In vitro Antioxidant and Cytoprotective Activities of the Extract of Dangyuja (Citrus grandis Osbeck) Leaves

  • Kim, Yun-Jung (Faculty of Biotechnology, College of Applied Life Sciences, Cheju National University) ;
  • Cho, Moon-Jae (Department of Medicine, Medical School, Cheju National University) ;
  • Kim Cho, So-Mi (Faculty of Biotechnology, College of Applied Life Sciences, Cheju National University)
  • Published : 2008.10.31

Abstract

The antioxidant activities of the extracts of dangyuja (Citrus grandis Osbeck) leaves were evaluated. The highest phenolic content was obtained from the ethyl acetate fraction (EF) (202.1$\pm$0.8 mg GAE/g dried extract) and it exhibited the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. The cytoprotective effects of EF on oxidative damage induced by tert-butyl hydroperoxide (t-BHP) in a human hepatoma cell line, HepG2 cells, were investigated to understand the intracellular antioxidant mechanisms. Treatment of HepG2 cells with EF prior to oxidative stress was found to inhibit reactive oxygen species (ROS) generation, lipid peroxidation, and DNA damage in a dose-dependent manner. Gas chromatography-mass spectrometry (GC-MS) studies on EF resulted in tentative identification of 19 compounds representing 94.3% of the total content. Taken together, these results demonstrated that EF has excellent antioxidant activities and thus dangyuja leaves have great potential as a source for natural antioxidant which can be applied in food products.

