Evaluation of Different Methods of Antioxidant Measurement

  • Yoo, Kyung-Mi (Department of Food Science and Technology, Cornell University) ;
  • Kim, Dae-Ok (Department of Food Science and Technology, Institute of Life Science and Resources, Kyung Hee University) ;
  • Lee, Chang-Yong (Department of Food Science and Technology, Cornell University)
  • Published : 2007.04.30


The beneficial effects of fruits, vegetables, and beverages on human health have been attributed to their antioxidant activities. Therefore, antioxidant activity of food products is recognized as one of the important parameters in determining their functional values. Until now, antioxidant activity has been measured by various chemical and biological methods; however, many factors confound the reliability and reproducibility of measurements of antioxidant activity of food. In vitro methods may provide a useful indication of antioxidant activity but their results may not translate to the human biological system, while in vivo tests are difficult to carry out due to the intricate processes of uptake, cellular transportation, and metabolism of individual antioxidant components. Therefore, as long as these limitations exist, our best option is to measure the antioxidant activity in food directly. This review briefly summarizes currently available methods for the measurement of antioxidant activity in food and examines their respective validity.


  1. 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
  2. Aruoma OI, Evans PJ, Kaur H, Sutcliffe L, Halliwell B. An evaluation of the antioxidant and potential pro-oxidant properties of food additives and of Trolox C, vitamin E, and probucol. Free Radical Res. Com. 10: 143-157 (1990) https://doi.org/10.3109/10715769009149883
  3. Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: a review of the epidemiological evidence. Nutr. Cancer 18: 1-29 (1992) https://doi.org/10.1080/01635589209514201
  4. Halliwell B, Gutteridge JM. Free radicals and antioxidant protection: mechanisms and significance in toxicology and disease. Hum. Toxicol. 7: 7-13 (1988) https://doi.org/10.1177/096032718800700102
  5. Halliwell B. Role offree radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drug Aging 18: 685-716 (2001) https://doi.org/10.2165/00002512-200118090-00004
  6. Halliwell B. How to characterize an antioxidant: an update. Biochem. Soc. Symp. 61: 73-101 (1995)
  7. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the degenerative diseases of aging. P. Natl. Acad. Sci. USA 90: 7915-7922 (1993) https://doi.org/10.1073/pnas.90.17.7915
  8. Halliwell B. Free radicals and antioxidants: a personal view. Nutr. Rev. 52: 253-265 (1994) https://doi.org/10.1111/j.1753-4887.1994.tb01453.x
  9. Halliwell B. Antioxidant characterization. Methodology and mechanism. Biochem. Pharmacol. 49: 1341-1348 (1995) https://doi.org/10.1016/0006-2952(95)00088-H
  10. Lemanska K, Szymusiak H, Tyrakowska B, Zielinski R, Soffers AEMF, Rietjens IMCM. The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones. Free Radical Bio. Med. 31: 869-881 (2001) https://doi.org/10.1016/S0891-5849(01)00638-4
  11. Nordberg J, Amer ESJ. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radical Bio. Med. 31: 1287-1312 (2001) https://doi.org/10.1016/S0891-5849(01)00724-9
  12. Fridovich I. The biology of oxygen radicals. Science 201: 875-880 (1978) https://doi.org/10.1126/science.210504
  13. Kim YC, Chung SK. Reactive oxygen radical species scavenging effects of Korean medicinal plant leaves. Food Sci. Biotechnol. 11: 407 -411 (2002)
  14. Halliwell B, Gutteridge JM. Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1: 1396-1397 (1984)
  15. Herrmann K. Occurrence and content of hydroxycinnamic and hydroxybenzoic acid compounds in foods. Crit. Rev. Food Sci. 28: 315-347 (1989) https://doi.org/10.1080/10408398909527504
