Journal of Nutrition and Health

The Korean Nutrition Society (한국영양학회)
권호 표지

Protective Effect of Flavonoids on Lymphocyte DNA Damage Using Comet Assay

Comet Assay를 이용한 Flavonoids와 항산화 비타민의 인체임파구 세포 DNA 손상 보호 효과

  • 박유경 (한남대학교 이과대학 식품영양학과) ;
  • 전은재 (한남대학교 이과대학 식품영양학과) ;
  • 강명희 (한남대학교 이과대학 식품영양학과)
  • Published : 2003.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

The present study was attempted to investigate and compare the antioxidant potency of several well-know flavonoids, antioxidant vitamin and commercially available popular beverages. The antioxidant potency was assessed by the effect on reducing oxidative DNA damage of human lymphocytes. Cellular oxidative DNA damage was measured by SCGE (single-cell gel electrophoresis), also known as comet assay. Lymphocytes were pre-treated for 30 minutes with wide ranges of doses of apigenin, kaempferol, luteolin, myricetin, rutin, quercetin, $\alpha$-tocopherol (10,25,50,100,200,500,1000 $\mu$M) ,green tea extract or grape juice (10,50,100,250,500,1000 $\mu$g/mL) followed by a $H_2O$$_2$(100 $\mu$M) treatment for 5 min as an oxidative stimulus. The physiological function of each antioxidant substance on oxidative DNA damage was analyzed as tail moment (tail length $\times$ percentage migrated DNA in tail) and expressed as relative DNA damage score after adjusting by the level of control treatment. Cells treated with $H_2O$$_2$alone (positive control) had an extensive DNA damage compared with cells treated with phosphate buffered saline (PBS, negative control) or pre-treated with all the tested samples. Of all the six flavonoids, quercetin was the most potent antioxidant showing the lowest $ED_{50}$/ of 8.5 $\mu$g/mL (concentration to produce 50% protection of relative DNA damage). The antoxidant potency of individual flavonoids were ranked as follows in a decreasing order; luteolin (18.4 $\mu$g/mL), myricetin (19.0 $\mu$g/mL) , rutin (22.2 $\mu$g/mL) , apigenin (24,3 $\mu$g/mL) , kaempferol (25.5 $\mu$g/mL). The protective effect of $\alpha$-tocopherol was substantially lower (highest $ED_{50}$value of 55.0 $\mu$g/mL) than all the other flavonoids, while the protective effect was highest in green tea and grape juice with low ED5O value of 7.6 and 5.3, respectively. These results suggest that flavonoids, especially quercetin, and natural compounds from food product, green tea and grape juice, produced powerful anti-oxidative activities, even stronger than $\alpha$-tocopherol. Taken together, supplementation of antioxidants to lymphocytes followed by oxidative stimulus inhibited damage to cellular DNA, supporting a protective effect against oxidative damage induced by reactive oxygen species.

