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1 J. Korean Soc. Appl. Biol. Chem. 51(3), (2008) Articles glutathione S-transferase y e w 1 Á 2 Á 1 Á½x 1 Á½ 3 Á 2 Áw 1, * 1 ûw tw, 2 ûw tw, 3 ûw yw Induction of Glutathione S-transferase Activity by the Extracts of Glycyrrhiza uralensis Fischer 1 Mi-Young Yoon 1, Kyung-Im Jun 2, Eun-Soon Son 1, Ji-Hyun Kim 1, Yongseong Kim 3, Eunju Park 2 and Hae-Ryong Park 1, * Department of Food Science and Biotechnology, Kyungnam University, Masan , Korea 2 Department of Food and Nutrition, Kyungnam University, Masan , Korea 3 Department of Chemistry, Kyungnam University, Masan , Korea Received May 22, 2008; Accepted July 14, 2008 In the present study, we investigated the glutathione S-transferase (GST) induction of medicinal plants using a cultured PC12 cells. The methanol extracts of Dendrobium nobile Lindley, Schizonepeta tenuifolia Briquet, Glycyrrhiza uralensis Fischer, Paenoia lactiflora Pall were tested. As the result, exposure of PC12 cells to G. uralensis resulted in the significant induction of GST. On the continuous experiment, G. uralensis was extracted with methanol, ethanol, and acetone. Among these extracts, methanol extracts showed the highest GST induction. The methanol extracts were further fractionated with hexane, diethyl ether, ethyl acetate, and water layer according to the degree of polarity. The diethyl ether layer showed the highest exhibited GST induction on PC12 cells. Based on these results suggest that the extracts of G. uralensis can be applicable for the development of a new antioxidant agent. Key words: G. uralensis Fischer, glutathione S-transferase, medicinal plants, PC12 cells y» vw» w ñ v w, v ƒ w y w y w y j w j. w y 1-3) superoxide dismutase(sod), glutathione, glutathione peroxidase, glutathione S-transferase(GST), catalase wy z w, k y wy z w ³x š. ù» ù ƒ Á 4,5) yw w y ƒ w s y p k, ü š sy,, DNA w k, ƒ q w y *Corresponding author Phone: ; Fax: parkhy@kyungnam.ac.kr w, qk ƒ w. 6-10) wy» wù glutathione S-transferase(GST) e (electrophilic metabolite) glutathione(gsh) ww w» j z phase II z w. sü 11-13) w, isozyme x š, ü GSH» w y q w ƒ l s yw w wš š ) wy w GST biomarker š. 17) q wy y ƒ s yw y ƒš š, x, š 18-19) PC12 s w GST y k w. l w GST y y w. (Glycyrrhiza uralensis Fischer) g w glycyrrhizin 6-14% w wš, 20) w 228

