus japonicus extract showed 59.14% inhibition of tyrosinase activity. On the clone M-3 cell meloanocyte, the mixture of Sanguisorba officinalis extract and Stichopus japonicus extract showed remarkable inhibition of melanogenesis. Also, those extracts were not irritable in ocular irritation test. It is concluded from these results that Sanguisorba officinalis and Stichopus japonicus have skin whitening effect. It could be used as natural depigmental material in cosmeceutical ingredients. Key words: Sanguisorba officinalis, Stichopus japonicus, herb, skin whitening, depigment, cosmeceutical ingredient Received 24 February 21; Revised version received 9 March 21; Accepted 27 April 21 s³ š w w š ƒ t, w, w y t x mw v y w š (Park et al., 24b; Cutler, 26; Lintner et al., 29). v w z ƒwš k v w»»ù t x ƒ š (Cutler, 26; Lintner et al., 29). v (skin whitening) w t» y t arbutin ascorbic acid ƒ š (Ikeda and Tsutsumi, 199; Lim et al., 29). ù arbutin ascorbic acid v n s *Corresponding authors: Jeho Song, Division of Beauty Design & Institute for Better Living, Wonkwang University, Shinyoungdong, Iksan , Korea Tel: Fax: sjhao@wku.ac.kr Okjin Kim, Center for Animal Resources Development, Wonkwang University, Shinyoung-dong, Iksan , Korea Tel: Fax: kimoj@wku.ac.kr ü š z w š (Qui et al., 1996).» y t ƒ š w wš w z k w» w y t w z y wš w. t(sanguisorb officinalis L.) (Rosaceae) w w t.»ƒ ¼,» š jƒ 1.5 m. jw ý ƒ ù» t š w k x ó 5~11 ƒ ½l š, ƒ 3 ƒ xk m ƒ š. 7~9 ¼ v. ¼ ƒ 2.5 cm š, e 4, 4. 4ƒx ú ƒ xk 1 (Park et al., 24a). w x w w ƒx,, z m, e (Park et al., 24a), vš wš e w, wš e e w š. 127

2 128 Min-Ok Lee et al. s, pomolic acid, k A tannin 17%ù w wš (Nguyen et al., 28). wr t k y,,,» t w w 8 v š (Nguyen et al., 28). t w w³ x v ³ Staphylococcus aureus, Staphylococcus epidermis ³ w w w³ z ƒ w, w y m w y t ƒ y w (Park et al., 24 b ). w w w w (Stichopus japonicus) v (Phylum Echinodermate) w (Class Holothuroidea) w, w, Ÿ. mx wš sw.» š ù š ù (Zhang et al., 21). w s s k š g» w wš e z ƒ «š, p e t w e x, š w z ƒ, x x»» yw, g p k ƒ w ù wx (Zhang et al., 21).» w w w š w g w w. ù w w t w y w ù v ƒ û w w z y w» (Bulgakov et al., 27). w t w z y wš Tyrosinase y Melanin w wwš y wš x w. h k x w t(sanguisorba officinalis L.) 29 2~5 w w. t ³ w» w ƒ 5~1 g w. w t 3 z w» w dry oven 4 o C, 24 z w. w (Stichopus japonicus) 29 2~5 ¾ û w w ü w. ü w z g e w. g 8% w w ü g ethanol 1 ml: w ü 2 g w. t w.1 g 99.9% k (Dae-Jung Co., Siheung, Korea) 1mL ƒw 65 o C 24 w, homogenizer w g k. þ w x w. w ü w w.1 g 99.9% k 1mL ƒw 65 o C 24 w homogenizer w g k. k þ w x w. 5ZSPTJOBTFm n l Tyrosinase y x Busca (1996) w xw. 96 well plate ƒ well