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1 KOREAN J. FOOD SCI. TECHNOL. Vol. 40, No. 6, pp. 696~701 (008) The Korean Society of Food Science and Technology HepG s p l p z w y z w s y z ½ xá x Á xká 1 Á v Áw * w t œw œ, 1 w BK1 ( p pf ), w w t ww Cytoprotective Effect by Antioxidant Activity of Codonopsis lanceolata and Platycodon grandiflorum Ethyl Acetate Fraction in Human HepG Cells Soo-Hyun Kim, Hyun-Jin Choi, Hyun-Taek Oh, Mi-Ja Chung 1, Cheng-Bi Cui, and Seung-Shi Ham* Department of Food Science and Biotechnology, School of Biotechnology, Kangwon National University 1 The Nutraceutical Bio Brain Korea 1 Project Group, Kangwon National University Department of Food Science and Engineering, Agricultural College of Yanbian University Abstract The objective of this study was to determine whether Codonopsis lanceolata or Platycodon grandiflorum ethyl acetate fraction (CLEA or PGEA) protect cells against sodium nitroprusside (SNP)-induced oxidative stress via the expression of various antioxidant systems. The HepG cells exposed for 4 hr to 0.5 mm SNP showed a reduction in the cell viability by an MTT assay. Pretreatment with CLEA and PGEA resulted in an inhibition of SNP-induced cell death. In addition, the effects of CLEA and PGEA on the expression of antioxidant systems via RT-PCR analyses was assessed. The levels of catalase (CAT), glucose-6-phosphate dehydrogenase (G6PD) and metallothionein (MT)-1A mrna were increased after 4 hr of CLEA exposure. The levels of Mn superoxide dismutase CAT, G6PD, MT-1A, and MT-A mrna were increased after PGEA treatment. In conclusion, CLEA and PGEA exert indirect antioxidant effects, perhaps via the induction of a variety of antioxidant systems which, may protect cells against oxidative stress. Key words: Codonopsis lanceolata, Platycodon grandiflorum, antioxidant, oxidative stress, antioxidant systems ù 65 ƒ ƒw y sw w ƒ š y (reactive oxygen species, ROS) w š. ü s ù y yw w superoxide radical anion(o ), hydrogen peroxide(h O ), hydroxyl radicals(oh ) ü s, DNA, y, w w, x y, x y y w x ƒ jš (1-7). ù ü w y l s yw» w ü l Cu/Zn superoxide dismutase(sod1), Mn superoxide dismutase, catalase(cat), glutathione peroxidase (GPx), glutathione reductase(gr), γ-glutamyl-cystein synthetase (GCS), human glutathione-s-transferase(hgsta1), glucose-6-phosphate-dehydrogenase metallothioneins(mt-1a, MT-A) w *Corresponding author: Seung-Shi Ham, Department of Food Science and Biotechnology, School of Biotechnology, Kangwon National University, Chunchon, Gangwon-do , Korea Tel: Fax: hamss@kangwon.ac.kr Received September 3, 008; revised October 1, 008; accepted October 31, 008 y z l tocopherol, vitamin C, carotenoid, flavonoid, glutathione š w w y BHA, BHT, Troxol-C (8-10). y y, ù y mw y w y w y k» j w y yw y» s ƒ š. SOD O ƒ w x superoxide anion hydrogen peroxide(h O ) y j w y jš, CAT x w H O H O y j ù hydrogen donor y w. GPx H O glutathione g y x glutathione(gsh) yx glutathione(gssg) y j z, GST(glutathione-S-transferase) GSH- Px e y j w y w. w s G6PD y glutathione y x w v NADPH w. s w» w s ü e ù y w w w. p k ƒ NADPH w w x G6PD v ƒw» j» w. GST w y z GSH sw w y g g yw. ü l ƒ g y w» w 696

