Journal of Food Science and Technology

, Volume 48, Issue 4, pp 412–422 | Cite as

Genesis and development of DPPH method of antioxidant assay

  • Sagar B. Kedare
  • R. P. SinghEmail author


α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging method offers the first approach for evaluating the antioxidant potential of a compound, an extract or other biological sources. This is the simplest method, wherein the prospective compound or extract is mixed with DPPH solution and absorbance is recorded after a defined period. However, with the advancement and sophistication in instrumental techniques, the method has undergone various modifications to suit the requirements, even though the basic approach remains same in all of them. This article presents a critical review on various developments to the DPPH method.


DPPH Absorbance Free radicals Antioxidant SIA 


  1. Abuin E, Lissi E, Ortiz P, Henriquez C (2002) Uric acid reaction with DPPH radicals at the micellar interface. Bol Soc Chil Quim 47:145–149Google Scholar
  2. Ancerewicz J, Miglavaca E, Carrupt PA, Testa B, Bree F, Zinin R, Tillement JP, Labidelle P, Goyot SD, Chauvet-Monges AM, Crevent A, Le Ridant A (1998) Structure property relationship of trimetadizine derivatives and model compounds as potential antioxidants. Free Rad Biol Med 25(1):113–120CrossRefGoogle Scholar
  3. Arnao MB (2000) Some methodological problems in the determination of antioxidant activity using chromogen radicals:a practice case. Tr Food Sc Tehnol 11(11):419–421CrossRefGoogle Scholar
  4. Bandoniene D, Murkovic M, Pfannhauser W, Venskutonis PR, Gruzdiene D (2002) Detection and activity evaluation of radical scavenging compounds by using DPPH free radical and on-line HPLC– DPPH methods. Euro Food Res Technol 214(2):143–147CrossRefGoogle Scholar
  5. Blois MS (1958) Antioxidant determinations by the use of a stable free radical. Nature 181:1199–1200CrossRefGoogle Scholar
  6. Bondet V, Brand-Williams W, Berset C (1997) Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Lebensmitt Wissenschaft Technologie Food Sci Technol 30:609–615CrossRefGoogle Scholar
  7. Brand-Williams W, Cuvelier ME, Berset C (1995) Use of a free radical method to evaluate antioxidant activity. Food Sci Technol 28:25–30Google Scholar
  8. Buijnster M, Bicanic D, Chirtoc M, Nicoli MC, Min-Kucience Y (2001) Evaluation of antioxidative activity of some antioxidants by means of combined opthothermal window and DPPH free radical colorimetry. Anal Sci 17:544–546Google Scholar
  9. Cerda V, Estela JM, Forteza R, Cladera A, Becerra E, Altimira P, Sitjar P (1999) An environmental friendly method for the automatic determination of hypochlorite in commercial products using multisyringe flow injection analysis. Talanta 50:695CrossRefGoogle Scholar
  10. Chandrasekar D, Madhusudhana K, Ramakrishna S, Diwan PV (2006) Determination of DPPH free radical scavenging activity by reversed-phase HPLC: A sensitive screening method for polyherbal formulations. J Pharm Biomed Anal 40(2):460–464CrossRefGoogle Scholar
  11. Changqing W, Feng C, Xi W, Yonnie W, Meidui D, Guoqing H, Ronald DG, Lilin H, Guohui H (2007) Identification of Antioxidant Phenolic Compounds in Feverfew (Tanacetum parthenium) by HPLC-ESI-MS/MS and NMR. Phytochem Anal 18:401–410CrossRefGoogle Scholar
  12. Chevion S, Berry EM, Kitrossky NK, Kohen N (1997) Evaluation of plasma low molecular weight antioxidant capacity by cyclic voltammetry. Free Radic Biol Med 22:411–421CrossRefGoogle Scholar
  13. Contreras-Guzman ES, Strong FC (1982) Determination of tocopherols (Vitamin E) by reduction of cupric ion. JAOAC 65:1215–1222Google Scholar
  14. Cui K, Luo X, Murthy MRV (2004) Role of oxidative stress in neurodegeneration: recent developments in assay methods for oxidative stress and nutraceutical antioxidants. Prog Neuropsychopharmacol Biol Psych 28:771–799CrossRefGoogle Scholar