  16. Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI, Halliwell B. Evaluation of the antioxidant activity of melatonin in vitro. Free Radical. Bio. Med. 21: 307-315 (1996) https://doi.org/10.1016/0891-5849(96)00046-9
  17. Rice-Evans C, Miller NJ. Total antioxidant status in plasma and body fluids. Method Enzymol. 234: 279-293 (1994) https://doi.org/10.1016/0076-6879(94)34095-1
  18. Long LH, Halliwell B. Antioxidant and prooxidant abilities of foods and beverages. Method Enzymol. 335: 181-190 (2001) https://doi.org/10.1016/S0076-6879(01)35242-4
  19. Amao MB, Cano A, Acosta M. Methods to measure the antioxidant activity in plant material. A comparative discussion. Free Radical Res. 31: S89-S96 (1999) https://doi.org/10.1080/10715769900301371
  20. Kim DO, Lee CY. Comprehensive study on vitamin C equivalent antioxidant capacity (VCEAC) of various polyphenolics in scavenging a free radical and its structural relationship. Crit. Rev. Food Sci. 44: 253-273 (2004) https://doi.org/10.1080/10408690490464960
  21. Gutteridge JM. Biological origin of free radicals, and mechanisms of antioxidant protection. Chem. -BioI. Interact. 91: 133-140 (1994) https://doi.org/10.1016/0009-2797(94)90033-7
  22. Patel MN. Oxidative stress, mitochondrial dysfunction, and epilepsy. Free Radical Res. 36: 1139-1146 (2002) https://doi.org/10.1080/1071576021000016391
  23. Husain SR, Cillard J, Cillard P. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry 26: 2489-2491 (1987) https://doi.org/10.1016/S0031-9422(00)83860-1
  24. Potapovich AI, Kostyuk VA. Comparative study of antioxidant properties and cytoprotective activity of flavonoids. Biochemistry Moscow 68: 514-519 (2003) https://doi.org/10.1023/A:1023947424341
  25. Farmer EH, Bloomfield GF, Sundralingam A, Sutton DA. The course and mechanism of autoxidation reactions in olefinic and polyolefinic substances, including rubber. T. Faraday Soc. 38: 0348-0355 (1942) https://doi.org/10.1039/tf9423800348
  26. Cheeseman KH, Slater TF. An introduction to free radical biochemistry. Brit. Med. Bull. 49: 481-493 (1993) https://doi.org/10.1093/oxfordjournals.bmb.a072625
  27. Cheeseman KH. Mechanisms and effects of lipid peroxidation. Mol. Aspects Med. 14: 191-197 (1993) https://doi.org/10.1016/0098-2997(93)90005-X
  28. Halliwell B. How to characterize a biological antioxidant. Free Radical Res. Com. 9: 1-32 (1990) https://doi.org/10.3109/10715769009148569
  29. Frankel EN, Meyer AS. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agr. 80: 1925-1941 (2000) https://doi.org/10.1002/1097-0010(200010)80:13<1925::AID-JSFA714>3.0.CO;2-4
  30. Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. J. Agr. Food Chem. 53: 1841-1856 (2005) https://doi.org/10.1021/jf030723c
  31. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin. Sci. 84: 407-412 (1993) https://doi.org/10.1042/cs0840407
  32. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio. Med. 26: 1231-1237 (1999) https://doi.org/10.1016/S0891-5849(98)00315-3
  33. Miller NJ, Rice-Evans CA. Factors influencing the antioxidant activity determined by the $ABTS^{*+}$ radical cation assay. Free Radical Res. 26: 195-199 (1997) https://doi.org/10.3109/10715769709097799
  34. Miller NJ, Rice-Evans C, Davies MJ. A new method for measuring antioxidant activity. Biochem. Soc. T. 21: 95S (1993)
  35. Long LH, Kwee DC, Halliwell B. The antioxidant activities of seasonings used in Asian cooking. Powerful antioxidant activity of dark soy sauce revealed using the ABTS assay. Free Radical Res. 32: 181-186 (2000) https://doi.org/10.1080/10715760000300181
  36. Kim DO, Lee KW, Lee HJ, Lee CY. Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J. Agr. Food Chem. 50: 3713-3717 (2002) https://doi.org/10.1021/jf020071c
  37. van den Berg R, Haenen GRMM, van den Berg H, Bast A. Applicability of an improved Trolox equivalent antioxidant capacity (TEAC) assay for evaluation of antioxidant capacity measurements of mixtures. Food Chem. 66: 511-517 (1999) https://doi.org/10.1016/S0308-8146(99)00089-8