2 HepG s p l p z w y z w s y z 697 w y w ƒ y w w š p, Oh(11) w y w wš 17, 15, k w DPPH radical y d w, Ko (1) w y t q w ü w y» y w. wr (Codonopsis lanceolata) l š w s» š ƒ, w,, w e š (13). w saponin, inulin flavonoid w wš š x z w yz ƒ y š (14-17). (Platycodon grandiflorum) w» wš š t triterpenoid s, w wš. w triterpenoid s x w,,,, w, x y w x û x w, g l, w y w z x (18-0). y ü ù w š y w y p w (1-3). s ü y t w ü w y w (4). s ü w y l ƒ k w y k s HepG s wš (5-7) 70% k w z x, j s, p l p, k š m zw s r w ƒ p l p z HepG sü ü l ƒ g y p l s yw w y x s d w. w w y y k mw» w» š w. x x w (Codonopsis lanceolata) (Platycodon grandiflorum) 007 ( )(Cheolwon, Korea) l xk œ 100 g 10 70% k ƒw 80 o C 8 y þƒw 3z z k w g. z x, j s, p l p, k d m zw z p l p d w þ š w x w. sü y p w z s HepG(hepatoblastoma, KCLB No ) Korea Cell Line Bank(Seoul, Korea) l w x w. HepG s 4 well plate cells/ml 1mL ƒ well ƒw 5% CO 37 o C 48 k z x mw s w e MEM 1 ml ƒw 4 w. z ƒ well d w z 0.5 mm SNP 1mL 4 w y p w SNP(sodium nitroprusside) w. óù s Chung (8) w MTT y w d w., ƒ well MTT (5 mg/ ml) FBS w ƒ ƒ MEM 10 1 ƒw š, 5% CO 37 C 4 w o MTT y g formazan ùƒ w. û w» w 30 ew z DMSO(dimethyl sulfoxide) w w k Micro-Reader(Molecular Devices, Sunnyvale, CA, USA) 570 nm Ÿ d w. Ÿ d œ DMSO w š, s w. s (%)=[ Ÿ / Ÿ ] 100 HepG s total RNA cells/ml s 4 well plate w ƒ µg/ml p l p z w w. óù z z TRIzol Reagent(Invitrogen, Carlsbad, CA, USA) 1 ml ƒw w k z TRIzol 1mL 0. ml chloroform š 15 z -3 g. 4 o C 15 (1,000 g)w d w p ¼ z isopropanol ƒw RNA e g. z 4 o C 10 (1,000 g) z d š e 75% DEPC(diethyl pyrocarbonate)-ethanol 1mL š z 4 o C 5 (1,000 g) w. w RNA z 0.1% DEPC 995 µl ww ŸŸ (GE healthcare, Pischaway, NJ, USA) Ÿ d w RT-PCR w. 60 nm 80 nm ƒ 1.6 ww. RT-PCR w total RNA w cdna w w Reverse Transcription System(Invitrogen) w., oligo(dt) 15 primer(500 µg/ml) 1 µl dntp mix(10 mm each) 1 µl PCR p š w RNA RNase-free vƒ 1 µlƒ š 65 C 5 o ò z þƒ g. 5 first-strand 4 µl, DTT(Dithiothreitol, 100 mm) µl, RNase-free 1µL Superscript TM II Reverse Transcriptase 1 µl ƒw z vr š. z 4 C o 50, 70 C 15 o g. ƒ x» w PCR w. x w primer» q w š(9), Table 1 ùkü. Table SOD1, SOD, CAT, GPx, GR, GCS, hgsta1, G6PD, MT-1A MT-A mrna x d w š, 18S ü t w. PCR 0.00% ethidium bromideƒ ƒ 1.% agarose gel 100V 30» w z image analysis

3 698 w t wz 40 «6y (008) Table 1. Primers used for RT-PCR* Gene Forward Reverse SOD1 5'-AAG GCC GTG TGC GTG CTG AA-3' 5'-CAG GTC TCC AAC ATG CCT CT-3' SOD 5'-GCA CAT TAA CGC GCA GAT CA-3' 5'-AGC CTC CAG CAA CTC TCC TT-3' CAT 5'-AAG GTT TGG CCT CAC AAG G-3' 5'-CGG CAA TGT TCT CAC ACA G-3' GPx 5'-GTG TAT GCC TTC TCG GCG CG-3' 5'-CGT TGC GAC ACA CCG GAG AC-3' GR 5'-CAG TGG GAC TCA CGG AAG AT-3' 5'-TTC ACT GCA ACA GCA AAA CC-3' GSC 5'-GGG GAA CCT GCT GAA CTG-3' 5'-GCT CCA AGG AAA GAT TAA CTC C-3' hgsta1 5'-TCC ATA TGG CAG GAG AGC CCA AGC-3' 5'-CCG AAT