Keywords

References

  1. Cancer Letter v.114 Vitamins and cancer Van Poppel G;Van den Berg H https://doi.org/10.1016/S0304-3835(97)04662-4
  2. Cancer and Metastasis Reviews v.16 Diet and the prevention of cancer Martinez ME;Giovannucci E https://doi.org/10.1023/A:1005860413247
  3. Cancer and Metastasis Reviews v.16 Dietary change as a strategy for preventing cancer Schatzkin A https://doi.org/10.1023/A:1005812514155
  4. Eur J Nutr v.38 Dietary flavonoids protect human colonocyte DNA from oxidative attack in vitro Duthie SJ;Dobson VL https://doi.org/10.1007/s003940050043
  5. Nutr Res v.19 The flavonoids, myricerin, quercetin and rutin, protect against cholestan-3 β, 5 α, 6 β-triol-induced toxicity in chinese hamster ovary cells in vitro Aheme SA;O'Brien NM https://doi.org/10.1016/S0271-5317(99)00037-8
  6. Nutr Res v.19 Antioxidant property of dietary phenolic agents in a human LDL-oxidation ex vivo model: interaction of protein binding activity Wang W;Goodman MT https://doi.org/10.1016/S0271-5317(98)00183-3
  7. Nutrition Research v.18 no.12 Flavonoids: Dietary Occurrence and biochemical activity Peterson J;Dwyer J.
  8. Korean Food Sci Technol v.28 no.2 Identification and Determination of Dietary Fibers and Flavonoids in Pulp and Peel of Korean Tangerine (Citus aurantium var) Eun JB;Jung YM;Woo GJ
  9. J Agric Food Chem v.40 Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands Hertog MGL;Hollman PCH;Katan MB
  10. J Agric Food Chem v.32 Varietal differences in distribution of quercetin and kaempferol in onion (Allium cepa L.) tissue Bilyk A;Cooper L;Sapers GM https://doi.org/10.1021/jf00122a024
  11. J Agric Food Chem v.33 Distribution of quercetin and kaempferol in lettuce, kale, chive, garlic chive, leek, horseradish, red radish, and red cabbage tissues Bilyk A;Sapers GM https://doi.org/10.1021/jf00062a017
  12. Proc Soc Exp Biol Med v.217 Evolution of the health benefits of soy isoflavones Barnes S
  13. Ann Rev Pharmacol Toxiol v.16 Chemicals, drugs and lipid peroxidation Plaa GL;Witschi H
  14. Biochem Biophys Res Commum v.123 Microgel electrophoretic study of radiation-induced DNA damages in individual mammalian cells Ostling O;Johanson KJ https://doi.org/10.1016/0006-291X(84)90411-X
  15. Exp Cell Res v.175 A simple technique for quantitation of low levels of DNA damage in individual cells Singh PN;McCoy MT;Tice RR;Schneider EL https://doi.org/10.1016/0014-4827(88)90265-0
  16. Mutat Res v.307 Microgel electorphoresis assay (comet assay) and SCE analysis in human lymphocytes from 100 normal subjects Betti C;Davini T;Giannessi L;Loprieno N;Barale R https://doi.org/10.1016/0027-5107(94)90306-9
  17. Korean J Nutrition v.35 no.2 Application of the alkaline comet assay for detecting oxidative DNA damage in human biomonitoring Park EJ;Kang MH
  18. Envioronmental Mutagens & Carcinogens v.22 no.2 Smoking related DNA damage in human lymphocytes assessed by the comet assay Sun SJ;Chung HW;Han JH
  19. Mutat Res v.288 The single cell gel electrophoresis assay (comet assay) : A european review Mckelvey-Martin VJ;Green MHL;Schemezer P;Pool-Zobel BL;De Meo MP;Collins A https://doi.org/10.1016/0027-5107(93)90207-V
  20. J Food Sci Nutr v.21 no.1 Antioxidative Effectiveness of Water Extract and Ether Extract in Wormwood (Artemisia montana Pampan) Yoon HS;Lee KE;Kim JS;Pae JO
  21. J Food Sci Nutr v.29 no.2 Originals: Physiological Activity and Antioxidative Effect of Methanol Extract from Onion (Allium cepa L) Kim HG;Kwon JH;Kwak HJ;Kwon YJ;Chung PH
  22. Antioxidant Methodology-in vivo and in vitro concept In vitro methods for characterizing potential prooxidant and antioxidant actions of nonnutritive substances in plant foods Aruoma OI;Halliwell B;Williamson G;Aruoma OI(ed.), Cuppet SL(ed.)
  23. Chem Phys Lipids v.79 The antioxidant activity of natural flavonoids is governed by number and location of their aromatic hydroxyl groups Chen ZY;Chan PT;Ho KY;Fung KP;Wang J https://doi.org/10.1016/0009-3084(96)02523-6
  24. Carcinogensis v.14 Radioprotective effect of antioxidative flavonoids in x-ray irradiated ice Shimoi K;Masuda S;Furugori M;Esaki S;Kinae N
  25. Phytochemistry v.25 Antioxidant activity of flavonoids and reactivity with peroxy radicals Torel J;Cillard J;Cillard P https://doi.org/10.1016/S0031-9422(00)85485-0
  26. Arch Intern Med v.155 no.4 Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study Hertog MG;Kromhout D;Aravanis C;Blackburn H;Buzina R;Fidanza F;Giampaoli S;Jansen A;MenottiA;Nedeljkovic S https://doi.org/10.1001/archinte.155.4.381
  27. Proc Soc Exp Biol Med v.220 no.4 Tea and Health: the underlying mechanism Weisburger JH https://doi.org/10.1046/j.1525-1373.1999.d01-46.x
  28. Ann Nutr Metab v.41 no.6 Effect of concentrated red grape juice consumption on serum antioxidant capacity and low-density lipoprotein oxidation Day AP;Kemp HJ;Bolton C;Hartog M;Stansbie D https://doi.org/10.1159/000178006
  29. J Nutr v.128 Grape juice but not orange or grapefruit juice inhibits platelet activity in dogs and monkeys Osmon H;Maalej N;Shanmuganayagam D;Folts JD
  30. IX Asian Congress of Nutrition New Delhi Changes in antioxidant status and lipid profiles after grape juice supplementation to patients with cardiovascular disease (CVD) Kim SM;Park YK;Seong IW;Kang MH
  31. Mutation Res v.496 Resveratrol, a natural phenolic compound inhibits cell proliferation and prevents oxidative DNA damage Sgambato A;ardito R;Faraglia B;Boninsegna A;Wolf FI;Cittadini A https://doi.org/10.1016/S1383-5718(01)00232-7
  32. Circulation v.91 Administration of wine and grape juice inhibits in vivo platelet activity and thrombosis in stenosed canine coronary arteries Demrow HS;Slane PR;Folts JD https://doi.org/10.1161/01.CIR.91.4.1182
  33. Mutat Res v.58 Comparative mutagensis of plant flavonoids in microbial systems Hardigree AA;Epler JL https://doi.org/10.1016/0165-1218(78)90014-9
  34. Food And Chem Toxicol v.40 Evaluation of the genotoxic effect of rutin and quercetin by comet assay and microuncleus test Silva J;Herrmann SM;Heuser V;Peres W;Possa Marroni;Gonzalez-Gallego J;Erdtmann B https://doi.org/10.1016/S0278-6915(02)00015-7
  35. Antioxidant Methodology-in vivo and in vitro concepts Application of the comet assay to study oxidative DNA damage in human cell Pool-Zobel BL;Bub A;Rechkemmer G;Aruoma OI(ed.);Cupper SL(ed.)
  36. Terat Carcinogen Mutagen v.17 Modulating effects of flavonoids on food mutagens in humans in human blood and sperm samples in the comet assay Anderson D;Basaran N;Dobrzynska MM;Basaran A;Yu T-W https://doi.org/10.1002/(SICI)1520-6866(1997)17:2<45::AID-TCM1>3.0.CO;2-E
  37. Mutagensis v.17 no.3 Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability Duthie SJ;Pirie L;Jenkinson AMcE;Narayanan S https://doi.org/10.1093/mutage/17.3.211