phosphate buffer (ph 6.8) 5, 1,, 1,5, 2, µg/ml w ƒƒ 7 µl š, mushroom tyrosinase (2, unit/mg) 2 µl 1 mm L- DOPA 1 µl ƒw z, 37 C 3 incubationw o microplate Reader (Infinite 2, Tecan Group Ltd., Switzerland) w 475 nm Ÿ d w w..fmbojo j n l Clone M-3 s(cloudman S91 melanoma, Mouse, KCLB, Korea) 1% fetal bovine serum (FBS, Gibco Co., USA), 1 µg/ml penicillin ƒ RPMI-164 w 37 o C, 5% CO 2 w. Cell T75 cm 2 flask (Falcon, USA) k z s t phosphate buffer saline (PBS) z.25% trypsin-edta š incubator 37 o C, 1 z 1,5 rpm, 5 w d trypsin-edta z s. k s 1% FBSƒ sw RPMI ml» w 37 o C, 5% CO 2» w. Melanin d Tsuboi (1998) w xw. Clone M-3 cell 6 well plate cells/well ƒ w z 37 o C, 5% CO 2 incubator 24 w z š PBS wš, PBS 2.5, 5, 1, 25, 5 µg/ml w ƒ well 1 µl ƒwš 72 w. óù z wš 1 PBS 2z w 1 N NaOH 5 µl ƒwš CO 2 incubator 2 g melanin

3 Skin whitening effects of Sanguisorba and Stichopus 129 w 96 well Microplate Reader (Infinite 2, Tecan Group Ltd., Switzerland) w 49 nm q Ÿ d w s w w. Ò 4 New Zealand white m mö( f) ( ) kg 2 w Ÿ w l x 1 y e z x œ w. m mö v x, w w v w w w t w x» ƒ x w sƒƒ w k. 23± 3 o C, 5±2% w, x» 3 2 w. x» m ö šx (Purina, Korea) w š w w. l t t š 25-6y t x» (25) 1 x xw w f New Zealand white mö 2 kw x 24 w z ƒ,, y k w. x ww 1, 2, 3 4 w t w z Ét l f x w z x.1 ml w 1 k g w. 1 PBS w š, 2 t, 3 w, 4 t w yw w. x 25 µg/ml PBS w w. 5 2z x w. x n z 1, 2, 3, 4 7 ƒ yk yk ƒ, y,, t x» (25) t w x n w w q w. x w Ÿ w x z x z z ww x w Ÿ w x ³ w w (Approval No.: WKU9-51). Mean±SD t»w. m w w w» w SPSS system w, x 4 ww w. y w w q ƒ w P.5 w. 5ZSPTJOBTFm n l t k w tyrosinase y z x Table 1. t k 5 µg/ml 17.48±2.56%, 1, µg/ml 3.6±4.81%, 1,5 µg/ml 49.11±3.67%, 2, µg/ ml 48.24±1.21% ùkù ƒ ƒw tyrosinase y z. w k 5 µg/ml 32.63±4.11%, 1, µg/ml ±.71%, 1,5 µg/ml 48.87±3.61%, 2,5 µg/ml 61.78±5.89% ùkù ƒ ƒw tyrosinase y z. t w k w ƒ 5, 1,, 1,5, 2, µg/ml ƒw 38.57±.79%, 56.11±4.62%, 54.27± 3.51%, 59.14± 2.68% tyrosinase y z ùk ü š, t k z w k û z (Table 1)..FMBOJO j n l t w Clone M-3 (Cloudman S91 melanoma, Mouse, KCLB, KOREA) 2.5, 5, 1, 25, 5 µg/ml w, t k ƒ 2.5, 5, 1, 25, 5 µg/ml ƒw w melanin w z 34.11±.46%, 36.19±3.48%, 35.41±2.96%, 44.69±.96%, 49.28±1.82 % ùkù melanin w z ƒ w ùkû ù š ƒ w melanin w z ƒ y. w k ƒ 2.5, 5, 1, 25, 5 µg/ml ƒw Table 1. Effects of Sanguisorba officinalis and Stichopus japonicus extracts of on the tyrosinase activity Concentration (mg/ml) ESL ESS MESS ± ± ±.79 1, 3.61± ± ±4.62 1, ± ± ±3.51 2, 48.24± ± ±2.68 Unit: %. C: Control. ESL: Ethanol extract of Sanguisorba officinalis. ESS: Ethanol extract of Stichopus japonicas. MESS: A mixture of Sanguisorba officinalis extract and Stichopus japonicus extract.

