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Youngdong University, Yeongdong, Korea 2 Department of Public Health, Keimyung University, Daegu, Korea In this study, we investigated the in vitro antioxidative effects, antimicrobial activities and

1 Lab. Anim. Res. 2010: 26(1), Investigations of Samwhang-sasimtang Extracts on Biological Activities In Vitro and Vivo In-Chul Lee 1, Bae-Hwan Kim 2 * and Mee-Kyung Kim 1 * 1 Senior Industry Cluster Agency, Youngdong University, Yeongdong, Korea 2 Department of Public Health, Keimyung University, Daegu, Korea In this study, we investigated the in vitro antioxidative effects, antimicrobial activities and single oral dose toxicity of the extracts from Samwhang-sasimtang to evaluate its use as a functional ingredient in cosmetics. In the antioxidative effect, the ethanol extract from Samwhang-sasimtang (SSE) had higher antioxidant values of 91.9% at 1,000 µg/ml than that of water extract from Samwhang-sasimtang (SSW, 77.0%) when evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization of SSE was 82.2%, higher than that of the SSW (55.0%) and the antioxidant protection factors (APF) of SSW and SSE were 1.64 and 1.62 at 1,000 µg/ml in concentration, respectively. This study was also undertaken to test the in vitro antimicrobial activity with the extracts of Samwhang-sasimtang. In general, the SSE showed the significant antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis. In single oral dose toxicity study, there were no differences in vivo were observed between control and treated groups in clinical signs, body weight gains, and gross finding. The results indicated that SSE did not show any toxic effects at 10 ml/kg in mice, and the LD 50 of SSE was found to be higher than 10 ml/kg in this experiment. In conclusion, the extracts from Samwhang-sasimtang may act as a natural subsistence for functional cosmetics. Key words: Samwhang-sasimtang, antioxidative activity, antimicrobial activity, single oral dose toxicity (Received 28 January 2010; Revised version received 5 March 2010; Accepted 12 March 2010) x ƒ y w w y, w ƒ y ü ü y (superoxide, nitric oxide, nitrogen dioxide, hydroxyl, peroxynitrite ) w yù w š (Wiseman, 1996). y y ƒ y w y w» w w y y w y g, *Corresponding authors: Bae-Hwan Kim, Department of Public Health, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu, , Korea Tel: Fax: kim9399@kmu.ac.kr Mee-Kyung Kim, Senior Industry Cluster Agency, Youngdong University, 12-1, Seolgye-ri, Yeongdong-eup, Yeongdong-gun, Chungbuk , Korea Tel: Fax: kim5179@youngdong.ac.kr or kim9399@kmu.ac.kr mgr, v, k w w y butylated hydroxyanisol (BHA) butylated hydroxytoluene (BHT) t, y t y w š, w w yy y w y w» w y w š (Masaki et al., 1995). p w w ù š ƒ w z w y, w³,, z w ƒ y w š (Masaki et al., 1995; Ham et al., 1997; Ryoo and Cha, 1998; Oh and Whang, 2003). y k( ) ( ˆ)», w šx, y, x,, ü e t (Oatsuka, 1963). y, y, y w y( ) w («) y ( ) w( ª) x( «) z, y ( ) w ( ) e w 15