2 . glycyrrhizin, glycyrrhetinic acid, liquilitin, isoliquilitin w z w m,, w,., m, w³ 21-23) GST y ¾ w ƒ. l GST y ƒ kw» w wy y ùkü w GST y š w. x. x š(dendrobium nobile Lindley), x (Schizonepeta tenuifolia Briquet), (Glycyrrhiza uralensis Fischer), (Paenoia lactiflora Pall) û () l œ w x w. GST y dw» w GST assay kit Sigma Chemical Co.(St. Louis, MO, USA) t w. s w v w Dulbecco s modified Eagle s medium(dmem), fetal bovine serum(fbs), horse serum(hs) penicillin-streptomycin Gibco-BRL(Grand Island, NT, USA) w, w. z. 5g methanol, ethanol, acetone 100 ml ƒw 3 eg w z, (5C. 110 mm, Advantec, Tokyo Roshi Kaish, Ltd., Tokyo, Japan) w. z»(eyela N-1000, Tokyo Rikakikai Co., Tokyo, Japan) w. Methanol z» w w zw., hexane yw z z w hexaned wš, w diethyl ether, ethyl acetate d zw ƒƒ z g w z. ƒ z 5mg/ml DMSO(dimethyl sulfoxide) w w x w. s. x s rat pheochromocytoma PC12 w sw(kclb) w. DMEM medium 10% FBS, 5% HS 100 unit/ml penicillin, 100 µg/ml streptomycin ƒw wš, 95% ƒ 37 o C, 5% CO 2 incubator(mco-18aic, SANYO, Osaka, Japan) w. sü GST y d. sü GST y glutathione S- transferase assay kit w dw. 24) 6- well plate 2 10 cells/ml 24 5 z, ƒ wš 24 w. š 5,000 rpm w š s 200 µl glutathione S-transferase y e w 229 phosphate buffered saline(pbs) ƒw. s wk z 50 µl 0.5% triton X-100 ƒwš microplate shaker w 300 rpm 10.» GST y d w [Dubecco s PBS, 200 mm glutathione (yx) 100 mm 1-chloro-2,4-dinitrobenzene(CDNB)] 180 µl ƒwš ƒ 20 µl ƒw 340 nm Ÿ dw. GST y µmol/ml/min w w GST y w w GST y ùkü. s xkw y. w PC12 s xkw w 6-well plate cells/well 24 w. methanol, acetone ƒƒ 50 µg/ml wš 24 z, phasecontrast microscope(ts 100-F, Nikon, Tokyo, Japan) ƒ well s xk wš 100 w. LDH d w s y. methanol, ethanol, acetone s e w d w» w lactate dehydrogenase(ldh) release assay w. PC12 s cells/ml z, 100 µl 96-well plate w CO 2 incubator 24 w, 10, 50 µg/ml w s w. 24 w, 96-well plate 50 µl wš, LDH reagent 50 µl ƒw ek z, 20 w. stop solution 1N HCl 100 µl ƒw k z, ELISA reader w 540 nm Ÿ dw. û s LDH d w w z, 0.5% Triton X µl ƒw 40 rpm 10 shakingg s Áp, LDH reagent 50 µl ƒw jš, óù, 540 nm Ÿ dw. LDH w s s LDH w l LDH w w ù kü. m. SPSS-PC+ m package w w. ƒ w s³e±tr (SD) wš ƒ s w» w one-way (ANOVA) ww F wš Scheffe s test w ƒ w. m 5% ü sƒw. š sü GST y. q üá w š 435ƒ e, r w y mw wy y y p k s y z ƒ 4 w š, w GST y dw. PC12 s 96 well plate 24 w

230 Á ÁÁ½xÁ½ÁÁw Table 1. Glutathione S-transferase activities of various methanol extracts from the medicinal plants Samples GST activity (Fold of increase) Control 1.00±0.

uralensis Fischer in PC12 cells. Cells were treated with 10 and 50 µg/ml of G. uralensis Fischer. GST activity was determined using glutathione S-transferase assay kit system. Significant vs.

