The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity Philip Molyneux
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: email@example.com 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• 
• 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  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)
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