2 16 In-Chul Lee et al.». y w( ª) z ƒ y, y wì w, x j z ƒ š x, z z. x, z ƒ». x mw y z g l z, y y y z ƒ š š(suzuki et al., 1991)w w y v w x š x, x x g l w š(suzuki et al., 1991)w. ¾ w w w z w š ù w z w» y t w w ƒ w. w wù y k k w y t» ƒ y w» w w yz w³y w w» w z x wì ww. x w y k ü w w, x ( ) w w z w. y k y(42.8 g), y (28.6 g), y (28.6 g) w 10 ƒw 85 o C 3 y þƒ w e w 3z w. k 70% k 10 ƒw 24 wš, z e w, w 3z w. ƒ, w z þ w w. l w³ x w œ ³ v ³ Staphylococcus aureus KCTC 1621, Staphylococcus epidermidis KCTC 1917 Escherichia coli KCTC 1039 w š, w œw l w. S. aureus tryptic soy broth (DB, USA), S. epidermidis E. coli nutrient broth (DB, USA) ƒƒ w. š» agar ƒw w. ³ slant ƒƒ ³ w 10 ml broth ³ wš ƒ ³ w w, ³ y w. k mn 1,1-diphenyl-2-picrylhydrazyl (DPPH) e d : DPPH radical w e y Blois (1958) xw d w. ƒ 0.5 ml 60 µm DPPH (Sigma, USA) 3 ml š vortexw z 15 ew 517 nm Ÿ d w. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation decolorization d : ABTS (Wako, Japan) d Pellegrin et al. (1999) w d w., 7 mm ABTS 5 ml 140 mm, K 2 S 2 O 8 88 µl œ 14~16 e k z, absolute ethanol 1: nm Ÿ 0.7±0.002ƒ w ABTS solution w. 50 µl ABTS solution 1 ml yww 30 kw z 2.5 jš 734 nm Ÿ d w. Antioxidant protection factor (APF) d : Antioxidant protection factor Andarwulan Shetty (1999) d w. 10 mg β-carotene (Sigma, USA) 50 ml chloroform 1mL evaporator» š 40 o C water bath chloroform k z 20 µl linoleic acid (Sigma, USA), 184 µl Tween ml H 2 O 2 ƒw emulsion š, 5 ml emulsion 100 µl yww vortex 50 o C 30 g þƒ k 470 nm Ÿ d w. k m w³y x disc (Higasi, 2000) w w., sq ³ 100 µl w ³ w, ³ disc paper (φ 8 mm) š 0.45 µm membrane filter ³w 50 µl jš, k z ƒ ³ 24 w disc clear zone y w wy w. ºm Ð l x : x w 5 ICR SPF (z, )

3 w 1» w kw x w. y 23±3 o C, 50±10%, y» z 15z/, 150~300 Lux,» 12 ( 8 ~ z 8 ) w. x f 5 ƒƒ w w x» (Purina Korea) g. Biological activity and oral toxicity of Samwhang-sasimtang 17 n : x x y k k x mw, (10% k ) 1% n w w 10 ml/kg n w ƒ 5 n w, 10% k 10 ml/kg w ƒ 5 n w. n 16 k z, n w n» 10 ml/kg 1z n w. x w s³ f 20.88±0.58 g, f 20.21± 0.59 g. w : x w n n z 6 w, n ú l 14 ¾ 1 1z k y, x w. w x x w y k k n, n z 1, 3, 7, 14 d w. x x l k z w xe w k ü» w. x 3z w s³ t w zn x š 1 xw» probit w LD 50 w w. x y student s t-test w w. m k mn x y k (10, 50, 100, 500, 1,000 µg/ml) 1,1-diphenyl-2-picrylhydrazyl (DPPH) w e y d w k ƒw w ùkü, 500~1,000 µg/ml Figure 1. Antioxidative effect of Samwhang-sasimtang extracts on DPPH radical scavenging activity. SSW, Samwhangsasimtang water extracts; SSE, Samwhang-sasimtang ethanol extracts. Data shown are mean values with SD (n=3). The value of each group statistically significant as compared with control (*P<0.05). Figure 2. Antioxidative effect of Samwhang-sasimtang extracts measured by ABTS radical cation decolorization. SSW, Samwhang-sasimtang water extracts; SSE, Samwhangsasimtang ethanol extracts. Data shown are mean values with SD (n=3). The value of each group statistically significant as compared with control (*P<0.05). 75.6~77.0%, k 91.8~91.9% ù kü (Figure 1). 2,2'-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) Figure 2 1,000 µg/ml k 82.2% w y ùkü 55.0% û w y ùkü. β-carotene linoleic acid emulsion ƒw y k w w y d w, k ƒw w ùkü, 10~1,000 µg/ ml antioxidant protection factor (APF)ƒ 1.20~ ~1.62 w w ùkü (Table 1).

