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1 J. Fd Hyg. Safety 7(2), 06 6 (2002) y s DNA w z Á *Áx w w w t t Protection of ROS-induced cytotoxicity and DNA damage by the extract of Alpinia officinarum Seung Cheol Lee, Kyoung Seung Shin*, Moon Young Heo $PMMFHFPGIBSNBDZ,BOHXPO/BUJPOBM6OJWFSTJUZ$IVODIPO,PSFB,ZVOH*O'%"*ODIPO,PSFB Abstract The 70% ethanol extract of Alpinia officinarum and its major flavonoid, galangin showed strong antioxidative effect on the lipid peroxidation of ethyl linolate with Fenton's reagent and free radical scavenging effect to DPPH radical generation. However, they did not reveal any pro-oxidant effect on bleomycin-fe(iii) dependent DNA degradation. They also showed the protective effect against H 2, K or UV-induced cytotoxicity in mammalian cells. They also showed the suppressive effect of DNA damage induced by H 2 or K with dosedependent manner in single cell gel electrophoresis(scge) assay. On the other hand, they have an anticlastogenic effect against adriamycin-induced micronucleated reticulocyte in peripheral blood of mice. These results suggest that the mechanism of inhibition by 70% ethanol extract of Alpinia officinarum and galangin against reactive oxygen species (ROS)-induced genotoxicity or cytotoxicity is due, at least partly, to their antioxidative and free radical scavenging properties without pro-oxidant effect. All these results indicate that 70% ethanol extract of Alpinia officinarum and galangin may be useful for protection against ROS-induced cytotoxicity and DNA damage. Key words ý Alpinia officinarum, galangin, reactive oxygen species, antioxidative effect, free radical scavenging effect, DNA damage, chemoprevention oxidative stress w, y l ü f š. ( ~) -4) (Zingiberaceae) Alpinia officinarum l w, m, m y, m, m, w w š. ƒ 0.5-%,8- cineol, methyl cinnamate, α-cadinene galangol, flavonoid galangin, kaempfride, alpinin w wš. 5) w galangin w y z free radical, pro-oxidant effect wš, y (reactive oxygen species, ROS) w z ³ w» w hydroxyl radical(. OH) H 2 superoxide(._) K UV-B, C s w z w. wr, H 2 Author to whom correspondence should be addressed. DNA w y z single cell gel electrophorsis(scge) w sƒw. w, superoxide anion adriamycin w w x w w z ³ w. w (Alpinia officinarum) 50 g ƒƒ, 30% p g, 50% p g, 70% p g, 00% p g, 80% p g, 00% p g, m, n- p g, j s, p l p, l, x, v v g, n-v v g 300 ml ƒw 7 wš œ w ƒƒ. galangin ƒƒ mg/ml ( w ) galangin(3,5,7-trihydroxy 06

2 SPUFDUJPOPG304JOEVDFEDZUPUPYJDJUZBOE%/"EBNBHFCZUIFFYUSBDUPG"MQJOJBPGGJDJOBSVN ± o C 55 ± 7% 7-0 k z w. pellet, wš, 2h/2h(L/D) cycle xw. Fig.. HPLC chromatogram of 70% ethanol extract of Alpinia officinarum(a) and galangin(b) flavonol) aldrich w w t t w. HPLC Shimadzu UV» 360 nm, f ODS(Shimadzu), A : 99% acetonitrile, B : water : acetonitrile(0-5 min : B 9%, 5-50 min : B 40%), ml/min w. Fig. HPLC chromatogram ùkü. x x w C57BL mice ( ) w x œ ü ³ s Chinese hamster lung(chl) cell NIH/3T3 cell w w x