Protective Effects of Chungkookjang Extract on High Glucose Induced Oxidative Stress in LLC-PK1 Cells

  • Yi, Na-Ri (Department of Food Science and Nutrition, Pusan National University) ;
  • Seo, Kyoung-Chun (Department of Food Science and Nutrition, Pusan National University) ;
  • Choi, Ji-Myung (Department of Food Science and Nutrition, Pusan National University) ;
  • Cho, Eun-Ju (Department of Food Science and Nutrition, Pusan National University) ;
  • Song, Young-Ok (Department of Food Science and Nutrition, Pusan National University) ;
  • Han, Ji-Sook (Department of Food Science and Nutrition, Pusan National University)
  • Published : 2008.06.30


This study was designed to investigate the protective effect of a methanol extract of Chungkookjang (CKJ) on high glucose induced oxidative stress in LLC-$PK_1$ cells (renal tubular epithelial cells), which are susceptible to oxidative stress. Freeze dried CKJ powder was extracted with methanol, and the extract solution was concentrated, and then used in this study. To determine the protective effect of CKJ extract, oxidative stress was induced by exposing of LLC-$PK_1$ cells to high glucose (30 mM) or normal glucose (5 mM) for 24 hr. Exposure of LLC-$PK_1$ cells to high glucose for 24 hr resulted in a significant (p<0.05) decrease in cell viability, catalase, SOD and GSH-px activity and a significant (p<0.05) increase in intracellular ROS level and thiobarbituric acid reactive substances (TBARS) formation in comparison to the cells treated with 5 mM glucose. CKJ extract treatment decreased intracellular ROS level and TBARS formation, and increased cell viability and activities of antioxidant enzymes including catalase, SOD and GSH-px in high glucose pretreated LLC-$PK_1$ cells. These results suggest that CKJ extract may be able to protect LLC-$PK_1$ cells from high glucose-induced oxidative stress, partially through the antioxidative defense systems.