Keywords

References

  1. Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and aging. Free Radical Biol. Med. 29: 222-230 (2000) https://doi.org/10.1016/S0891-5849(00)00317-8
  2. Halliwell B, Aeschbach R, Loliger J, Aruoma OI. The characterization of antioxidants. Food Chem. Toxicol. 33: 601-617 (1995) https://doi.org/10.1016/0278-6915(95)00024-V
  3. Lopaczynski W, Zeisel SH. Antioxidants, programmed cell death, and cancer. Nutr. Res. 21: 295-307 (2001) https://doi.org/10.1016/S0271-5317(00)00288-8
  4. Cui K, Luo X, Xu K, Ven Murthy MR. Role of oxidative stress in neurodegeneration: Recent developments in assay methods for oxidative stress and nutraceutical antioxidants. Prog. Neuro-Psychoph. 28: 771-799 (2004) https://doi.org/10.1016/j.pnpbp.2004.05.023
  5. Ballinger SW. Mitochondrial dysfunction in cardiovascular disease. Free Radical Biol. Med. 38: 1278-1295 (2005) https://doi.org/10.1016/j.freeradbiomed.2005.02.014
  6. Sgambato A, Ardito R, Faraglia B, Boninsegna A, Wolf FI, Cittadini A. Resveratrol, a natural phenolic compound, inhibits cell proliferation, and prevents oxidative DNA damage. Mutat. Res. - Gen. Tox. En. 496: 171-180 (2001) https://doi.org/10.1016/S1383-5718(01)00232-7
  7. Barlow SM. Toxicological aspects of antioxidants used as food additives. pp. 253-307. In: Food Antioxidants. Hudson BJF (ed). Elsevier Science Publishers Ltd., Barking, UK (1990)
  8. Finkel T, Holbrook NJ. Oxidants, oxidative stress, and the biology of ageing. Nature 408: 239-247 (2000) https://doi.org/10.1038/35041687
  9. Loguercio C, Federico A. Oxidative stress in viral and alcoholic hepatitis. Free Radical Bio. Med. 34: 1-10 (2003) https://doi.org/10.1016/S0891-5849(02)01167-X
  10. Vitaglione P, Morisco F, Caporaso N, Fogliano V. Dietary antioxidant compounds and liver health. Crit. Rev. Food Sci. 44: 575-586 (2004) https://doi.org/10.1080/10408690490911701
  11. Yoo MA, Kim JS, Chung HK, Park WJ, Kang MH. The antioxidant activity of various cultivars of grape skin extract. Food Sci. Biotechnol. 16: 884-888 (2007)
  12. Hwang IG, Woo KS, Kim DJ, Hong JT, Hwang BY, Lee YR, Jeong HS. Isolation and identification of an antioxidant substance from heated garlic (Allium sativum L.). Food Sci. Biotechnol. 16: 963-966 (2007)
  13. Shin SR, Hong JY, Yoon KY. Antioxidant properties and total phenolic contents of cherry elaegnus (Elaeagnus multiflora Thunb.) leaf extracts. Food Sci. Biotechnol. 17: 608-612 (2008)
  14. Gorinstein S, Milena C, Machackova I, Haruenkit R, Park YS, Jung ST, Yamamoto K, Ayala AL, Katrich E, Trakhtenberg S. Characterization of antioxidant compounds in Jaffa sweeties and white grapefruits. Food Chem. 84: 503-510 (2004) https://doi.org/10.1016/S0308-8146(03)00127-4
  15. Sun CD, Chen KS, Chen Y, Chen QJ. Contents and antioxidant capacity of limonin and nomilin in different tissues of Citrus fruit of four cultivars during fruit growth and maturation. Food Chem. 93: 599-605 (2005) https://doi.org/10.1016/j.foodchem.2004.10.037
  16. Mokbel MS, Hashinaga F. Evaluation of the antioxidant activity of extracts from buntan (Citrus grandis Osbeck) fruit tissues. Food Chem. 94: 529-534 (2006) https://doi.org/10.1016/j.foodchem.2004.11.042
  17. Lim HK, Yoo ES, Moon JY, Jeon YJ, Cho SK. Antioxidant activity of extracts from dangyuja (Citrus grandis Osbeck) fruits produced in Jeju Island. Food Sci. Biotechnol. 15: 312-316 (2006)
  18. Lima CF, Fernandes-Ferreira M, Pereira-Wilson C. Phenolic compounds protect HepG2 cells from oxidative damage: Relevance of glutathione levels. Life Sci. 79: 2056-2068 (2006) https://doi.org/10.1016/j.lfs.2006.06.042
  19. 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. Pharm. 212: 110-118 (2006) https://doi.org/10.1016/j.taap.2005.07.014
  20. Kim HY. Distribution, taxonomy, horticultural characters of the local Citrus spp. in Cheju and the genetic markers among them. PhD thesis, Chonnam National University, Gwangju, Korea (1988)
  21. Cheung LM, Cheung PC, Ooi VE. Antioxidant activity and total phenolics of edible mushroom extracts. Food Chem. 81: 249-255 (2003) https://doi.org/10.1016/S0308-8146(02)00419-3
  22. Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64: 555-559 (1999) https://doi.org/10.1016/S0308-8146(98)00102-2
  23. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 181: 1199-1200 (1958) https://doi.org/10.1038/1811199a0
  24. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Bio. Med. 27: 612-616 (1999) https://doi.org/10.1016/S0891-5849(99)00107-0
  25. Datta L, Babbar P, Srivastava T, Sinha S, Chattopadhyay P. p53 Dependent apoptosis in glioma cell lines in response to hydrogen peroxide induced oxidative stress. Int. J. Biochem. Cell B 34: 148-157 (2002) https://doi.org/10.1016/S1357-2725(01)00106-6
  26. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95: 351-358 (1979) https://doi.org/10.1016/0003-2697(79)90738-3
  27. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35: 206-221 (2000) https://doi.org/10.1002/(SICI)1098-2280(2000)35:3<206::AID-EM8>3.0.CO;2-J
  28. Hansen MB, Nielsen SE, Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods 119: 203-210 (1989) https://doi.org/10.1016/0022-1759(89)90397-9
  29. Rice-Evans CA, Miller NJ, Paganga G. Antioxidant properties of phenolic compounds. Trends Plant Sci. 2: 152-159 (1997) https://doi.org/10.1016/S1360-1385(97)01018-2
  30. Luo XD, Basile MJ, Kennelly EJ. Polyphenolic antioxidants from the fruits of Chrysophyllum cainito L. (star apple). J. Agr. Food Chem. 50: 1379-1382 (2002) https://doi.org/10.1021/jf011178n
  31. Veglioglu YS, Mazza G, Gao L, Oomah BD. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agr. Food Chem. 46: 4113-4117 (1998) https://doi.org/10.1021/jf9801973
  32. Jayaprakasha GK, Patil BS. In vitro evaluation of the antioxidant activities in fruit extracts from citron and blood orange. Food Chem. 101: 410-418 (2007) https://doi.org/10.1016/j.foodchem.2005.12.038
  33. Brand-Williams W, Cuvelier ME, Berset C. Use of a free-radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 28: 25-30 (1995) https://doi.org/10.1016/S0023-6438(95)80008-5
  34. Alia M, Ramos S, Mateos R, Bravo L, Goya L. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J. Biochem. Mol. Toxic. 19: 119-128 (2005) https://doi.org/10.1002/jbt.20061
  35. Nieminen AL, Byrne AM, Herman B, Lemasters JJ. Mitochondrial permeability transition in hepatocytes induced by t-BuOOH:-NAD(P)H and reactive oxygen species. Am. J. Physiol.-Cell Ph. 272: 1286-1294 (1997) https://doi.org/10.1152/ajpcell.1997.272.4.C1286
  36. Amoroso S, Gioielli A, Cataldi M, Di Renzo G, Annunziato L. In the neuronal cellline SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular $Ca^{2+}$ increase. Biochim. Biophys. Acta 1452: 151-160 (1999) https://doi.org/10.1016/S0167-4889(99)00110-X
  37. 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. 62: 705-712 (2001) https://doi.org/10.1016/S0006-2952(01)00704-3
  38. Korsmeyer SJ. Regulators of cell death. Trends Genet. 11: 101-105 (1995) https://doi.org/10.1016/S0168-9525(00)89010-1
  39. Rich T, Watson CJ, Wyllie A. Apoptosis: The germs of death. Nat. Cell Biol. 1: 69-71 (1999) https://doi.org/10.1038/9045
  40. Labieniec M, Gabryelak T, Falcioni G. Antioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus. Mutat. Res. -Gen. Tox. En. 539: 19-28 (2003) https://doi.org/10.1016/S1383-5718(03)00115-3