Dpph Method

Topics: Antioxidant, Vitamin C, Chemical reaction Pages: 14 (4901 words) Published: May 6, 2013

The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity Philip Molyneux

Molyneux, P.

The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity Songklanakarin J. Sci. Technol., 2004, 26(2) : 211-219
The use of the stable free radical diphenylpicrylhydrazyl (DPPH) to estimate the activity of antioxidants is reviewed. Current applications of the method are examined, particularly the use of the parameter EC50 (substrate concentration to produce 50% reduction of the DPPH). Some recommendations are made as to the most suitable ways of carrying out this assay and evaluating the data produced.

Key words : DPPH, diphenylpicrylhydrazyl, free radical, antioxidant activity Ph.D.(Polymer Chemistry), Macrophile Associates, 9 Brewery Lane, Salisbury, Wiltshire, SP1 2LJ, U.K. E-mail: molyneux@easynet.co.uk Received, 8 June 2003 Accepted, 15 December 2003

Songklanakarin J. Sci. Technol. Vol. 26 No. 2 Mar.-Apr. 2004


Use of DPPH to estimate antioxidant activity Molyneux, P.

There is an increasing interest in antioxidants, particularly in those intended to prevent the presumed deleterious effects of free radicals in the human body, and to prevent the deterioration of fats and other constituents of foodstuffs. In both cases, there is a preference for antioxidants from natural rather than from synthetic sources (Abdalla and Roozen, 1999). There is therefore a parallel increase in the use of methods for estimating the efficiency of such substances as antioxidants ′ (S a nchez-Moreno, 2002; Schwarz, et al., 2001). One such method that is currently popular is based upon the use of the stable free radical diphenylpicrylhydrazyl (DPPH). The purpose of this paper is to examine the basis of this method, and to further examine the use of the parameter “EC50” (equivalent concentration to give 50% effect) which is currently used in the interpretation of experimental data from the method. It should be noted that the present paper is not concerned with the correlation between the results of the DPPH method and the actual activity of the substance in autoxidation reactions (Schwarz, et al., 2001); neither is it concerned with the actual efficiency of these substances either as antioxidants or as “life-style enhancers” in humans (Wanjek, 2001). Basis of the Method 1. DPPH - free radical and reduced form The molecule of 1,1-diphenyl-2-picryl-

hydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH: 1) is characterised as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecules do not dimerise, as would be the case with most other free radicals. The delocalisation also gives rise to the deep violet colour, characterised by an absorption band in ethanol solution centred at about 520 nm. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form (2) with the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present). Representing the • DPPH radical by Z and the donor molecule by AH, the primary reaction is Z• + AH = ZH + A• [1]

• where ZH is the reduced form and A is free radical produced in this first step. This latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorised) by one molecule of the reductant. The reaction [1] is therefore intended to provide the link with the reactions taking place in an oxidising system, such as the autoxidation of a lipid or other unsaturated substance; the DPPH • molecule Z is thus intended to represent the free radicals formed in the system whose activity is to

1: Diphenylpicrylhydrazyl (free radical)

2: Diphenylpicrylhydrazine (nonradical)

Songklanakarin J. Sci. Technol....

References: Abdalla, A.E. and Roozen, J.P. 1999. Effect of plant extracts on the oxidative stability of sunflower oil and emulsion, Food Chemistry, 64: 323-329. Blois, M.S. 1958. Antioxidant determinations by the use of a stable free radical, Nature, 181: 11991200. Bondet, V., Brand-Williams, W. and Berset, C. 1997. Kinetics and mechanisms of antioxidant activity using the DPPH• free radical method, Lebensmittel-Wissenschaft und -Technologie/Food Science and Technology, 30: 609-615. Brand-Williams, W., Cuvelier, M.E. and Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity, Lebensmittel-Wissenschaft und -Technologie/Food Science and Technology, 28: 25-30.
Songklanakarin J. Sci. Technol. Vol. 26 No. 2 Mar.-Apr. 2004