4 13 Min-Ok Lee et al. Table 2. Melanin inhibitory activity of Sanguisorba officinalis and Stichopus japonicus extracts in Clone M-3 cells Concentration (mg/ml) ESL ESS MESS C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.87 Unit: %. C: Control. ESL: Ethanol extract of Sanguisorba officinalis. ESS: Ethanol extract of Stichopus japonicas. MESS: A mixture of Sanguisorba officinalis extract and Stichopus japonicus extract. w melanin w z ƒ ƒƒ ±1.76%, 48.23±5.76%, 49.41±4.21%, 47.69±3.75% 51.34±4.11% melanin w z ƒ y. t w k w ƒ 2.5, 5, 1, 25, 5 µg/ml ƒw w melanin w z ƒ 52.9±3.69%, 55.99±2.41%, 58.17± 3.66%, 63.8±3.43%, 68.7±3.87% ùkù t k, w k w melanin w z l w melanin w z ƒ y. x t w k w 5 µg/ml w 68.7±3.87% ƒ w w z ƒ ùkû, w k 5 µg/ml 51.34% ùkü Clone M-3 s w y w (Table 2). l t w y w» w mö w x ww. x x» mw s n w w. w mö x z 7 w x ƒ y. n z 1, 2, 3, 4 7 ƒƒ y, w Table 3 ùkù. n 1, 2, 3, 4 7 t w w z w w p w w. mö w w, q. w» w y t ql, ey ey w y t w w y t w ƒ y w š (Park et al., 24b; Lintner et al., 29). y t y t yw w y, y, v ƒ š š (Berne et al., 1994). yw w w y t ƒ w v w» y t v š, v ƒ š» y t v Table 3. Eye irritation score of New Zealand White male rabbit treated with Sanguisorba officinalis and Stichopus japonicus extracts Group Tissue Scores Days Total Score Cornea A B 5 /8 Iris A 5 /1 Conjunctiva (A+B+C) 2 (++) 2 /2 Cornea A B 5 /8 Iris A 5 /1 Conjunctiva (A+B+C) 2 (++) 2 /2 Cornea A B 5 /8 Iris A 5 /1 Conjunctiva (A+B+C) 2 (++) 2 /2 Cornea A B 5 /8 Iris A 5 /1 Conjunctiva (A+B+C) 2 (++) 2 /2 A: Redness, B: Chemosis, C: Discharge. Group 1: control, 2: treated with Sanguisorba officinalis extract, 3: treated with Stichopus japonicus extract, 4: treated with the mixture of Sanguisorba officinalis extract and Stichopus japonicuss extract. M.O.I.

5 (skin whitening) z y t (Chen et al., 29). Tyrosinase melanine w j w z. x y t z w w y j. tyrosinase wy w (Kang et al., 28). Kim (29) p l p z 94% tyrosinase y šw Kang (28) y 6 k 8% 4.2% tyrosinase y šw. t w k tyrosinase y y w w k, t w k w t k 61.78%, 59.14% 48.24% tyrosinase y y w. v v e ƒ x s s w z w» w w ƒ w (Jeong et al., 28). Park (29) y 5% w z šw, Jeong (28) 55.23%, Chang (27) 83.7% w z šw. t w k w z y w t w k w, w k t k ƒƒ 68.7%, 51.34% 49.28% w z y w. x ¾ t w z w w ƒ. t w z wš tyrosinase y z melanoma cell w z y w. w x mw w ww t w y w. w ww t w k tyrosinase y k z z w q. t w z ùkü y» wz v» y t y ƒe ƒ. q 29 Ÿ w w,. Skin whitening effects of Sanguisorba and Stichopus 131 k Berne, B., Lundin, A. and Malmros, P. E. (1994) Side effects of cosmetics and toletries in relation to use: a retrospective study in a Swedish population. Eur. J. Dermatol. 4(3), Bulgakov, A.A., Eliseikina, M.G., Petrova, I.Y., Nazarenko, E.L., Kovalchuk, S.N., Kozhemyako, V.B. and Rasskazov, V.A. (27) Molecular and biological characterization of a mannan-binding lectin from the holothurian Apostichopus japonicus. Glycobiology 17(12), Busca, R., Bertolotto, C., Ortonne, J.P. and Ballotti, R. (1996) Inhibition of the Phosphatidylinositol 3-Kinase/p7S6-kinase Pathway Induces B16 Melanoma Cell Differentiation. J. Biol. Chem. 271(5), Chang, M.S., Kim, M.H., Yang, W.M., Kim, D.R., Park, E.H., Ko, E.B., Choi, M.J., Kim, H.Y., Oh, J.H., Shim, K.J. and Yoon, J.W. (27). Inhibitory effects of nelumbo