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1 Lab. Anim. Res. 2010: 26(1), 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 * 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 1 This study is designed to apply for cosmeceutical ingredients with Sanguisorba officinalis and Stichopus japonicus distributed all over the country. The antioxidizing effect of the ethanol extract of Stichopus japonicus displayed stronger inhibition than the Sanguisorba officinalis ethanol extract and mixture of Sanguisorba officinalis and Stichopus japonicus extract. They used in the experiment as 63.28% with the density of 50 µg/ml, and compared with the ascorbic acid used as positive counterpart. Since the physiological activating ingredient generating the antioxidizing effect was confirmed with the Sanguisorba officinalis and Stichopus japonicus with the experiment results, the possibility for natural antioxidants was implied. Key words: Sanguisorba officinalis, Stichopus japonicus, antioxidant, herb, cosmetic (Received 11 February 2010; Revised version received 9 March 2010; Accepted 12 March 2010) œw y w j jš, w wš w ƒ f y w š. w w ƒ w w y ƒ w z ƒ w y š (Park et al., 2004). k E, flavonoids, phenolic acid, carotenoid w y w w y k y w š. (Kim et al., 1996) l y (Park, 2003), yy(beak et al., 1998), (Bang et al., 1999), w (Lee et al., 2002) w *Corresponding authors: Jeho Song, Division of Beauty Design & Institute for Better Living, Wonkwang University, Shinyoung- Dong, 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 ƒ š š, v, ù, (Park et al., 2009). t(sanguisorba 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 3ƒxk m ƒ š. 7~9 ¼ v. ¼ ƒ 2.5 cm š, e 4, 4. 4ƒx ú ƒ xk 10 (Park et al., 2004 a ). w x w w ƒx,, z m, e (Park et al., 2004 a ), vš wš e w, wš e e w š. s, pomolic acid, k A tannin 17%ù w wš (Nguyen et al., 2008). wr t k y,,,» t w w 8 v š 31

2 32 Min-Ok Lee et al. (Nguyen et al., 2008). t w w³ x v ³ Staphylococcus aureus, Staphylococcus epidemis ³ w w w³ z ƒ w, w y mw y t ƒ y w ((Park et al., 2004 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., 2010). w s s 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 w (Zhang et al., 2010).» w w w š w g w w. ù w w t w y w ù v ƒ û w w w y y w» (Bulgakov et al., 2007). y tannin w w z ƒ w t v w w ü w y y wš w» y t w š, v x mw w» y t y ƒ w m w. h k x w t(sanguisorba officinalis L.) ~5 w w. t ³ w» w ƒ 5~10 g w. w t 3 z w» w dry oven 40 o C, 24 z w. w (Stichopus japonicus) ~5 ¾ û w w ü w. ü w z g e w. g 80% w w ü g ethanol 100 ml: w ü 20 g w. t w 0.01 g 99.9% k (Dae-Jung Co., Siheung, Korea) 1 ml ƒw 65 o C 24 w, homogenizer w g k. þ w x w. w ü w w 0.01 g 99.9% k 1mL ƒw 65 o C 24 w homogenizer w g k. k þ w x w %11)BTTBZ j'sff3bejdbm Ñm Free Radical w Blois (Blois, 1958) xw t w w y sƒw. 96 well plate k 0.2 mm 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma, USA) 1mL k w t, w 2.5, 5, 10, 25, 50 µg/ml ƒƒ 20 µl ƒwš Ÿ k 30 k z microplate Reader (Infinite 200, Tecan Group Ltd., Switzerland) w 517 nm Ÿ d w w. y j» (control) w..55bttbz j$fmmwjbcjmjuz ƒ z s d x w» w MTT assay ww. Clone M-3 s(cloudman S91 melanoma, Mouse, KCLB, Korea) 10% fetal bovine serum (FBS, Gibco Co., USA), 100 µg/ml penicillin ƒ RPMI-1640 w 37 o C, 5% CO 2 w. w Clone M-3 cell 3 Ü 10 5 cells/well 96 well 100 µl wì w, 37 o C, 5% CO 2 incubator 24 w z š PBS 2 z w. PBS 5 µg/ml 250 µg/ml¾ w ƒ well w 72 w. óù» 4 PBS 5 mg/ml MTT [3-(4,5-dimethylthiazol- 2yl)-2,5-diphenyl-2H-tetrazolium bromide] 20 µl ƒ well ƒwš y Ÿ k z 4 37 o C, 5% CO 2 incubator w z wš DMSO 200 µl ƒw 37 o C, CO 2 incubator 1 k z Microplate Reader (Infinite 200, Tecan Group Ltd., Switzerland) w 570 nm Ÿ d w w.