  15. Dan T, Hui-Jun L, Jun C, Chao-Wei G, Ping L (2008) Rapid and simple method for screening of natural antioxidants from Chinese herb Flos Lonicerae Japonicae by DPPH-HPLC-DAD-TOF/MS. J Sep Sci 31(20):3519–3526CrossRefGoogle Scholar
  16. Dapkevicius A, van Beek TA, Niederlander HAG, de Groot AE (1999) Evaluation and comparison of two improved techniques for the on-line detection of antioxidants in liquid chromatography eluates. Anal Chem 71:736–740CrossRefGoogle Scholar
  17. David MR, Christopher O, Slavko K, Georgie F, Alexander P, Peter IA, Yoichiro I, Ilya R (2007) Preparative isolation and identification of tyrosinase inhibitors from the seeds of Garcinia kola by high-speed counter-current chromatography. J Chromato A 1151(1–2):45–50Google Scholar
  18. Gil MI, Thomas Barberan FA, Hess-Pierce B, Hplcroft DM, Kader AA (2000) Antioxidant activity of pomegranate juice and its relation ship with phenolic composition and processing. J Agri Food Chem 48:4581–4589CrossRefGoogle Scholar
  19. Glavind J, Holmer G (1967) Thin-Layer Chromatographic Determination of Antioxidants by the Stable Free Radical Diphenyl-picrylhydrazyl. Ibid 44:539–542Google Scholar
  20. Gomez-Alonso S, Fregapane G, Salvador MD, Gordon MH (2003) Changes in phenolic composition and antioxidant activity of virgin olive oil during frying. J Agri Food Chem 51:667–672CrossRefGoogle Scholar
  21. Guo JT, Lee HL, Chiang SH, Lin FI, Chang CY (2001) Antioxidant properties of the extracts from different parts of broccoli in Taiwan. J Food Drug Anal 9(2):96–101Google Scholar
  22. Gutzeit D, Wray VP, Winterhalter P, Jerz G (2007) Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. sp. rhamnoides) by High-Speed Counter-Current Chromatography. Chromatographia 65(1–2):1–7Google Scholar
  23. Halliwell B (1996) Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in humans. Free Rad Res 25(1):57–74CrossRefGoogle Scholar
  24. Halliwell B (1997) Antioxidants and human disease: a general introduction. Nutr Rev 5:544–552Google Scholar
  25. Ionita G, Em Sahini V, Semenescu G, Ionita P (2000) Kinetics of oxidation of amino acids by some free stable hydrazyl radicals. Act Chim Slov 47:111–119Google Scholar
  26. Karunakar V, Prabhakar MC, Krishna V (2003) Determination of antioxidant activity of some drugs using high-pressure liquid chromatography. Arzneim Forsch Drug Res 53:254–259Google Scholar
  27. Kim JK, Noh JH, Lee S, Choi JS, Suh H, Chung HY, Song YO, Choi WC (2002) The first total synthesis of 2, 3, 6-tribromo-4, 5-dihydroxybenzyl methyl ether (TDB) and its antioxidant activity. Bull Korean Chem Soc 23(5):661–662CrossRefGoogle Scholar
  28. Koleva II, Niederlander HAG, van Beek TA (2000) An on-line HPLC method for detection of radical scavenging compounds in complex mixtures. Anal Chem 72:2323–2328CrossRefGoogle Scholar
  29. Kosar M, Dorman HJD, Bachmayer O, Baser KHC, Hiltunen R (2003) An improved on-line HPLC-DPPH method for the screening of free radical scavenging compounds in water extracts of Lamiaceae plants. Plant Chem Nat Comp 39:161–166CrossRefGoogle Scholar
  30. Lebeau J, Furman C, Bernier JL, Duriez P, Teissier E, Cotelle N (2000) Antioxidant properties of di-tert-butylhydroxylated flavonoids. Free Radic Biol Med 29(9):900–912CrossRefGoogle Scholar
  31. Leitao GG, Leitao SG, Vilegas W (2002) Quick preparative separation of natural naphthoquinones with antioxidant activity by high-speed counter-current chromatography. Z Naturforsch 57:1051–1055Google Scholar
  32. Li P, Anu H, Jari S, Teijo Y, Heikki V (2005). TLC method for evaluation of free radical scavenging activity of rapeseed meal by video scanning technology,, 2005 (Accessed on September 2008).
  33. Lu Y, Foo LY (2000) Antioxidant and radical scavenging activities of polyphenols from apple pomace. Food Chem 68:81–85CrossRefGoogle Scholar