3 230 Á ÁÁ½xÁ½ÁÁw Table 1. Glutathione S-transferase activities of various methanol extracts from the medicinal plants Samples GST activity (Fold of increase) Control 1.00±0.01 š(dendrobium nobile Lindley) 1.48±0.16 x (Shizonepeta tenuifolia Briquet) 2.20±0.61 (Glycyrrhiza uralensis Fischer) 3.13±1.29 (Paenoia lactiflora Pall) 1.14±0.04 Fig. 1. Glutathione S-transferase activities of the methanol and acetone extracts from the G. uralensis Fischer in PC12 cells. Cells were treated with 10 and 50 µg/ml of G. uralensis Fischer. GST activity was determined using glutathione S-transferase assay kit system. Significant vs. untreated control cells, **: p<0.01; ***: p< methanol s 2% w GST y dw., Table 1 ùkü w w š 1.48, x 2.20 GST y š, GST y. GST y w, ƒ GST y, w GST y w y kw» w w GST y r., Fig. 1 methanol 10 µg/ml 50 µg/ml w 1.21, 2.23 GST y ù kû, ethanol 10 µg/ml 50 µg/ml 0.60, 1.05(data not shown), acetone 10 µg/ml 50 µg/ml 1.07, 1.71 GST y yw. methanol PC12 s GST y, ethanol acetone methanol z GST y ùkü yw (Fig. 1). s. s y w w s w s xkw yƒ ùkù. PC12 s xkw y e w Fig. 2. Morphological changes and cytotoxicity of the extracts from the G. uralensis Fisher in PC12 cells. (A) The Cells were exposed to extracts of G. uralensis (50 µg/ml) for 24 h (a: control cells, b: methanol extract, c: acetone extract). Photographs were taken with a phase-contrast microscope at 100 magnification. (B) LDH release assay. Data were normalized to the activity of LDH release from vehicle-treated cells (100%) and expressed as percentage of the control (obtained separate plating).» w Ÿw x w w(fig. 2A)., w methanol acetone ƒƒ 50 µg/ml w s xkw j yƒ y w. š s yw wù LDH(lactate dehydrogenase) dw 23.1%, methanol 10 µg/ml 50 µg/ml 20.8%, 21.2% LDH y w, acetone 10 µg/ml 50 µg/ml 22.1%, 22.1% LDH yw (Fig. 2B). l k m ùkù ù 10 µg/ml 50 µg/ml s w, p GST y ƒk. methanol z sü GST y. GST y kùü p yw» w ƒ GST y ƒ methanol hexane, diethyl ether, ethyl acetate, water zw z (Fig. 3). ƒ z w z, 10, 50 µg/ml w PC12 s w GST y w. Table 2 kùü z GST y hexane

4 glutathione S-transferase y e w 231 Fig. 3. Fractionation of the methanol extracts from G. uralensis Fischer. The extract was fractionated in sequence with hexane, diethyl ether, ethyl acetate, and aqueous layer according to degree of polarity. Table 2. Glutathione S-transferase activities of the each fractions of methanol extracts from G. uralensis Fischer Fractions Concentration (µg/ml) d 10 µg/ml 50 µg/ml 2.26, 3.11, ether d 10 µg/ml 50 µg/ml 5.52, 3.33, ethyl acetate d 10 µg/ml 50 µg/ml 1.62, 1.67 y ùkü. l yw GST y wš. wy y ƒ s y z ƒš š, x, GST y z xw., ƒ GST y ùkü. methanol, ethanol, acetone z w GST y r, methanol 50 µg/ml w 2.23 ƒ GST y ùkû. š ƒ s xkw y w, LDH release assay m w û s yw. GST y p» w methanol zw GST y y GST activity (Fold of increase) Control ±0.02 Hexane Diethyl Ether Ethyl Acetate ± ± ± ± ± ±0.03 w, diethyl etherd z ƒ GST y ùký yw. Key words: G. uralensis Fischer, glutathione S-transferase, medicinal plants, PC12 cells 2006 (w») w w w (No. R ) šx 1. Yoon, M. Y., Lee, B. B., Kim, J. Y., Kim, Y., Park, E., Lee, S. C. and Park, H. R. (2007) Antioxidant activity and neuroprotective effect of Psoralea corylifolia Linne extracts. Kor. J. Pharmacogn. 38, Hyun, S. H., Jung, S. K., Jwa, M. K., Song, C. K., Kim, J. H. and Lim, S. (2007) Screening of antioxidants and cosmeceuticals from natural plant resources in jeju island. Korean J. Food Sci. Technol. 39, Cha, B. C. and Lee, E. H. (2007) Antioxidant activities of flavonoids from the leaves of Smilax china Linne. Kor. J. Pharmacogn. 38, Ellis, E. M. (2007) Reactive carbonyls and oxidative stress: potential for therapeutic intervention. Pharmacol Ther. 115, Scandalios, J. G. (2005) Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz. J. Med. Biol. Res. 38, Shi, Q. and Gibson, G. E. (2007) Oxidative stress and transcriptional regulation in Alzheimer disease. Alzheimer Dis. Assoc. Disord. 21, Szeto, H. H. (2006) Mitochondrial-targeted peptide antioxidants: novel neuroprotective agents. AAPS J. 18, E521-E531.