w. s 0% FBS(GIBCO), % glutamine(gibco), % penicillin-streptomycin(gibco) w w MEM, DMEM (GIBCO) ƒƒ CHL NIH/3T3 s w. wr, primary cell culture w C57BL mouse spleen lymphocyte w xw. C57BL l ³ w z 50 ml ³ plunger w s k z cell countw 20x0 6 cell/ml. sxk 0.5 ml ƒw xw. Complete medium 5% heat inactivated fetal bovine serum(gibco No ), % sodium heparin(,000 unit, Invenex No.33-), % penicillin-streptomycin(gibco No ), 2% concanavalin A(Con A, Sigma No. C-5275), 0.0% β- mercaptoethanol(0.05m, Sigma No. M-6250) RPMI 640(GIBCO) culture medium w w. 37 o C, 5% C». v e DPPH(,-Diphenyl-2-picryl hydrazil) ü radical w wš free radical ww w complex. DPPH(0.47 g/ml) 2 ml sample 2ml ƒwš 5 š 30 e z 520 nm d w. 6) y z y z d w» w Fenton's reagent w w. sodium dodecyl sulfate 2% ƒ w w jš 0.79 mmol potassum chloride, 0.25 mmol Trizma(pH 7.4), µmol FeCl 2 ƒ wš mm H 2 w g. 0 µl ethyl linoleate ƒw z w stock solution 0. ml w. ƒ sample 5mlƒ w. sample vortex mixer ywwš aluminum foil 6h 55 C o incubation k. ƒƒ sample 50 µl 4% BHT ethanol solution w y k

3 08 4FVOH$IFPM-FFFUBM w y TBA d w. 7,8) Pro-oxidant effect Bleomycin-Fe(III) dependent DNA degradation e galangin w antioxidants pro-oxidant z 00 µg/ml calf thymus DNA 50 µg/ml bleomycin, mm FeCl 3, 5 mm MgCl 2, 30 mm KH 2 PO 4 -KOH buffer(ph 7.0), mlƒ ƒ w hr 37 C incubation k z o % TBA g 532 nm Ÿ d w. 9) s (cytoprotective activity) H 2 K s w z CHL cell spleen lymphocyte H 2 K s w z MTT 0) microplate reader d w. CHL cell well 25,000 wš 80 µl 24 z H 2 K 0 µl 0 µl ƒwš C incubator 20 w z MTT 5 µl ƒwš 4 z DMSO 200 µl ƒwš z 570 nm Ÿ d w. wr, mouse spleen lymphocyte well 750,000 wš w w xw, 24 w. UV s w z NIH/3T3 cell UV-B, C s w z MTT 0) microplate reader d w. w UV v UV-B(Model XX-5B, medium wavelength: 32 nm, Spectroline, Westbury, NY, USA) UV-C (Model XX-5F, medium wavelength: 254 nm) w DRC-00X digital radiometer (Spectroline) w d w. s 90 µl 24 z 0 µl ƒwš UV(.0mW/ cm2) 0 z, C incubator 20 w z MTT 6 µl ƒwš 4 z DMSO 200 µl ƒwš z 570 nm Ÿ d w. DNA z CHL cell w SCGE x w. ) H 2 K w. w ù n w DNA z sƒw. x 4 CHL cell 5 0 culture dish(0 cm) š, 24 hz H 2 K 2 O 0 µl DMSO 0 µl n w. 24 hz culture dish trypsin 2 ml cell harvestw test tube w. 2,000 rpm z PBS z 2,000 rpm w. d š ƒ ƒ 0.5%-LMPA(low melting point agarose) ml ƒ w ƒƒ. 0.65%-NMPA(normal melting point agarose) 30 µl slide(fully frosted) 75 µl z cover slide. þ š 5 cover slide wš 0.5%- LMPA 00 µl z cover slide š þ š 5. Cover slide w z lysis buffer(2.5 M NaCl, 00 mm Na 2 EDTA, 0 mm Tris, ph 0, 0% DMSO, % Triton X-00) ƒ 30 w k. z» (300 mm NaOH, mm Na 2 EDTA, ph 3) 5 ƒ ew.» e slide w 25 V, 250 ma 5» w. Éü 0.4 M tris(ph7.5) 5 ƒ yw. Tray z ethidium bromide(20 µg/ml) 50 µl ƒƒ z nm excitation filter 590 nm barrier filter w xÿx w image analyzer COMET 3.(Kinetic image, England) w 25 cell w. w x Õ w w x w. 2) superoxide adriamycin w. galangin, n wš adriamycin 5 mg/kg w oxidative DNA damage 48 hrz Õ w ù x acridine orange gq š š e š 2 z w w. xÿx (blue excitation, 488 nm & yellow filter, 55 nm) 000 w. galangin w Table ùkü ƒ galangin w ethyl acetate acetone ƒƒ 6.84% w ùkü. g ƒ galangin w. p g 30% 0.38%, 50% 2.8%, 70% 4.44%, 00% 9.66% p g 80% 5.8%, 00%