TCT CCA TGA CTG CGT TAT TA-3' G6PD 5'-CCG GAT CGA CCA CTA CCT GGG CAAG-3' 5'-GTT CCC CAC GTA CTG GCC CAG GAC CA-3' MT-1A 5'-CTC GAA ATG GAC CCC AACT-3' 5'-ATA TCT TCG AGC AGG GCT GTC-3' MT-A 5'-CCG ACT CTA GCC GCC TCTT-3' 5'-GTG GAA GTC GCG TTC TTT ACA-3' 18S rrna 5'-CGG CTA CCA CAT CCA AGG AA-3' 5'-GCT GGA ATT ACC GCG GCT GC-3' *Abbreviations: Cu/Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl-cystein synthetase (GCS), human glutathione-s-transferase (hgsta1), glucose-6-phosphate dehydrogenase (G6PD), metallothioneins (MT-1A, MT-A) Table. Amplification conditions for RT-PCR* (unit: o C) Gene SOD1 SOD CAT GPx GR GCS hgsta1 G6PD MT-1A MT-A 18S rrna Initial denaturation Further denaturation Annealing Extension Final extension Cycle *Abbreviations: Cu/Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl-cystein synthetase (GCS), human glutathione-s-transferase (hgsta1), glucose-6-phosphate dehydrogenase (G6PD), metallothioneins (MT-1A, MT-A) wokrstation(slimline TM series, Spectra Services Inc., Ontario, NY, USA) w x. SigmaGel(Jandel Scientific, San Rafael, CA, USA) vp w w. m x mean±sd t w SPSS v (Version 10.0, SPSS, Chicago, IL, USA) w (ANOVA) w š, ƒ d s³ p<0.05 f x (Duncan s multiple range test) w. š p l p w sü y p z 70% k z x, j s, p l p, k d m zw s r w d w p l p d ƒ w wš ( ). s r w ƒ p l p d x w. š 70% k ƒƒ 37.5% 44.7% p l p z ƒƒ 1.9% 1.4%. Fig. 1 HepG s p l p w z sü NO j SNP w y p g s e w ùkü. w, SNP w š p l p z w x w. s 100% š, s 5.1% ùkû p l p z 50 µg/ml 100 µg/ml ƒƒ s w HepG s 77.5% 91.4% ƒw. p l p z 50 µg/ml 100 µg/ml w ƒƒ 76.% 80.8% s ƒw (p<0.05). p l p ƒ y p l s yw š, z w ùkþ ù, š z y p w s w. Lee (30) tert-butylhydroperoxide w y p ƒ rat primary s m LDH ƒ, sü GSH y ƒ s w ù w s µg/ml n w, s w š šw, Lee (31) LLC-PK1 s H O y p w s Rg µm n w, s w š šw š Kim (3) H O w V79-4 s y p z x w, z w yw 00 µg/ml w 80% s ƒƒ š šw. Chung (5,33), caffeic acid š Trolox w y s z SNP sü NO

4 HepG s p l p z w y z w s y z 699 Fig. 1. Effects of Codonopsis lanceolata and Platycodon grandiflorum ethyl acetate fraction against SNP (sodium nitroprusside)-induced cytotoxicity. HepG cells were incubated with 0.5 mm SNP (4 hr) and/or with C. lanceolata (CLEA) and P. grandiflorum ethyl acetate fraction (PGEA) and their viability was assessed by MTT assay. 1) Results are from three experiments and are expressed as mean±sd. a-d Means with the different letters are significantly different (p<0.05) by Duncan s multiple range test. g y p w w y ƒ y p ww s š š w x w w. p l p w w y x e w p l p sü w y l 4 z (SOD1, SOD, CAT GPx), glutathione w z (GR, GCS, hgsta1 G6PD) w y (MT-1A MT-A) mrna x e w RT-PCR Fig. -4 ùkü. p p p z 100 µg/ml n CAT mrna ƒw w ƒ w ù SOD1, SOD GPx mrna w. ù p l p z, 50 µg/ml 100 µg/ml n w SOD CAT mrna ƒw (Fig. ). p l p z glutathione w z x w w e r, GR, GCS hgsta1 mrna x w ù G6PD 50 µg/ ml 100 µg/ml s n w w ƒw (Fig. 3). w p l p z 50 µg/ml 100 µg/ml s n w MT-1A MT- A mrna ƒw ù p l p z 50 µg/ml w MT-1A mrna ƒw (Fig. 4). w Lee (0) l p l 8: yww