  34. Luıs MM, Marcela AS, Salette R, Lima JLFC (2006) Automatic method for determination of total antioxidant capacity using 2, 2-diphenyl-1-picrylhydrazyl assay. Anal Chim Acta 558:310–318CrossRefGoogle Scholar
  35. Masahiro N, Masahiro K, Minemitsu N, Akio K, Yoshimi N (2005) Non-reductive Scavenging of 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) by Peroxyradical: A Useful Method for Quantitative Analysis of Peroxyradical. Chem Pharm Bull 53(6):714–716CrossRefGoogle Scholar
  36. Milardovic S, Ivekovic D, Rumenjak V, Bozidar SG (2005) Use of DPPH./DPPH redox couple for biamperometric determination of antioxidant activity. Electroanalysis 17(20):1847–1853CrossRefGoogle Scholar
  37. Min DB (1998) Lipid oxidation of edible oil. In: Akoh K, Min DB (eds) Food Lipids: Chemistry, Nutrition and Biotechnology. Marcel Dekkar, New York, pp 283–296Google Scholar
  38. Molyneux P (2004) The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J Sci Technol 26(2):211–219Google Scholar
  39. Nuengchamnong N, de Jong CF, Bruyneel B, Niessen WMA, Irth H, Ingkanina K (2005) HPLC Coupled On-line to ESI-MS and a DPPH-based assay for the rapid identification of antioxidants in Butea superba. Phytochem Anal 16:422–428CrossRefGoogle Scholar
  40. Olga NP, Svetlana AI, Alexander NS, Valery GM (2007) Separation and evaluation of free radical-scavenging activity of phenol components of Emblica officinalis extract by using an HPTLC-DPPH method. J Sep Sci 30(9):1250–1254CrossRefGoogle Scholar
  41. Ozcelik B, Lee JH, Min DB (2003) Effects of light, oxygen, and pH on the absorbance of 2, 2-Diphenyl-1-picrylhydrazyl. J Food Sci 68(2):487–490CrossRefGoogle Scholar
  42. Parry J, Su L, Luther M, Zhou KQ, Yurawecz MP, Whittaker P, Yu LL (2005) Fatty acid composition and antioxidant properties of cold-pressed marionberry, boysenberry, red raspberry, and blueberry seed oils. J Agric Food Chem 53:566–573CrossRefGoogle Scholar
  43. Perez-Bonilla M, Salido S, van Beek TA, Linares-Palomino PJ, Altarejos J, Nogueras M, Sanchez A (2006) Isolation and identification of radical scavengers in olive tree (Olea europaea) wood. J Chromato A 1112(1–2):311–318CrossRefGoogle Scholar
  44. Polasek M, Skala P, Opletal L, Jahodar L (2004) Rapid automated assay of anti-oxidation/radical-scavenging activity of natural substances by sequential injection technique (SIA) using spectrophotometric detection. Anal Bioanal Chem 379:754–758CrossRefGoogle Scholar
  45. Prakash A (2001) Antioxidant activity. Med Lab Anal Prog 19(2):1–6Google Scholar
  46. Prior RL, Wu X, Schaich K (2005) Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agri Food Chem 53:4290–4302CrossRefGoogle Scholar
  47. Pukalskas A, van Beek TA (2005) Development of a triple hyphenated HPLC–radical scavenging detection–DAD–SPE–NMR system for the rapid identification of antioxidants in complex plant extracts. J Chromato A 1074(1–2):81–88CrossRefGoogle Scholar
  48. Pukalskas A, van Beek TA, Venskutonis RP, Linssen JPH, van Veldhuizen A, de Groot A (2002) Identification of radical scavengers in sweet grass (Hierochloe odorata). J Agric Food Chem 50:2914–2919CrossRefGoogle Scholar
  49. Rocha FRP, Reis BF, Zagatto EAG, Lima JLFC, Lapa RAS, Lapa SJLM (2002) Multicommutation in flow analysis: concepts, applications and trends. Anal Chim Acta 468:119CrossRefGoogle Scholar
  50. Ruiz MA, Reviejo AJ, Parrado C, Pingarron JM (1996) Development of an amperometric enzyme biosensor for the determination of the antioxidant tert-butylhydroxyanisole in a medium of reversed micelles. Electroanalysis 8(6):529–533CrossRefGoogle Scholar
  51. Sanchez-Moreno C (2002) Methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Sci Technol Int 8(3):121–137Google Scholar
  52. Sanchez-Moreno C, Larrauri JA, Saura-Calixto F (1998) A procedure to measure the antiradical efficiency of polyphenols. J Sci Food Agri 79:270–276CrossRefGoogle Scholar