4 SPUFDUJPOPG304JOEVDFEDZUPUPYJDJUZBOE%/"EBNBHFCZUIFFYUSBDUPG"MQJOJBPGGJDJOBSVN 09 Table. Inhibitory activity of lipid peroxidation and free radical scavenging activity by various solvent extracts of Alpinia officinarum % Inhibition(at 25 µg/ml) 2 Extract Galangin content(%) Free radical Lipid peroxidation Water Not Detected % ethyl alcohol % ethyl alcohol % ethyl alcohol % ethyl alcohol % methyl alcohol % methyl alcohol acetone n-butyl alcohol chloroform ethyl acetate ether hexane isopropyl alcohol n-propyl alcohol ,2 n=3 Table 2. Free radical scavenging effect of Alpinia officinarum extracts and galangin Treatment OD 520nm (/ml or M) Mean S.D. % Inhibition Alpinia officinarum (70% EtOH) 0 µg/ml ± µg/ml ± ** 7.8 µg/ml ± 0.04 * µg/ml ± ** µg/ml ± ** µg/ml ± ** 48.9 Alpinia officinarum (80% MeOH) 0 µg/ml ± µg/ml ± * 3.5 µg/ml ± µg/ml ± ** µg/ml ± ** µg/ml ± ** 49.7 dl--tocopherol 0 M ± M ± ** M ± ** M ± 0.04 ** M ± * 36.9 Galangin 0 M ± M ± ** M ± ** M ± ** M 0.29 ± 0.02 ** 38.8 n=3, Significantly different from the positive control group ( Student's t-test ) * p< 0.05, ** P <0.0 2 % Inhibition =[ OD positive - OD ]/ OD sample-blank positive 00

5 0 4FVOH$IFPM-FFFUBM Table 3. Inhibitory effect of lipid peroxidation of Alpinia officinarum extracts and galangin. Treatment (/ml or M) OD 535nm Mean ± S.D. % Inhibition Alpinia officinarum (70% EtOH) 0 µg/ml 0.25 ± µg/ml 0.22 ± µg/ml 0.20 ± µg/ml 0.08 ± µg/ml 0.04 ± 0.02 * 6.8 Alpinia officinarum (80% MeOH) 0 µg/ml 0.44 ± µg/ml 0.24 ± 0.02 * µg/ml ± * µg/ml ± ** µg/ml ± ** 68.7 BHT 0 M 0.57 ± M 0.2 ± * M 0.03 ± ** M 0.06 ± 0.06 ** M ± 0.00 ** 8.5 Galangin 0 M 0.57 ± M 0.52 ± 0.03 * M 0.48 ± M 0.20 ± * M 0.08 ± * 3. n=3, Significantly different from the positive control group ( Student's t-test ) * P < 0.05, ** P < % Inhibition = [ OD positive OD ] / OD sample-blank positive % ùkü. g galangin w wš ùkû. wr, 25 µg/ml v e 70, 00% p g, 80, 00% p g f g y f. w, y z 80, 00% p g f n- p g, j s j r j ƒ y f. v e Table 2 ùkü 70% p g, 80% p g, galangin dl-α-tocopherol v e ùkü 70% p g 80% p g y w. ù w dlα-tocopherol y. SC 50 (µg/ml) w 80% p g = 70% p g < galangin < dl-a-tocopherol f. x DPPH free radical w x DPPH radical. OH w w galangin w wš w v e q. y z Table 3 ùkü 70% p g, 80% p g, galangin BHT y z ùkü. 80% p g 70% p g y { f. ù w BHT y û. IC 50 (µg/ml) w BHT < 80% p g < galangin < 70% p g f. x ethyl linoleate w Fenton reaction w OH y w z. w ùkù galangin w wš w y z ƒ q. Pro-oxidantz Fig. 2 ùkü galangin pro-