5 232 Á ÁÁ½xÁ½ÁÁw 8. Wittgen, H. G. and Van Kempen, L. C. (2007) Reactive oxygen species in melanoma and its therapeutic implications. Melanoma Res. 17, Fran, J., Nakamura, H. and Iyer, A. K. (2007) Tumor-targeted induction of oxystress for cancer therapy. J. Drug Target. 15, Jackson, M. J. (2008) Free radicals generated by contracting muscle: by-products of metabolism or key regulators of muscle function?. Free Radic. Biol. Med. 15, Hayes, J. D., Flanaqan, J. U. and Jowsey, I. R. (2005) Glutathione transferase. Annu. Rev. Pharmacol Toxicol. 45, Bora, P. S., Spilburg, C. A. and Lange, L. G. (1989) Metabolism of ethanol and carcinogens by glutathione transferases. Proc. Natl. Acad. Sci. 86, Wattenberg, L. W. and Bueding, E. (1986) Inhibitory effect of 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) on carcinogenesis induced by benzo(a)pyrene, diethlnitrosamine, and uracil mustard. Carcinogenesis. 7, Nam, K. S., Kim, H. G. and Shon, Y. H. (2003) Effect of ethanol extract from Thesium chinense Tunczaninove on chemopreventive enzymes of breast cancer. Kor. J. Pharmacogn. 34, Prochaska, H. J., Delong, M. J., Delong, M. J. and P. Talalay. (1985) On the mechanism of induction of cancer-protective enzymes: a unifying proposal. Proc. Natl. Acad. Sci. 82, Shon, Y. H., Cho, H. J., Chang, H. W., Son, K. H. and Nam, K. S. (2006) Chemopreventive potential of Salvia miltiorrhiza fraction extracts. J. Life Sci. 16, Kang, B. S. and Lee, K. D. (2002) Cytotoxic activities and antioxidative activities against liver cancer cell of Albizzia root. J. Applied Pharmacol. 10, Yoon, M. Y., Kim, J. Y., Hwang, J. H., Cha, M. R., Lee, M. R., Jo, K. J. and Park, H. R. (2007) Protective effect of methanol extracts from Dendrobium nobile Lindl. on H 2 O 2 - induced neurotoxicity in PC12 cells. J. Korean Soc. Appl. Biol. Chem. 50, Yoon, M. Y., Lee, H. J., Lee, S. M., Kim, J. Y., Kim, Y., Park, E. and Park, H. R. (2007) Protective effect of Schizonepeta tenuifolia Briquet extracts on oxidative DNA damage in human leucocytes and on hydrogen peroxide-induced cytotoxicity in PC12 cells. Food Sci. Biotechnol. 16, Chung, W. T., Lee, S. H., Cha, M. S., Sung, N. S., Hwang, B. and Lee, H. Y. (2001) Biological activities in roots of Glycyrrhiza uralensis Fisch. Korean J. Medicinal Crop Sci. 9, Wang, Z. Y. and Nixon, D. W. (2001) Licorice and cancer. Nutr. Cancer. 39, Demizu, S., Kajiyama, K., Takahashi, K., Hiraga, Y., Yamamoto, S. and Tamura, Y. (1998) Antioxidant and antimicrobial constituents of licorice: Isolation and structure elucidation of a new benzofuran derivative. Chem. Pharm. Bull. 36, Friis-Møller, A., Chen, M., Fuurasted, K., Christensen, S. B. and Kharazmi, A. (2002) In vitro antimycobacterial and antilegionella activity of licochalcone A from Chinese licorice roots. Planta Med. 68, Habig, W. H., Pabst, M. J. and Jakoby, W. B. (1974) Glutathione S-transferases. J. Biol. Chem. 249,

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