  53. Sanchez-Moreno C, Larrauri JA, Saura-Calixto F (1999a) Free radical scavenging capacityand inhibition of wines, grape juices and related polyphenolic constituents. Food Res Int 32:407–412CrossRefGoogle Scholar
  54. Sanchez-Moreno C, Larrauri JA, Saura-Calixto F (1999b) Free radical scavenging capacity of selected red, rose and white wines. J Sci Food Agri 79:1301–1304CrossRefGoogle Scholar
  55. Schwarz K, Bertelsen G, Nissen LR, Gardner PT, Heinonen MI, Hopia A, Huynh-Ba T, Lambelet P, McPhail D, Skibsted LH, Tijburg L (2001) Investigation of plant extracts for the protection of processed foods against lipid oxidation. Comparison of antioxidant assays based on radical scavenging, lipid oxidation and analysis of the principal antioxidant compounds. Eur Food Res Technol 212:319–328CrossRefGoogle Scholar
  56. Sendra JM, Sentandreu E, Navarro JL (2006) Reduction kinetics of the free stable radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH•) for determination of the antiradical activity of citrus juices. Eur Food Res Technol 223:615–624CrossRefGoogle Scholar
  57. Shuyun S, Honghao Z, Yuping Z, Kelong H (2008) Hyphenated HSCCC–DPPH for rapid preparative isolation and screening of antioxidants from Selaginella moellendorffii. Chromatographia 68:173–178CrossRefGoogle Scholar
  58. Singh S, Singh RP (2008) In vitro methods of assay of antioxidants: An overview. Food Rev Int 24(4):392–415CrossRefGoogle Scholar
  59. Sung-Kun Y, Su-Jung Y, Chul-Ho Y (2004) A continuous spectrophotometric assay for NADPH-cytochrome P450 reductase activity using 1, 1-diphenyl-2-picrylhydrazyl. J Biochem Mol Biol 37:629–633CrossRefGoogle Scholar
  60. Takao T, Kitatani F, Watanabe N, Yagi A, Sakata KA (1994) Simple Screening Method for Antioxidants and Isolation of Several antioxidants Produced by Marine Bacteria from Fish and shellfish. Biosci Biotechnol Biochem 58:1780–1783CrossRefGoogle Scholar
  61. Teijo Y, Li P, Jari S, Anu H, Heikki V (2003) Free radical scavenging activity of phenolics by reversed phase TLC. JAOCS 80(1):9–14CrossRefGoogle Scholar
  62. Ukeda H (2004) Flow-injection analytical system for the evaluation of antioxidative activity. Bunseki Kagaku 53:221CrossRefGoogle Scholar
  63. Ukeda H, Adachi Y, Sawamura M (2002) Flow injection analysis of DPPH radical based on electron spin resonance. Talanta 58:1279CrossRefGoogle Scholar
  64. Vít K, Daniel J, Lubos O, Ludek J, Kamil K (2007) Assay of radical scavenging activity of antidotes against chemical warfare agents by DPPH test using sequential injection technique. J Appl Biomed 5:81–84Google Scholar
  65. Yamaguchi T, Takamura H, Matoba T, Terao J (1998) HPLC method for evaluation of the free radical scavenging activity of foods by using 1,1,-Diphenyl-2-Picrylhydrazyl. Biosci Biotech Biochem 62:1201–1204CrossRefGoogle Scholar
  66. Yepez B, Espinosa M, Lopez S, Bolanos G (2002) Producing antioxidant fractions from herbaceous matrices by supercritical fluid extraction. Fluid Phase Equil 194:879–884CrossRefGoogle Scholar
  67. Yoichiro I (1981) Efficient preparative counter-current chromatography with a coil planet centrifuge. J Chromato 214(1):122–125CrossRefGoogle Scholar
  68. Yu LL (2001) Free radical scavenging properties of conjugated linoleic acids. J Agric Food Chem 49:3452–3456CrossRefGoogle Scholar
  69. Zhihong C, Moore J, Liangli Y (2006) High-Throughput Relative DPPH Radical Scavenging Capacity Assay. J Agric Food Chem 54:7429–7436CrossRefGoogle Scholar
  70. Zhu QY, Hackman RM, Ensunsa JL, Holt RR, Keen CL (2002) Antioxidative activities of oolong tea. J Agric Food Chem 50:6929–6934CrossRefGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2011

Authors and Affiliations

  1. 1.Human Resource DevelopmentCentral Food Technological Research Institute (CSIR)MysoreIndia

Personalised recommendations