6 SPUFDUJPOPG304JOEVDFEDZUPUPYJDJUZBOE%/"EBNBHFCZUIFFYUSBDUPG"MQJOJBPGGJDJOBSVN Fig. 2. Pro-oxidant effect of flavonoids on bleomycin-fe() dependent DNA degradation. oxidant z ùkü ù dl-α-tocopherol ascorbic acid 0 M 4 prooxidantz ùkü. wr, BHT x pro-oxidant y ùkü. ù galangin flavonol quercetin prooxidant z ùkü 0 M ƒ y ù 5 kü š 0 M û biphasicw w ùk 4 ü. wr, 70% p g x pro-oxidant y ùkü.(data not shown) s z Fig. 3 ùkü H 2 s w CHL cell mouse spleen lymphocyte 70% p g, galangin dl-α-tocopherol s ùkü. H 2 0 M n w 4 s 00% wš 0 µg/ml w galangin < 70% p g <dl-α-tocopherol CHL cell mouse spleen lymphocyte w y w. H 2 OH. w s ùkü, galangin w wš H 2 s w ùkü. w, Fig. 3 ùkü K s w Fig. 3. Cytoprotective effect of 70% ethanol extract of Alpinia officinarum(ao) and galangin against H 2 (0-4 M) or K (0-3 M)- induced cytotoxicity in chinese hamster lung cell (CHL) and mouse spleen lymphocytes.

7 2 4FVOH$IFPM-FFFUBM Fig. 4. Cytotoxicity of 70% ethanol extract of Alpinia officinarum (AO) and galangin in chinese hamster lung cell (CHL) and spleen lymphocytes CHL cell mouse spleen lymphocyte 70% p g, galangin dl-α-tocopherol s ùkü. K 0 M n w 3 s 00% wš 0 µg/ml w H 2 s ƒ galangin < 70% p g <dl-αtocopherol CHL cell mouse spleen lymphocyte w y w. K._ w s ùkü, galangin w wš H 2 s w wì K s w ùkü. wr, Fig. 4 s w s 00% w w s y ùkü. 70% p g galangin dl-α-tocopherol ƒ CHL cell Fig. 5. Cytoprotective effect of 70% ethanol extract of Alpinia officinarum (AO) and galangin against ultraviolet B or C-indued cytotoxiciy in NIH/3T3 fibroblast mouse spleen lymphocyte s x ùkü ƒ s z ùkþ. w dl-αtocopherol ƒ f. w s ƒ, galangin dl-α-tocopherol ƒ e s ww w» q. Fig. 5 NIH/3T3 fibroblast UV-B, UV- C s w z ùkü. UV-B, C s w 70% p g, galangin dl-α-tocopherol s ùkü. 70% p g galangin y dl-αtocopherol û. UV-B, C UV s ùkü, galangin w w š v e y

8 SPUFDUJPOPG304JOEVDFEDZUPUPYJDJUZBOE%/"EBNBHFCZUIFFYUSBDUPG"MQJOJBPGGJDJOBSVN 3 Table 4. Protective effect of Alpinia officinarum and galangin on H 2 (5 0-4 M) or K (0-3 M)-induced oxidative DNA damage in CHL cells H 2 K Treatment (µg/ml or M) Tail DNA(%) Tail length(µm) Tail DNA(%) Tail length(µm) Alpinia ± ± ± ± 5.2 officinarum ± ± ± ± % EtOH ± ± ± ± (µg/ml) ± ± ± ± 25.0 * ± 4.94 ** ± 4.49 * 6.70 ± 2.57 * ± 5.53 * Galangin ± ± ± ± 5.2 ( M ) ± ± ± ± ± ± ± ± ± ± ± 3.5 * ± 2.28 * ± 4.09 ** 86.2 ± * 3.00 ± 2.32 ** ± * n=3, Mean ± S.D. *,** Significantly different from the control group at p < 0.05 and p < 0.0, respectively ( Student's t-test ). j ùkü UV-B, C s w ùkü. wr NIH/3T3 fibroblast 70% p g, galangin dl-αtocopherol ƒ s ùkü š, ƒ s ƒ ù kü (Data not shown). y DNA e w Single cell gel electrophoresis(scge) w 70% p g galangin y DNA y Table 4 ùkü. H 2 K DNA w galangin y ùkü. H 2 w 500 µg/ml tail DNA tail length ƒƒ 78.4%, 54.6% y ùkü. Galangin 0-5 M(= 2.7 µg/ml) tail DNA tail length ƒƒ 66.2%, 38.0% y ùkü. wr, K w 500 µg/ml tail DNA tail length ƒƒ 65.7%, 44.2% y ùkü. Galangin 0-5 M(= 2.7 µg/ml) tail DNA tail length ƒƒ 73.3%, 43.5% y ùkü. galangin.. OH w DNA single strand break w DNA y w. w x Adriamycin w galangin w w y Table 5 ùkü. galangin y ùk Table 5. Effect of Alpinia officinarum extract and galangin on the frequency of MNRETs by adriamycin (5mg/kg, i.p.) Treatment MNRETs /,000 RETs (mg/kg, p.o.) Ind. Value Mean ± S.E. Alpinia ±.73 officinarum ± 5.39 (70 % EtOH) ± ±.40 ** ± 0.97 ** Galangin ± ± ± ± ± 4.3 *, ** Significantly different from the control group (0mg/kg) at p < 0.05 and p < 0.0, respectively ( Student's t-test ). MNRET : Micronucleated reticulocyte, RET : Reticulocyte ü. wš mg/ kg n y ùkü 400 mg/kg 33.5% y ùkü. w galangin 00 mg/kg 3.6% y ùkü. wš y w ùkü.. w ùkü adriamycin w w w. š galangin w š. p 3) galangin w yy w š low oxidation

9 4 4FVOH$IFPM-FFFUBM potential(.5v) š y z ƒ w š wš4), ƒ flavonoid ƒ LDL y z ƒ f š šw. 5) w, galangin CCl 4 microsomal lipid peroxidation w jš š šw. 6) wr, galangin y murin hepatoma cell anticarcinogenic marker enzyme quinone reductase y ƒ k š. Galangin 7) human breast cancer cell wš 8), sulfation induced carcinogenesis w 9), multidrugresistant breast cancer cell p-glycoprotein mediated efflux ƒ g ƒ effluxƒ DMBA burden g cancer risk k š. 20) wr, t v s (propolis)ƒ galanginw 2), propolis w l 22), w 23), w 24) š š. (Alpinia officinarum) 00% p g galangin 9.6%, 00% p g 8.27%, m 6.84%, p l p 6.84% w (Table ). galangin sƒ. p, l w t 70% p g w galangin y y (Table 2) v e (Table 3) ùkü, H 2, K UV-B, C s w w s y ùkü (Fig. 3). wr, H 2 K DNA w galangin y ùkü (Table 4). w, superoxide anion w ùkü adriamycin w w w (Table 5). Galangin w w v e z y z hydroxyl peroxy radical wš, Fe Fenton reaction w q. w galangin 25) superoxide flavonoid one-electron transfer mechanism 26), converted mechanism w superoxide w q. 27) Table 4 ùkü quercetin flavonoid Fe 3+ -bleomycin-dna complex 2+xk Fe y g bleomycin-dependent DNA damage k. r yw yw 28) pro-oxidantz ƒ. ù w galangin quercetin x pro-oxidantz ùkü. wr, UV-B, C s y mw DNA. w 29) v w. 30) UV-B, C w s w w y photo-carcinogenesis w z ƒ š. 3) galangin UV Ÿ w y ƒ q. ROS w oxidative DNA damage y ¾. 32) point mutation ù deletion sww nuclear DNA j mitochodrial DNA oxidative damage y s v g ù ù. 33) DNA v s jš s carcinogenesis j w. y DNA mw ù ù w, w y mw s ü, DNA, DNA ù DNA» ù w» š. 34) w galangin y OH.._ w SCGE DNA single strand breakage y p w y wš. w w x w adriamycin._ w w galangin z ùkü. w galangin y DNA ù mw w y ƒ š q. ww l w 70% p g w galangin v e y y ù kü, H 2 K s w w s yz ùkü. w, DNA single strand breakage w galangin z ùkü. v e w y w» y w w y p m w w y, ƒ wz» t ƒ q. 999 w w w (KRF F0030)

10 SPUFDUJPOPG304JOEVDFEDZUPUPYJDJUZBOE%/"EBNBHFCZUIFFYUSBDUPG"MQJOJBPGGJDJOBSVN 5 (Alpinia officinarum, 70% ethanol extract) w galangin v e y y ùkü, H 2 K s w w s yz ùkü. DNA single strand breakage y p w y wš. w, w x w adriamycin superoxide w w z ùkü. galangin z w v e w y w» y p w w y, ƒ wz» t ƒ q. š x. Ductile, G. and Crozier, A.: Plant-derived phenolic antioxidants. Curr. Opin. Clin. Nutr. Metab. Care, 3, (2000). 2. Marnett, L.J.: Oxyradicals and DNA damage. Carcinogenesis, 2, (2000). 3. Lin, J.K. and Tsai, S.H.: Chemoprevention of cancer and cardiovascular disease by resveratrol. Proc. Natl. Sci. Counc. Repub. China, 23, (999). 