This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types:  assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include th...

The Chemistry behind Antioxidant Capacity Assays

Methods available for the measurement of antioxidant capacity are reviewed, presenting the general chemistry underlying the assays, the types of molecules detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and dietary supplement industries. From evaluation of data presented at the First International Congress on Antioxidant Methods in 2004 and in the literature, as well as consideration of potential end uses of antioxidants, it is proposed that procedures and applications for three assays be considered for standardization: the oxygen radical absorbance capacity (ORAC) assay, the Folin-Ciocalteu method, and possibly the Trolox equivalent antioxidant capacity (TEAC) assay. ORAC represent a hydrogen atom transfer (HAT) reaction mechanism, which is most relevant to human biology. The Folin-Ciocalteu method is an electron transfer (ET) based assay and gives reducing capacity, which has normally been expressed as phenolic contents. The TEAC assay represents a second ET-based method. Other assays may need to be considered in the future as more is learned about some of the other radical sources and their importance to human biology.

https://faculty.missouri.edu/~glaserr/3700s14/Antioxidant-Capacity_jf0502698.pdf

Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements

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

https://rdo.psu.ac.th/sjstweb/journal/26-2/07-DPPH.pdf

The use of the stable free radical diphenylpicrylhydrazyl (dpph) for estimating antioxidant activity

Three methods widely employed in the evaluation of antioxidant activity, namely 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, static headspace gas chromatography (HS-GC) and β-carotene bleaching test (BCBT), have been compared with regard to their application in the screening of plant extracts. The strengths and limitations of each method have been illustrated by testing a number of extracts, of differing polarity, from plants of the genus Sideritis, and two known antioxidants (butylated hydroxytoluene and rosmarinic acid). The sample polarity was important for the exhibited activity in the BCBT and HS-GC methods but not for the DPPH method. The complex composition of the extracts and partition phenomena affected their activity in each assay. The value of the BCBT method appears to be limited to less polar samples. Although slow, the HS-GC method is preferable for assessing the antioxidant inhibitory properties on the formation of unwanted secondary volatile products. Being rapid, simple and independent of sample polarity, the DPPH method is very convenient for the quick screening of many samples for radical scavenging activity. Copyright © 2001 John Wiley & Sons, Ltd.

Screening of Plant Extracts for Antioxidant Activity: a Comparative Study on Three Testing Methods

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.

/pdf/genesis-and-development-of-dpph-method-of-antioxidant-assay-vb625t39xs.pdf

Genesis and development of DPPH method of antioxidant assay

The reaction mechanisms of three antioxidants are proposed in order to explain experimental results obtained from a kinetic study using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH . ) method, previously adapted in our laboratory. In its radical form, DPPH . shows an absorbance maximum at 515 nm which disappears upon reduction by an antiradical compound. BHT, a synthetic antioxidant, slowly reacts with DPPH . reaching a steady state within 5 h. This 2.8-stoichiometric complete reaction follows a 1.5-order with respect to DPPH . and 0.5 to BHT. The kinetic rate constant, k, is estimated to be 5.0 L/(mol·s) at 20 °C and the energy of activation, Ea , is equal to 35 kJ/mol in methanol. Eugenol reacts with DPPH . reaching a steady state within 2 h. This 1.9-stoichiometric reaction follows a 2-order with respect to both DPPH . and eugenol, k and Ea are estimated to be 5.4 × 10 10 L 3 /(mol 3 ·s) at 20 °C and 30 kJ/mol, respectively. The eugenol mechanism may involve a dimerization between two phenoxyl radicals. The reaction with isoeugenol is rapid and reversible, with a stoichiometry of 1.1. It is first order with respect to isoeugenol with k (direct reaction) equal to 8.9 × 10 −2 s −1 at 10 °C. This reaction is consistent with a pseudo-monomolecular mechanism.

V. Bondet

Papers

Kinetics and Mechanisms of Antioxidant Activity using the DPPH.Free Radical Method