Protective Effects of Punicalagin on Caco-2 Intestine Cell Line under Oxidative Stress Caused by Tert-butyl hydroperoxide


  • Ulfat Omar University of Surrey, Guildford, Surrey, GU2 7XH, UK
  • Akram Aloqbi University of Surrey, Guildford, Surrey, GU2 7XH, UK
  • Marwa Yousr University of Surrey, Guildford, Surrey, GU2 7XH, UK
  • Nazlin Howell University of Surrey, Guildford, Surrey, GU2 7XH, UK



 Polyphenols, Malondialdehyde, Glutathione, Reactive oxygen species, morphology.


Hydrolysable tannin polyphenols from pomegranate (punicalagin) have been reported to show a wide range of health properties correlated to their high antioxidant and free radical scavenging activities. The objective of the current study was to investigate the protective effect of punicalagin on cell viability and redox status of cultured Caco-2 cells exposed to oxidative stress induced by tert-butyl hydroperoxide. The production of malondialdehyde, and total glutathione levels, as well as the generation of reactive oxygen species were used as markers of cellular oxidative status. Pretreatment of Caco-2 cells with 5 and 10 µM punicalagin for 24 hours significantly protected cell viability after exposure to tert-butyl hydroperoxide IC50 = 3 mM for 2 hours. The examined doses prevented the decrease of total glutathione and the increase of malondialdehyde induced by tert-butyl hydroperoxide in Caco-2 cells. Reactive oxygen species generation provoked by tert-butyl hydroperoxide was significantly reduced at the same concnetrations. Finally, cell morphology with treatments before and after induction by tert-butyl hydroperoxide showed irreversible effect of the oxidizing agent. The results of the biomarkers analyzed showed that treatment of Caco-2 cells with the natural dietary antioxidant punicalagin protected the cells against oxidative stress.


Wijeratne SSK, Cuppett SL, Schlegel V. Hydrogen peroxide induced oxidative stress damage and antioxidant enzyme response in caco-2 human colon cells. J Agric Food Chem 2005; 53: 8768-8774.

Devasagayam TP, Boloor KK, Ramasarma T. Methods for estimating lipid peroxidation: an analysis of merits and demerits. Indian J Biochem Biophys 2003; 40: 300-8.

Castell JV, Gomez-lechon MJ, Ponsoda X, Bort R. In vitro investigation of the molecular mechanisms of hepatotoxicity. Arch Toxicol Suppl 1997; 19: 313-21.

Kedderis GL. Biochemical basis of hepatocellular injury. Toxicol Pathol 1996; 24: 77-83.

Lima CF, Fernandes-ferreira M, Pereira-wilson C. Phenolic compounds protect hepg2 cells from oxidative damage: relevance of glutathione levels. Life Sci 2006; 79: 2056-68.

Steinmetz KA, Potter JD. Vegetables, fruit, and cancer prevention: a review. J Am Diet Assoc 1996; 96: 1027-39.

Arts IC, Jacobs DR, Harnack lJ, Gross M, Folsom AR. Dietary catechins in relation to coronary heart disease death among postmenopausal women. Epidemiology 2001; 12: 668-75.

Gil MI, Tomas-barberan FA, Hess-pierce B, Holcroft DM, Kader AA. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agric Food Chem 2000; 48: 4581-9.

Cotelle N, Bernier JL, Catteau JP, Pommery J, Wallet JC, Gaydou EM. Antioxidant properties of hydroxy-flavones. Free Radic Biol Med 1996; 20: 35-43.

Van acker SA, Van den berg DJ, Tromp MN, et al. Structural aspects of antioxidant activity of flavonoids. Free Radic Biol Med 1996; 20: 331-42.

Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic Biol Med 1997; 22: 749-60.

Jouvet P, Rustin P, Taylor DL, et al. Branched chain amino acids induce apoptosis in neural cells without mitochondrial membrane depolarization or cytochrome c release: implications for neurological impairment associated with maple syrup urine disease. Mol Biol Cell 2000; 11: 1919-1932.

Waterfield CJ, Westmoreland C, Asker DS, Murdock JC, George E, Timbrell JA. Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations. Archives of Toxicology 1998; 72: 588-596.

Lima CF, Valentao PC, Andrade PB, Seabra RM, Fernandes-ferreira M, Pereira-wilson C. Water and methanolic extracts of salvia officinalis protect hepg2 cells from t-bhp induced oxidative damage. Chem Biol Interact 2007; 167: 107-15.

Liu J, Yeo HC, Doniger SJ, Ames B. N. Assay of aldehydes from lipid peroxidation: gas chromatography-mass spectrometry compared to thiobarbituric acid. Anal Biochem 1997; 245: 161-6.

Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351-8.

Osseni RA, Debbasch C, Christen MO, Rat P, Warnet JM. Tacrine-induced reactive oxygen species in a human liver cell line: the role of anethole dithiolethione as a scavenger. Toxicol In Vitro 1999; 13: 683-8.

Suttnar J, Cermak J, Dyr JE. Solid-phase extraction in malondialdehyde analysis. Anal Biochem 1997; 249: 20-3.

Suttnar J, Masova l, Dyr JE. Influence of citrate and edta anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 2001; 751: 193-7.

Alia M, Ramos S, Mateos R, Granado-serrano AB, Bravo l, Goya l. Quercetin protects human hepatoma hepg2 against oxidative stress induced by tert-butyl hydroperoxide. Toxicol Appl Pharmacol 2006b; 212: 110-8.

Peng IW, Kuo SM. Flavonoid structure affects the inhibition of lipid peroxidation in caco-2 intestinal cells at physiological concentrations. J Nutr 2003; 133: 2184-7.

Brown JE, Khodr H, Hider RC, Rice-evans CA. Structural dependence of flavonoid interactions with cu2+ ions: implications for their antioxidant properties. Biochem J 1998; 330: 1173-8.

Yokomizo A, Moriwaki M. Effects of uptake of flavonoids on oxidative stress induced by hydrogen peroxide in human intestinal caco-2 cells. Biosci Biotechnol Biochem 2006; 70: 1317-24.

Alia M, Mateos R, Ramos S, Lecumberri E, Bravo l, Goya l. Influence of quercetin and rutin on growth and antioxidant defense system of a human hepatoma cell line (hepg2). Eur J Nutr 2006a; 45: 19-28.

Martin C, Martinez R, Navarro R, Ruiz-sanz JI, Lacort M, Ruiz-larrea MB. Tert-butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals. Biochem Pharmacol 2001; 62: 705-12.

Vergauwen H, Tambuyzer B, Jennes K, Degroote J, Wang W, De smet S, Van ginneken C. Trolox and ascorbic acid reduce direct and indirect oxidative stress in the ipec-j2 cells, an in vitro model for the porcine gastrointestinal tract. Plos one 2015; 10(3).




How to Cite

Ulfat Omar, Akram Aloqbi, Marwa Yousr, & Nazlin Howell. (2015). Protective Effects of Punicalagin on Caco-2 Intestine Cell Line under Oxidative Stress Caused by Tert-butyl hydroperoxide. Journal of Pharmacy and Nutrition Sciences, 5(4), 249–256.