  38. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensm. -Wiss, Technol. 28: 25-30 (1995) https://doi.org/10.1016/S0023-6438(95)80008-5
  39. Koleva, II, van Beek TA, Linssen JP, de Groot A, Evstatieva LN. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochem. Analysis 13: 8-17 (2002) https://doi.org/10.1002/pca.611
  40. Kim SJ, Kim GH. Quantification of quercetin in different parts of onion and its DPPH radical scavenging and antibacterial activity. Food Sci. Biotechnol. 15: 39-43 (2006)
  41. Sanchez-Moreno C, Larrauri JA, Saura-Calixto F. A procedure to measure the antiradical efficiency of polyphenols. J. Sci. Food Agr. 76: 270-276 (1998) https://doi.org/10.1002/(SICI)1097-0010(199802)76:2<270::AID-JSFA945>3.0.CO;2-9
  42. Awika JM, Rooney LW, Wu XL, Prior RL, Cisneros-Zevallos L. Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. J. Agr. Food Chem. 51: 6657-6662 (2003) https://doi.org/10.1021/jf034790i
  43. Ou B, Huang D, Hampsch-Woodill M, Flanagan JA, Deemer EK. Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study. J. Agr. Food Chem. 50: 3122-3128 (2002) https://doi.org/10.1021/jf0116606
  44. Ou B, Hampsch- Woodill M, Flanagan J, Deemer EK, Prior RL, Huang DJ. Novel fluorometric assay for hydroxyl radical prevention capacity using fluorescein as the probe. J. Agr. Food Chem. 50: 2772-2777 (2002) https://doi.org/10.1021/jf011480w
  45. Prior RL, Cao G. Analysis of botanicals and dietary supplements for antioxidant capacity: a review. J. AOAC Int. 83: 950-956 (2000)
  46. Cao G, Alessio HM, Cutler RG. Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Bio. Med. 14: 303-311 (1993) https://doi.org/10.1016/0891-5849(93)90027-R
  47. Cao G, Sofie E, Prior RL. Peroxyl radical absorbing antioxidant activities of flavonoids. FASEB J. 10: 4745-4745 (1996)
  48. Wang H, Cao GH, Prior RL. Total antioxidant capacity of fruits. J. Agr. Food Chem. 44: 701-705 (1996) https://doi.org/10.1021/jf950579y
  49. Sofie E, Cao G, Prior RL. Antioxidant capacity of tea and vegetables. FASEB J. 10: 1148-1148 (1996)
  50. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of 'antioxidant power': the FRAP assay. Anal. Biochem. 239: 70-76 (1996) https://doi.org/10.1006/abio.1996.0292
  51. Boo HO, Chon SU, Kim SM, Pyo BS. Antioxidant activities of colored sweet potato cultivars by plant parts. Food Sci. Biotechnol. 14: 177-180 (2005)
  52. Davalos A, Gomez-Cordoves C, Bartolome B. Extending applicability of the oxygen radical absorbance capacity (ORAC-fluorescein) assay. J. Agr. Food Chem. 52: 48-54 (2004) https://doi.org/10.1021/jf0305231
  53. Mathew S, Abraham TE. In vitro antioxidant activity and scavenging effects of cinnamomum verum leaf extract assayed by different methodologies. Food Chem. Toxicol. 44: 198-206 (2006) https://doi.org/10.1016/j.fct.2005.06.013
  54. 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)
  55. Park YK, Lee WY, Park SY, Ahn JK, Han MS. Antioxidant activity and total phenolic content of Callistemon citrinus extracts. Food Sci. Biotechnol. 14: 212-215 (2005)
  56. Herrmann K. Flavonols and flavones in food plants: a review. J. Food Technol. 11: 433-448 (1976) https://doi.org/10.1111/j.1365-2621.1976.tb00743.x
  57. Miller NJ, Sampson J, Candeias LP, Bramley PM, Rice-Evans CA. Antioxidant activities of carotenes and xanthophylls. FEBS Lett. 384: 240-242 (1996) https://doi.org/10.1016/0014-5793(96)00323-7
  58. Roginsky V, Alegria AE. Oxidation of tea extracts and tea catechins by molecular oxygen. J. Agr. Food Chem. 53: 4529-4535 (2005) https://doi.org/10.1021/jf040382i
  59. Chun OK, Kim DO, Smith N, Schroeder D, Han JT, Lee CY. Daily consumption of phenolics and total antioxidant capacity from fruit and vegetables in the American diet. J. Sci. Food Agr. 85: 1715-1724 (2005) https://doi.org/10.1002/jsfa.2176