w ƒ w w y ùk ü w Han Cho(15) yk n w n w xanthine oxidase, GST y w surperoxide dismutase y ƒ k š šw, Chung (5,8,33,34) w y genistin, caffeic acid Trolox s Fig.. Effect of Codonopsis lanceolata and Platycodon grandiflorum ethyl acetate fraction on the expression of SOD1, SOD, CAT and GPx mrna. 1) Results are from three experiments and are expressed as mean±sd. a-b Means with the different letters are significantly different (p<0.05) by Duncan s multiple range test. NS=Not significantly (p<0.05) by Duncan s multiple range test. Abbreviations: SOD1=Cu/Zn superoxide dismutase, SOD=Mn superoxide dismutase, CAT=catalase, GPx=glutathione peroxidase. Fig. 3. Effect of Codonopsis lanceolata and Platycodon grandiflorum ethyl acetate fraction on the expression of GR, 1) GCS, hgsta1 and G6PD mrna. Results are from three experiments and are expressed as mean±sd. a-b Means with the different letters are significantly different (p<0.05) by Duncan s multiple range test. NS=Not significantly (p<0.05) by Duncan s multiple range test. Abbreviations: GR=glutathione reductase, γ- glutamyl-cystein synthetase (GCS), hgsta1=human glutathione-stransferase, G6PD=glucose-6-phosphate dehydrogenase. HepG š» ƒ s w G6PD, MTs š GST mrna w x ƒw š, w y ƒƒ y p l s y w š w. x p l p z w sü G6PD, CAT MT-1A mrna x ƒw š,

5 700 w t wz 40 «6y (008) x Fig. 4. Effect of Codonopsis lanceolata and Platycodon grandiflorum ethyl acetate fraction on the expression of MT-1A and MT-A mrna. 1) Results are from three experiments and are expressed as mean±sd. a-c Means with the different letters are significantly different (p<0.05) by Duncan s multiple range test. Abbreviations: Metallothioneins (MT-1A, MT-A). p l p z SOD, CAT, G6PD, MT- 1A MT-A mrna x ƒw. p l p z sü w y l ƒ j y p l s yw. p l p z l ƒ w sü y z ƒ w Á y ƒ y t w» ƒ wš. p l p z (CLEA ù PGEA) w w y l ƒ g sodium nitroprusside(snp) w y p ww s yw w. 0.5 mm SNP HepG s 4 g s w ù, CLEA PGEA w SNP w s ƒ w. w w y l x e CLEA PGEA w RT-PCR. Catalase, glucose-6-phosphate dehydrogenase(g6pd) š metallothionein (MT)-1A mrna CLEA s 4 w z ƒw š, Mn superoxide dismutase, catalase, G6PD, MT-1A MT- A mrna PGEA w ƒw. CLEA PGEAƒ w w y l ƒ j w y z ùkü, z y p l s yw w. 008 w. 1. Yu BP. Aging and oxidative stress: Modulation by dietary restriction. Free Radical Bio. Med. 1: (1996). Harman D. Free radical theory of aging. Mutat. Res. 75: (199) 3. Biesalski HK. Free radical theory of aging. Curr. Opin. Clin. Nutr. 5: 5-10 (00) 4. Oh MH, Chung HY, Yang HS, Kim KW, Hikokichi O, Takako Y. Effects of ginsenoside Rb on the antioxidants in SAM-R/1 mice. Korean Biochem. J. 5: (199) 5. Kim SK, Lee HJ, Kim MK. Effect of water and ethanol extracts of persimmon leaf and green tea different conditions on lipid metabolism and antioxidative capacity in 1-month-old rats. Korean J. Nutr. 34: (001) 6. Oh HM, Kim MK. Effect of dried leaf powders, water, and ethanol extracts of persimmon and green tea leaves on lipid metabolism and antioxidative capacity in 1-month-old rats. Korean J. Nutr. 34: (001) 7. Sung IS, Park EM, Lee MK, Han EK, Jang JY, Cho SY. Effects of acorn extracts on the antioxidative enzyme system. J. Korean Soc. Food Sci. Nutr. 6: (1997) 8. Block G, Langseth L. Antioxidant vitamins and disease prevention. Food. Technol. 