4. Inagake, M., Yamane, T., Kitao, Y., Oya, K., Matsumoto, H., Kikuoka, N., Nakatani, H., Takahashi,T., Nishimura, H. and Iwashima, A.: Inhibition of,2-dimethylhydrazine-induced oxidative DNA damage by green tea extract in rat. Japan J. Cancer Res. 86, 06- (995). 5. w r: 2«, w w» q, w, pp 782 (985). 6. Fugita,Y., Uera, I., Morimoto, Y., Nakajima,M., Hatano, C. and Okuda,T.: Studies on inhibition mechanism of autooxidation by tannins and flavonoids. Yakugaku Zasshi, 08, (988). 7. Ohtka,H., Ohishi, N. and Yaki, k.: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal.Biochem., 95, (979). 8. Kitta, K., Hagiwara, Y. and Shibamoto, T.: Antioxidative activity of an isoflavonoid, 2"-o-glycosylisovitexin isolated from green barley leaves. J. Agric. Food Chem., 40, 843 (992). 9. Okezie, I. K., Antoniam M., Johnm B. and Barry H.: Evaluation of the antioxidant and pro-oxidant actions of gallic acid and its derivatives. J. Agric. Food Chem., 4, (993). 0. Cole, S. P. C.: Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother. Pharmacol., 7, 259 (986).. Singh, N.P., McCoy, M.T., Tice, R.R. and Schneider, E.L.: A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research, 75, 84-9(988). 2. Hayashi, M., Morita, T., Kodama, T., Sofuni, T. and Ishidate, Jr, M.: The micronucleus assay with peripheral blood reticulocytes using acridine orange-coated slides. Mutation Res., 278, (990). 3. Heo, M.Y., Sohn, S.J. and Au, W.W.: Anti-genotoxicity of galangin as a cancer chemopreventive agent candidate. Mutat. Res., 488, 35-50(200). 4. Rapta, P., Misik, V., Stasko, A. and Vrabel, I.: Redox intermediates of flavonoids and caffeic acid esters from propolis: an EPR spectroscopy and cyclic voltammetry study. Free Radic. Biol. Med., 8, (995). 5. Myara, I., Pico, I., Vedie, B. and Moatti, N.: A method to screen for the antioxidant effect of compounds on low-density lipoprotein(ldl): illustration with flavonoids. J. Pharmacol. Toxicol. Methods, 30(2), (993). 6. Cholbi, M. R., Paya, M. and Alcaraz, M. J.: Inhibitory effects of phenolic compounds on CCl 4 -induced microsomal lipid peroxidation. Experientia, 47(2), (99). 7. Uda, Y., Price, K. R., Williamson, G. and Rhodes, M. J.: Induction of the anticarcinogenic marker enzyme, quinone reductase, in murine hepatoma cells in vitro by flavonoids. Cancer Lett., 20(2), (997). 8. So, F.V., Guthrie, N., Chambers, A.F. and Caroll, K.K.: Inhibition of proliferation of estrogen recepter-positive MCF- 7 human breast cancer cells by flavonoids in the presence and absence of excess estrogen. Cancer Lett., 2(2), 27-33(997). 9. Eaton, E. A., Walle, U. K., Lewis, A. J., Hudson, T., Wilson, A. A. and Walle, T.: Flavonoids, potent inhibitors or the human P-form phenolsulfotransferase. Potential role in drug metabolism and chemoprevention. Drug Metab. Dispos., 24(2), (996). 20. Phang, J. M., Poore, C. M., Lopaczynska, J. and Yeh, G... : Flavonol-stimulated efflux of 7,2-dimethylbenz(a)anthracene in multidrug-resistant breast cancer cells, Cancer Res., 53(24),