3 Antioxidant activities of Sanguisorba and Stichopus 33 Mean±SD t»w. m w w w» w SPSS system w, x 4 ww w. y w w q ƒ w P 0.05 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, 50±20% w, x» 3 2 w. x» m ö šx (Purina, Korea) w š w w. j l t t š y t x» ( ) 10 x xw w f New Zealand white mö 2 x 24 v l»»» cm ƒ ù w. x ƒƒ 2 (, 2.5 cm 2.5 cm j») w, d z e z, d z e z w x w. v e w tv š x ù v ƒ ü. v 4 v 4 ù š, n z z š jš en s g. z e w. n 24 z s q wš w x w» w ƒ w. v sƒ w t t - t x» w q w. 24 x w z q w 24 (q z) 72 (q z 48 ) y (erythema) ƒv(scar), (edema) w. w q Draize v (Primary skin irritation index, PII) (Draize et al., 1994). h k ¼jk mn t w DPPH t k 2.5 µg/ml 1.67±0.17%, 5 µg/ml 4.54± 0.24%, 10 µg/ml 7.34±0.71%, 25 µg/ml ±1.68%, 50 µg/ml 34.69±1.52% ùkù ƒ ƒw ùkü š, w k 2.5 µg/ml 3.59±0.27%, 5 µg/ml 8.66±1.22%, 10 µg/ml 18.39±1.41%, 25 µg/ml 45.91±5.44%, 50 µg/ml 63.28±6.1% ùkù ƒ ƒw ùkþ. t w k w ƒ 2.5, 5, 10, 25, 50 µg/ml ƒw 2.24±0.23%, 10.81±1.48%, 16.9±1.91%, 28.17±3.01%, 41.6±4.35% ùkü š, t k ùkþ w k û. ascrobic acid w t k 2.5, 5, 10, 25, 50 µg/ml ascrobic acid w û DPPH ùkþš, w k 2.5, 5, 10, 25 µg/ml ascrobic acid w û ùkþ ù 50 µg/ml DPPH ùkþš, t w k w 2.5, 5, 10, 25, 50 µg/ml ascrobic acid w û DPPH ùk þ (Table 1). gð ² k t k w k 5, 10, Table 1. DPPH free radical scavenging activities in various concentrations of Sanguisorba officinalis and Stichopus japonicus extracts Concentration (µg/ml) ESL ESS MESS C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±4.35 Ascorbic acid 50 µg/ml 58.45±0.78 Unit: % C: control ESL: ethanol extract of Sanguisorba officinalis ESS: ethanol extract of Stichopus japonicus MESS: mixture ethanol extract of Sanguisorba officinalis and Stichopus japonicus

4 34 Min-Ok Lee et al. Table 2. Cell viability measured by MTT assay Concentration (µg/ml) ESL ESS MESS C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±4.21 Unit: % C: control ESL: ethanol extract of Sanguisorba officinalis ESS: ethanol extract of Stichopus japonicus MESS: mixture ethanol extract of Sanguisorba officinalis and Stichopus japonicus 25, 50, 100, 250 µg/ml w z 24 w s w, t k 5, 10, 25, 50, 100, 250 µg/ml 93.1±5.71%, 90.22±5.48%, 87.35±4.31%, 85.05±3.97%, 81.6±4.16%, 75.86±1.26% w 5µg/mL 93.1±5.71% ùkü, w k 5, 10, 25, 50, 100, 250 µg/ml 95.4±6.01%, ±4.18%, ±5.46%, ±3.87%, 93.1±3.88%, 83.9± 2.47% w 10 µg/ml ±5.46% ùkü. t w k w 5, 10, 25, 50, 100, 250 µg/ml 97.7±0.72%, 93.67±5.67%, 89.65±4.83%, 85.63±2.49%, 74.13±3.66%, 56.32±4.21% w., t k 5~ 100 µg/ml 80% ùkþ, w k 93% ùkþ, t w k w 5~100 µg/ml 74% (Table 2). j l x z sƒw e z w ù x e z w v. w k t k, t w k w v 3 0 ùkü v x, w k t k, t w k w v 3 t k w k w y ùkû.» w Draize v sƒw, w t v 0.05 sƒ (Table 3). w y t (Lee et al., 2007), (Kim et al., 2009), (Beak and Lee, 2008) w ƒ š. y t y t yw w y, y, v ƒ š (Berne et al., 1994). Table 3. Results of skin reaction of rabbits treated with extracts of Sanguisorba officinalis and Stichopus japonicus Sites Control site Test site Change Erythema & Eschar Edema Erythema & Eschar Edema Phases Intact Abraded Intact Abraded Intact Abraded Intact Abraded Time (h) Animal No Mean score Total score Mean 0.05 Animal No: 1-Root of Sanguisorba officinalis extract. 2-Stichopus japonicus extract, 3-mixture ethanol extract of Sanguisorba officinalis and Stichopus japonicus Erythema was scored as follows: no erythema=0, very slight erythema (barely perceptible)=1, well-defined erythema=2, moderate to severs erythema=3, and severe erythema (beet redness) to slight eschar formation (injuries in depth)=4. Edema formation was scored as follows: no edema=0, very slight edema (barely perceptible)=1, slight edema (edges of area well defined by definite raising)=2, moderate edema (raised approximately 1 mm)=3, and severe edema (raised more than 1 mm and extending beyond area of exposure)=4. PII (Primary irritation index)=total score/4