4 18 In-Chul Lee et al. Table 1. Antioxidative effect of Samwhang-sasimtang extracts by antioxidant protection factor Sample Concentration (µg/ml) ,000 BHT 1.55±0.08 a 1.67±0.07 a 1.78±0.11 ab 1.78±0.08 ab 1.79±0.07 ab SSW 1.20±0.11 a 1.24±0.13 a 1.42±0.09 ab 1.53±0.05 ab 1.64±0.05 c SSE 1.21±0.01 a 1.27±0.07 a 1.40±0.08 ab 1.56±0.01 bc 1.62±0.02 bc BHT, butylated hydroxytoluene; SSW, Samwhang-sasimtang water extracts; SSE, Samwhang-sasimtang ethanol extracts. a,b Values are mean±standard deviations of triplicate determinations, different superscripts within a same row are significant differences at P<0.05. k m Disc diffusion method w y k Table 2. Antimicrobial activities of Samwhang-sasimtang extracts against several microorganisms Strains Staphylococcus aureus Staphylococcus epidermidis Escherichia coli Concentration (mg/8 mm paper disc) Clear zone on plate (mm) 1 SSW SSE SSW, Samwhang-sasimtang water extracts; SSE, Samwhangsasimtang ethanol extracts. 1 Diameter. 2 No inhibitory zone was formed. w³ z mw» w v ³ S. aureus, S. epidermidis E. coli w wy x w Table 2 y k 3 ³ w w³y ùkü. k S. aureus S. epidermidis w w³y j ùkû S. aureus 1, 2, 4 mg/disc k w ƒƒ 9.0, 12.0, 16.3 mm wy, S. epidermidis k w ƒƒ 9.0, 10.0, 11.0 mm wy y w. ù E. coli w w³y ùkü. ºm Ð y k k n x» x y k k» w (Table 3), p w w ùkù (Table 4). y k k (1%) z n w e 10 ml/kg. Table 3. Mortality of male and female mice orally treated with Samwhaing-sasimtang ethanol extract (1%) Material Sex Injected volume (ml/kg) Hours after treatment Days after treatment Final mortality Male /5 Con 1 Female /5 Male /5 SSE 2 Female /5 1 Con, 10% ethanol. 2 SSE, ethanol extract from Samwhang-sasimtang (1%). Values are expressed as number of dead animals/total animals (n=5). Table 4. Clinical signs of male and female mice orally treated with 10 ml/kg of Samwhaing-sasimtang ethanol extract (1%) Hours after treatment Days after treatment Material Sex Clinical signs Male Normal Con 1 Female Normal Male Normal SSE 2 Female Normal Con, 10% ethanol. 2 SSE, ethanol extract from Samwhang-sasimtang (1%). Values are expressed as number of animals observed clinical signs (n=5).

5 Biological activity and oral toxicity of Samwhang-sasimtang 19 Table 5. Body weights of male and female mice orally treated with 10 ml/kg of Samwhaing-sasimtang ethanol extract (1%) Material Sex Number of animals Days after treatment Male ± ± ± ± ±1.88 Con 1 Female ± ± ± ± ±2.43 Male ± ± ± ± ±1.89 SSE 2 Female ± ± ± ± ± Con, 10% ethanol. 2 SSE, ethanol extract from Samwhang-sasimtang (1%). Values are expressed as means±sd (g). Table 6. Gross findings of necropsy in male and female mice orally treated with 10 ml/kg of Samwhaing-sasimtang ethanol extract (1%) Material Sex Observation Frequency Male NGL 3 5/5 Con 1 Female NGL 5/5 Male NGL 5/5 SSE 2 Female NGL 5/5 1 Con, 10% ethanol. 2 SSE, ethanol extract from Samwhangsasimtang (1%). 3 NGL, no gross lesion. Values are expressed as animal numbers. ºm Ð m á x» mw d y k k n» w x w ƒ w ƒ ùkù (Table 5). x z w y k k n ü» (Table 6). w y w,»k y w y p w y y w w w t, w t,» t. x» w ƒ Ÿ w w š (Kim et al., 2006; Shin, 2007) p, w w ù w w y z w ƒ š (Masaki et al., 1995; Ham et al., 1997; Ryoo and Cha, 1998; Oh and Whang, 2003). ù w z w» y t w w ƒ w. w wù y k w y t» ƒ y w» w w yz w³y w» w z x w. 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3- ethylbenzothiazoline-6- sulfonic acid) (ABTS) antioxidant protection factor (APF) w yy ùkü DPPH w e g, mgr, w BHA w y k flavonoids phenol w w y t š (Jeong et al., 2005). e ü ww y j» r yw e y j ù j w ü y w w (Torel et al., 1986). š APF lipoprotein ey ü w singlet oxygen z w β-carotene linoleic acid emulsion ƒ w w w y d w. x y k w y z d w 1,000 µg/ml k DPPH w e 91.9%, ABTS 82.2% w w y ùkü, APF k 1.64, 1.62 w y ùkü. w z y, y y p w y, y z ú t, t, y t z y wš. y k k w y z» w q w ü ù y p y w. v ³ S. aureus, S. epidermidis E. coli w y k w³z d w» w ³ wy x w 3 ³ w w³y ùkü, k S. aureus w w³y 1~4 mg/disc 9.0~16.3 mm wy, S. epidermidis w w³y 9.0~11.0 mm wy ùkü ù E. coli w w³y ùkü