11 6 4FVOH$IFPM-FFFUBM (993). 2. Park, Y. K., Koo, M. H., Abreu, J. A., Ikegaki, M., Cury, J.A. and Rosalen, P. L.: Antimicrobial activity of propolis on oral microorganisms. Curr. Microbiol., 36(), (998). 22. Afolayan, A. J. and Meyer, J. J.: The antimicrobial activity of 3,5,7-trihydroxyflavone isolated from the shoots of Helichrysum aureonitens. J. Ethnopharmacol., 57(3), 77-8 (997). 23. Meyer, J. J., Afolayan, A. J., Taylor, M. B. and Erasmus, D.: Antiviral activity of galangin isolated from the aerial parts of Helichrysum aureonitens. J. Ethnopharmacol., 56(2), 65-69(997). 24. Krol, W., Scheller, S., Czuba, Z., Matsuno, T., Zydowicz, G., Shani, J. and Mos, M.: Inhibition of neutrophils' chemiluminescence by ethanol extract of propolis(eep) and its phenolic components. J. Ethnopharmacol., 55(), 9-25 (996). 25. Hiramoto, K., Ojima, N., Sako, K. and Kikugawa, K.: Effect of plant phenolics on the formation of the spin-adduct of hydroxyl radical and the DNA strand breaking by hydroxyl radical. Biol. Pharm. Bull., 9, (996). 26. Noda, Y., Anzai, K., Mori, A., Kohno, M., Shinme, M. and Packer, L.: Hydroxyl and superoxide anion radical scavenging activities of natural source antioxidants using the computerized JES-FR30 ESR spectrometer system. Biochem. Mol. Biol. Int., 42(), 35-44(997). 27. Afanas'ev, I.B. and Polozova, N.I.: One electron oxidation of p- and o-dihydroxy benzenes by oxygen radical anion in aprotic medium. Zh. Organ. Khim., 26, 03-06(978). 28. Miranda, J.L., Barry, H., Patricia, J.E. and Robin, S.H.: Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossypol and myricetin. Biochemical Pharmacology, 38(7), (989). 29. Cadet, J., Douki, T., Pouget, J.P., Ravanat, J.L. and Sauvaigo, S.: Effects of UV and visible radiations on cellular DNA. Curr. Probl. Dermatol., 29, 62-73(200). 30. Hemminki, K, Xu, G. and Le Curieux, F.: Ultraviolet radiationinduced photoproducts in human skin DNA as biomarkers of damage and its repair. IARC Sci Publ., 54, (200). 3. Heo, M.Y., Kim, S.H., Yang, H.E., Lee, S.H., Jo, B.K. an Kim, H.P.: Protection against ultraviolet B- and C-induced DNA damage and skin carcinogenesis by the flowers of Prunus persica extract. Mutat. Res., 496(-2), (200). 32. Ames, B.N.: Endogeneous DNA damage as related to cancer and aging. Mutation Res. 24, 4(989). 33. Ames, B.N.: Dietary carcinogens and anticarcinogens. Oxygen radical and degenerative diseases. Science, 22, 256 (983). 34. Flora, S., Bronzetii, G. and Sovels, F.H.: Assesment of antimutagenicity and anticarcinogenicity. Mutation Res. 267, (992).

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82-01.fm w y wz 8«( 2y) 57~61, 2005 J. of the Korean Society for Environmental Analysis p w w Á Á w w» y l Analysis of Influence Factors and Corrosion Characteristics of Water-pipe in Potable Water System Jae Seong