5 w y y w ù t y, q, w ù k» ƒ yw ew, ƒ š w y r yw œw butylated hydroxyanisole (BHA), butylated hydroxytolune (BHT), tertiary butylhydroquinone (TBHQ) š, w z w ƒ k Cù tocopheral w w š (50 mg/kg/day) w (Branen, 1975) w wš w y š (Satto et al., 1979). w w y, w³, w, w z y š y ƒ y (reactive oxygen species) e(free radical) w x (Saha et al., 2004), ƒ ù ww free radical z ƒ ù y y ƒ w š š (Kim et al., 1996; Oh and Lee, 2003; Saha et al., 2004). w ƒ y ƒ t s r yw, k, flavonoids l e š š (Halliwell et al., 1998) w w y z w y z ùkü š (Lee et al., 2005). t(sanguisorba officinalis L.) (Rosaceae) w w t ù s, jw ý ƒ ù» t (Park et al., 2004 a ).» (sanguisorbienin) k, t k y,,,» t w w 8 v š (Park et al., 2004 b ). w (Stichopus japonicus) v (Phylum Echinodermate) w (Class Holothuroidea) w (Zhang et 2010), w s s(macrophage) š g» w wš e z ƒ, w ü wš k w,, z š m, x{,,, e w š š (Zhang et al., 2010). w y y k ƒ š DPPH y w, y 4 (,, Õ», ƒ ) DPPH radical y 33.33~70.52% š (Shin et al., 2006). Antioxidant activities of Sanguisorba and Stichopus 35 al., t w k w w y z w k t k, t w k w w ùkþ. ascrobic acid w 4.8% w y z ùkü w w y ùkü. ww t w w y z ùkü» y t wz z» w w z ƒ v w,» y t y ƒeƒ q. q 2008 Ÿ w w,. k Bang, M.H., Song, J.C., Lee, S.Y., Park, N.K. and Beak, N.I. (1999) Isolation and structure determination of antioxidants from the root of Paeonia lactiflora. J. Korean Soc. Aric. Chem. Biotechnol. 42(2), Beak, D.J. and Lee, J. C. (2008) A study on the antioxidative and depigmentation activities of the ethanol extract of Saururus herba. Kor. J. Herbology 23(2), Beak, N.I., Kim, Y.H., Ahn, E.M., Bang, M.H., Nam, J.Y. and Kwon, B.M. (1998) Isolation of biologically active compounds from the flower petals of Carthamus tinctorius L. Agricultural Chemistry and Biotechnology. 41(2), 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. European Journal of Dermatology. 4(3), Blois, M.S. (1958) Antioxidant determination by the use of a stable free radical. Nature. 181, Branen, A.L. (1975) Toxicalogy and biochemistry of butylated hydroxyanisole and butylated hydroxytolune. J. Am. Oil Chem. Soc. 52, Bulgakov, A.A., Eliseikina, M.G., Petrova, I.Y., Nazarenko, E.L., Kovalchuk, S.N., Kozhemyako, V.B. and Rasskazov, V.A. (2007) Molecular and biological characterization of a mannan-binding lectin from the holothurian Apostichopus japonicus. Glycobiology. 17(12), Draize, J.H., Woodard, G. and Calvery, H. (1994) Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharm. Exp. Ther. 82, Halliwell, B., Hoult, R.J. and Blake D.R. (1998) Oxidants inflammation, and anti-inflammatory drugs. FASEB J. 2, Kim, C.J., Kang, B.H., Ryo, I.J., Park, D.J., Lee, H.S., Kim, Y.H. and Yoo, I.D. (1996) Screening of biologically active compounds from various weeds. Agricultural Chemistry and Biotechnology. 39(5), Kim, K.Y., Lee, S.J., Bak, E.Y., Jang, G.S. and Hwang, W.G. (2009) Effects of Galla rhois extracts and fractions on antioxidative acitivity and inhibition of melanin synthesis by melanoma cell. J. Kor. Soci. Cosm. 15(3), Lee, J.J., Han, J.U. and Lee, M.R. (2007) Antioxidaive activity of

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