48(7): (1994) 9. Fukuzawa K, Takaishi Y. Antioxidants. J. Act. Oxyg. Free Rad. 1: (1990) 10. Hatano T. Constituents of natural medicines with scavenging effects on active oxygen species-tannins and related polyphenols. Nat. Med. 49: (1995) 11. Oh HS. Studies on biological activities of the plant resources of Gangwon-do: Focused on the Acer ginnala Max. PhD thesis, Gangwon National University, Chunchon, Korea (005) 1. Ko MS, Shin KM, Lee MY. Effects of Hijikia fusiforme ethanol extract on antioxidative enzymes in ethanol-induced hepatotoxicity of rat liver. J. Korean Soc. Food Sci. Nutr. 31: (00) 13. Lee YE, Hong SH. Natural Food Material, Kyomunsa Publishers Inc., Seoul, Korea. pp (003) 14. Choi MS, Choi PS. Plant regeneration and saponin contents in Codonopsis lanceolata L. Korean J. Med. Crop Sci. 7: (1999) 15. Han EG, Cho SY. Effects of Codonopsis lanceolata water extract on the activities of antioxidative enzymes in carbon tetrachloride treated rats. Korean J. Soc. Food Sci. Nutr. 6: (1997) 16. Han EG, Sung IS, Moon HG, Cho SY. Effects of Codonopsis lanceolata water extract on the level of lipid in rats fed high fat diet. Korean J. Soc. Food Sci. Nutr. 7: (1998) 17. Maeng YS, Park HK. Antioxidant activity of ethanol extract from Ddodok (Codonopsis lanceolata). Korean J. Food Sci. Technol. 3: (1991) 18. Kim KS, Osamu E, Shinji I, Hiroshige I. Effects of Platycodon grandiflorum feeding on serum and liver lipid concentrations in rats with diet-induced hyperlipidemia. J. Nutr. Sci. Vitaminol 41: (1995) 19. Lee JY, Hwang WI, Lim ST. Effect of Platycodon grandiflorum DC extract on the growth of cancer cell lines. Korean J. Food Sci. Technol. 30: 13-1 (1998) 0. Lee JY, Hwang WI, Lim ST. Antioxidant and anticancer activities of organic extracts from Platycodon grandiflorum A. De Candolle roots. J. Ethnopharmacol. 93: (004) 1. Bartsch H, Nair J. Chronic inflammation and oxidative stress in the genesis and perpetuation of cancer: Role of lipid peroxidation, DNA damage, and repair. Langenbeck Arch. Surg. 391: (006). Van Houten B, Woshner V, Santos JH. Role of mitochondrial DNA in toxic responses to oxidative stress. DNA Repair 5: (006) 3. Comelli MC, Mengs U, Schneider C, Prosdocimi M. Toward the definition of the mechanism of action of silymarin: Activities related to cellular protection from toxic damage induced by chemotherapy. Integr. Cancer Ther. 6: (007) 4. Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74: (004)

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Æ¯ÇãÃ» '' - 1 - - 2 - - 3 - - 4 - - 5 - ' - 6 - - 7 - - 8 - - 9 - - 10 - - 11 - Super-Bio Co., Ltd. KWON, Suk-Tae Thermostable DNA Polymerase-encoding Gene from Thermus sp. X-1 an d Amino Acid Sequence