nucifera on tyrosinase activity and melanogenesis in Clone M-3 melanocyte cells. Kor. J. Herbology 22(4), Chen, L.G., Chang, W.L., Lee, C.J., Lee, L.T., Shih, C.M. and Wang, C.C. (29) Melanogenesis inhibition by gallotannins from Chinese galls in B16 mouse melanoma cells. Biol. Pharm. Bull. 32(8), Cutler, R.G. (26) Antioxidants, aging and longevity, free radical in biology. Academic Press., 6, Ikeda, T and Tsutsumi, T. (199) Function and depigmental activity of crude drugs. Fragrance J. 6, 56. Jeong, H.S., Han, J.H., Ha, J.H., Kim, Y., Oh, S.H., Kim, S.S., Jeong, M.H., Choi, G.P., Park, U.Y. and Lee, H.Y. (29) Antioxidant activities and skin-whitening effects of nanoencapsulated water extract from Rubus coreanus miquel. The Korean Society of Medicinal Crop Science 17(2), Kang, J.R., Lee, M.K. and Kang, S.M. (28) Anti-oxidant property and tyrosinase inhibition activity of various extracts from plants in composite plants. Kor. J. Med. Crop. Sci. 51(4), Kim, K.Y., Lee, S.J., Bak, E.Y., Jang, G.S. and Hwang, W.G. (29) Effects of Galla rhois extracts and fractions on antioxidative acitivity and inhibition of melanin synthesis by melanoma cell. J. Kor. Soci. Cosm. 15(3), Lim, Y.J., Lee, E.H., Kang, T.H., Ha, S.K., Oh, M.S., Kim, S.M., Yoon, T.J., Kang, C., Park, J.H. and Kim, S.Y. (29) Inhibitory effects of arbutin on melanin biosynthesis of alphamelanocyte stimulating hormone-induced hyperpigmentation in cultured brownish guinea pig skin tissues. Arch. Pharm. Res. 32(3), Lintner, K., Mas-Chamberlin, C., Mondon, P., Peschard, O. and Lamy, L. (29) Cosmeceuticals and active ingredients. Clin. Dermatol. 27(5), Nguyen, T.T., Cho, S.O., Ban, J.Y., Kim, J.Y., Ju, H.S., Koh, S.B., Song, K.S. and Seong, Y.H. (28) Neuroprotective effect of Sanguisorbae radix against oxidative stress-induced brain damage: in vitro and in vivo. Biol. Pharm. Bull. 31(11), Park, K.H., Koh, D., Kim, K., Park, J. and Lim, Y. (24a) Antiallergic activity of a disaccharide isolated from Sanguisorba officinalis. Phytother. Res. 18(8), Park, S.Y., Lee, K.J. and Yi, T.H. (29) Effect of Buddleja officinalis MAXIM, on melanin biosynthesis and anti-oxidative activity. J. Beau. Tricho. 5(1), 1-7. Park, S.G., Kim, S.N., Lee, J.C., Kim, H.S., Kim, Y.J., Lee, B.G. and Jang, I.S. (24b) Anti-aging effect on skin with Jaeum- Dan (JED). Kor. J. Herbology 19(1), Qui, F., Komatsu, K.I., Satio, K.I., Kawasaki, X. and Yao, Y. (1996) Kano Pharmacological properties of traditional medicine. XII. Pharmacokinetic study of mulberroside A and its metabolites

6 132 Min-Ok Lee et al. in rat, Biol. Pharm. Bull. 19(11), Tsuboi, T., Kondoh, H., Hiratsuka, J. and Mishima, Y. (1998) Enhanced melanogenesis induced by tyrosinase gene-transher increases boron-uptake and killing effect of boron neutron capture therapy for amelanotic melanoma. Pigment Cell Res. 11(5), Zhang, Y., Song, S., Song, D., Liang, H. and Wang, W. and Ji, A. (21) Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus. J. Biosci. Bioeng. 19(1),

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105.fm Lab. Anim. Res. 2010: 26(1), 31-36 Antioxidant Activities of Sanguisorba officinalis and Stichopus japonicus Min-Ok Lee 1, Hong-Geun Oh 2, Sang-Ho Park 3, Hyun-A Lee 4, Jeho Song 1 * and Okjin Kim 4 *