Use of DPPH to estimate antioxidant activity Molyneux, P.
G O mez-Alonso, S., Fregapane, G., Salvador, M.D. and Gordon, M.H. 2003. Changes in phenolic composition and antioxidant activity of virgin olive oil during frying, J. Agric. Food Chem., 51: 667672. Guo, J.-T., Lee, H.-L., Chiang, S.-H., Lin, F.I. and Chang, C.-Y. 2001. Antioxidant properties of the extracts from different parts of broccoli in Taiwan, J. Food Drug Anal., 9(2): 96-101. Kim, J.-K., Noh, J.H., Lee, S., Choi, J.S., Suh, H., Chung, H.Y., Song, Y.-O. and Choi, W.C. 2002. The first total synthesis of 2,3,6-tribromo-4,5dihydroxybenzyl methyl ether (TDB) and its antioxidant activity, Bull. Korean Chem. Soc., 23(5): 661-662. Lebeau, J., Furman, C., Bernier, J.-L., Duriez, P., Teissier, E. and Cotelle, N. 2000. Antioxidant properties of di-tert-butylhydroxylated flavonoids, Free Radic. Biol. Med., 29(9): 900-912. ˜ o, G.G., Leit a o, S.G. and Vilegas, W. 2002. ˜ Leit a Quick preparative separation of natural naphthoquinones with antioxidant activity by high-speed counter-current chromatography, Z.Naturforsch., 57c: 1051-1055. Lu, Y. and Foo, L.Y. 2000. Antioxidant and radical scavenging activities of polyphenols from apple pomace, Food Chemistry, 68: 81-85. ′ S a nchez-Moreno, C. 2002. Review: methods used to evaluate the free radical scavenging activity in foods and biological systems, Food Sci. Tech. Int., 8(3): 121-137.
′ S a nchez-Moreno, C., Larrauri, J.A. and Saura-Calixto, F. 1998. New parameter for evaluation of free radical scavenging capacity of polyphenols, 2nd International Electronic Conference on Synthetic Organic Chemistry (ESCOC-2), http://www. mdpi.org/escoc/, September 1-30, 1998 [dp130]; http://ecsoc2.hcc.ru/DP_TOP1/dp130/ dp130. htm. ′ S a nchez-Moreno, C., Larrauri, J.A. and Saura-Calixto, F. 1999. Free radical scavenging capacity and inhibition of lipid oxidation of wines, grape juices and related polyphenolic constituents, Food Res. Int., 32: 407-412. Schwarz, K., Bertelsen, G., Nissen, L.R., Gardner, P.T., Heinonen, M.I., Hopia, A., Huynh-Ba, T., Lambelet, P., McPhail, D., Skibsted, L.H. and 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: 319-328. Wanjek, C. 2001. Mixed messages: antioxidants may in some cases do more harm than good. The Washington Post, 7 Aug, p HE01. ′ ˜ Yepez, B., Espinosa, M., L O pez, S. and Bola n os, G. 2002. Producing antioxidant fractions from herbaceous matrices by supercritical fluid extraction, Fluid Phase Equil., 194-197: 879-884. Zhu, Q.Y., Hackman, R.M., Ensunsa, J.L., Holt, R.R. and Keen, C.L. 2002. Antioxidative activities of oolong tea, J. Agric. Food Chem., 50: 6929-6934.
Continue Reading

Please join StudyMode to read the full document

You May Also Find These Documents Helpful

  • Essay on Research Methods
  • The Effect of the Method of Sorting on Response Time Essay
  • Quantitative research methods Essay
  • social science research method Essay
  • Determination Of The Amount Of Dissolved Oxygen In A Water Sample By Iodonmetry The Winkler S Method Research Paper
  • Methods of Research Research Paper
  • Empirical Methods Essay
  • Forecasting Methods Essay

Become a StudyMode Member

Sign Up - It's Free