6 20 In-Chul Lee et al.. y k k y t v ³ w w³ ƒ w. w w w ùkú t y t» w» w y w v ƒ ƒ. š n 10 ml/kg w z w y k k n w x x» y k k w š w yù. y k k 10 ml/kg ¾ z x w w, LD 50 w 10 ml/ kg. x y k k in vitro z x 1,000 µg/ml w yz v ³ w w³y ùkü, ICR m z n 10 ml/kg w w w y t» ƒ w ƒ. k Andarwulan, N. and Shetty, K. (1999) Phenolic content in differentiated tissue cultures of untransformed and Agrobacterium transformed roots of anise (Pimpinella anisum L.). J. Agric. Food Chem. 47(4), Blois, M.S. (1958) Antioxidant determinaion by the use of stable free radical. Nature 81, Pellegrin, N., Roberta, K., Min, Y. and Catherine, R.E. (1999) Screening of dietary carotenoid and carotenoid-rich fruit extracts for antioxidant activities applying 2,2'-azino-bis(3- ethylenebenzothiazoline-6-sulfonic acid) radical cation decolorization assay. In Method in Enzymology, 299, pp , Academic Press, New York and London. Ham, S.S., Oh, D.H., Hong, J.K. and Lee, J.H. (1997) Antimutagenic effects of juices from edible Korean wild herbs. J. Food Sci. Nutr. 2(2), Higashi, G.S. (2000) Appraisement of antioxidative activity from vegetables. Jpn. J. Food Ind. 57(1), Jeong, J.H., Wee, J.J., Shin, J.Y., Cho, J.H. and Jung, D.H. (2005) Antioxidative effect of crude saponin fraction prepared from culture product of basidiomycota cultured with fresh ginseng as substrate. Kor. J. Food Sci. Technol. 37(1), Kim, H.J., Lim, H.W., Choi, S.W. and Yoon, C.S. (2006) Antimicrobial effect of ethanol extract of Dryopteris crassirhizoma nakai on Propionibacterium acnes. J. Soc. Cosmet. Scientists Kor. 32(3), Masaki, H., Sakaki, S., Atsumi, T. and Sakurai, H. (1995) Activeoxygen scavenging activity of plants extracts. Biol. Pharm. Bull. 18(1), Oatsuka, K. (1963) Practical treatment of herbal medicine for various symptoms, pp. 17, 453, 653, 679, Nanjantou, Tokyo, Japan. Oh, M.H. and Whang, H.J. (2003) Chemical composition of server herb plants. Kor. J. Food Sci. Techol. 35(1), 1-6. Ryoo, J.W. and Cha, B.C. (1998) Mineral content and antioxidative activity in some herb plants. Kor. J. Med. Crop. Sci. 6(1), Shin, H.J. (2007) A trend in research and development of natural gardenia pigments. Kor. J. Biotechnol. Bioeng. 22(5), Suzuki, Y., Inoue, T. and Fukuda, H. (1991) The Japenese pharmacopoeia, 12th ed., pp. D-116, D-132, D-576, Hirokawa Shyoudden, Tokyo, Japan. Torel, J., Gillard, J. and Gillard, P. (1986) Antioxidant activity of flavonoids and reactivity with peroxy radical. Phytochemistry 25, Wiseman, H. (1996) Dietary influences on membrane function: Important in protection against oxidative damage and disease. J. Nutr. Biochem. 7(1), 2-6.

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16(1)-3(국문)(p.40-45).fm w wz 16«1y Kor. J. Clin. Pharm., Vol. 16, No. 1. 2006 x w$btf3fqpsu'psn û w m w Department of Statistics, Chonnam National University Eunsik Park College of Natural Sciences, Chonnam National University