  1. Willcox JK, Ash SL, Catignani GL. 2004. Antioxidants and prevention of chronic disease. Crit Rev Food Sci Nutr 44: 275-295
  2. Kawanish S, Hiraku Y, Murata M, Oikawa S. 2005. The role of metals in site-specific DNA damage with reference to carcinogenesis. Free Radic Biol Med 32: 822-832
  3. Sachidanandame K, Fagan SC, Ergul A. 2005. Oxidative stress and cardiovascular disease: Antioxidants and unresolved issues. Cardiovasc Drug Rev 23: 115-132
  4. Kim KS, Lee S, Lee YS, Jung SH, Park Y, Shin KH, Kim BK. 2003. Anti-oxidant activities of the extracts from the hurbs of Artemisia apiacea. J Ethnopharmaco 85: 69-72
  5. Hou YZ, Zhao GR, Yang J, Yuan YJ, Zhu GG, Hiltunrn R. 2004. Protective effect of Ligusticum chuanxiong and Angelica sinensis on endothelial cell damage induced by hydrogen peroxide. Life Sci 75: 1775-1786
  6. Gulter RG, Packer LB, Eartum J, Mori A. 1995. Oxidative stress, antioxidant, aging and disease. In Oxidative stress and aging. Birkhauser Verlag, Basel, Switzerland. p 125-163
  7. Rosen P, Nawroth PP, King G, Moller W, Tritschler HJ, Packer L. 2001. The role of oxidative stress in the onset and progression of diabetes and its complications: A summary of a Congress Series sponsored by UNESCOMCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 17: 189-212
  8. Wiernsperger NF. 2003. Oxidative stress: The special case of diabetes. Biofactors 19: 11-18
  9. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, Yorek MA, Beebe D, Oates PJ, HAmmes HP, Giardino I, Brownlee M. 2000. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycemic damage. Nature 404: 787-790
  10. Beckman JA, Goldfine AB, Gordon MB, Creager MA. 2001. Ascorbate restores endothelium-dependent vasodilation impaired by acute hyperglycemia in humans. Circulation 103: 1618-1623
  11. Tan KC, Chow WS, Ai VH, Metz C, Bucala R, Lam KS. 2002. Advanced glycation end products and endothelial dysfunction in type 2 diabetes. Diabetes Care 25: 1055-1059
  12. Giuglino D, Ceriello A, Paolisso G. 1996. Oxidative stress and diabetic vascular complications. Diabetes Care 19: 257-267
  13. Baynes JW, Thorpe SR. 1999. Role of oxidative stress in diabetic complications: A new perspective on an old paradigm. Diabetes 48: 1-9
  14. Diabetes Control and Complications Trial Research Group. 1993. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Eng J Med 329: 977-986
  15. Kang SM, Lee CS, Yoo CK, Seo WS. 1998. Purification and characterization of fibrinolytic enzyme excreted by Bacillus subtilis K-54 isolated from Chung Guk Jang. Kor J Appl Microbiol Biotechnol 26: 507-515
  16. Ryu SH. 2001. Studies on antioxidative effects antioxidative components of soybean and Chongkukjang. PhD Dissertation. Inje University, Korea
  17. Kim JI, Kang MJ, Kwon TW. 2003. Antidiabetic effect of soybean and Chongkukjang. Korea Soybean Digest 20: 44-53
  18. Kim Y, Cho JY, Kuk JH, Moon JH, Cho JI, Kim YC, Park KH. 2004. Identification and antimicrobial activity of phenylacetic acid produced by Bacillus licheniforms isolated from fermented soybean, Chungkook-Jang. Curr Microbiol 48: 312-317
  19. Yang JL, Lee SH, Song YS. 2003. Improving effect of powders of cooked soybean and chongkukjang on blood pressure and lipid metabolism in spontaneously hypertensive rats. J Korean Soc Food Sci Nutr 32: 899-906
  20. Lee BY, Kim DM, Kim KH. 1991. Physico-chemical properties of viscous substance extracted from chungkook-jang. Korean J Food Sci Technol 23: 599-604
  21. Jing Y, Waxman S. 1995. Structural requirements for differentiation-induction and growth-inhibition of mouse erythroleukemia cells by isoflavones. Anticancer Res 15: 1147-1152
  22. Shamsuddin AM. 1995. Inositol phosphates have novel anticancer function. J Nutr 125: 725s-732s
  23. Rao AV, Sung MK. 1995. Saponins as anticarcinogens. J Nutr 125: 717s-724s
  24. Ferguson IR, Chavan RR, Harris PJ. 2001. Changing concepts of dietary fiber: Implications for carcinogenesis. Nutr Cancer 39: 155-169
  25. Nicholson Ml, Neoptolemons JP, Clayton HA, Talbot IC, Bell PRF. 1990. Inhibition of experimental colorectal carcinogenesis by dietary N-6-polyunsaturated fats. Carcinogenesis 11: 2191-2197
  26. Barnes S, Sfakianos J, Coward L, Kirk M. 1996. Soy isoflavonoids and cancer prevention. Underlying biochemical and pharmacological issues. Adv Exp Med Biol 401: 87-100
  27. Lee JJ, Lee DS, Kim HB. 1999. Fermentation patterns of Chungkookjang and Kanjang by Bacillus licheniformis B1. Kor J Microbiol 35: 296-301
  28. Carmichael J, Degraff WG, Gazdar AF, Minna JD, Mitchell JB. 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res 47: 936-942
  29. Fraga CG, Leibovita RM, Roeder RG. 1988. Lipid peroxidation measured as thiobarbituric-reactive substances in tissue slices: Characterization and comparision with ho homogenates and microsomes. Free Radic Biol Med 4: 155-161
  30. Wang H, Joseph JA. 1999. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27: 612-616
  31. Nath J, Powledge A. 1997. Modulation of human neutrophil inflammatory responses by nitric oxide: Studies in unprimed and LPS-primed cells. J Leukocyte Biol 62: 805-816
  32. Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Ann Biochem 72: 248-254
  33. Marklund S, Marklund G. 1974. Involvement of the superoxide anion radical in antioxidant of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47: 469-474
  34. Aebi H. 1984. Catalase in vitro. Methods Enzymol 105: 121-126
  35. Lawrence RA, Burk RF. 1976. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 71: 952-958
  36. Sevanian A, Hochstein P. 1985. Mechanism and consequence of lipid peroxidation in biological systems. Annu Rev Nutr 5: 365-390
  37. Hochstein P, Jain SK. 1981. Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging. Fed Proc 40: 183-188
  38. Pacifici RE, Davies KJ. 1991. Protein, lipid and DNA repair systems in oxidative stress: The free radical theory of aging revisited. Gerontology 37: 166-180
  39. Chen CF, Tsai SY, Ma MC, Wu MS. 2003. Hypoxic preconditioning enhances renal superoxide dismutase levels in rats. J Physiol 552: 561-569
  40. Dong J, Ramachandiran S, Tikoo K, Jia Z, Lau SS, Monks TJ. 2004. EGFR-independent activation of p38 MAPK and EGFR-dependent activation of ERK1/2 is required for ROS-induced renal cell death. Am J Physiol Renal Physiol 87: F1049-F1058
  41. Lee HC, Yin PH, Lu CY, Chi CW, Wei YH. 2000. Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochemical J 348: 425-432
  42. Beckman JS, Koppenol WH. 1996. Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and ugly. Am J Physiol 271: C1424-C1437
  43. Corbett JA, McDaniel ML. 1992. Does nitric oxide mediate autoimmune destruction on $\beta$-cells? Possible therapeutic interventions in IDDM. Diabetes 41: 897-903
  44. Husain K, Somani SM. 1998. Interaction of exercise training and chronic ethanol ingestion on testicular antioxidant system in rat. J Appl Toxicol 18: 421-429<421::AID-JAT532>3.0.CO;2-R
  45. Runnegar MTC, Andrews J, Gerdes RG, Falconer IR. 1987. Injury to hepatocytes induced by a peptide toxin from the cyanobacterium Microcrystis aeruginosa. Toxicon 25: 1235-1239
  46. Alptekin N, Seckin S, Dogru-Abbasoglu S, Yelkenci F, Kocak-Toker N, Toker G, Uysal M. 1996. Lipid peroxides, glutathione, gamma-glutamylcysteine synthetase and gamma-glutamyltranspeptidase activities in several tissues of rats following water-immersion stress. Pharmacol Res 34: 167-169

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