Assessing the activity of natural food antioxidants - Oxidation in Foods and Beverages and Antioxidant Applications - page 344

Typically, the test is carried out in methanol or ethanol at room temperature, and
results are expressed as percent radical scavenging activity [(%RSA) [(Abs t=0 ÿ
Abs t )/Abs t=0 ] 100], or in terms of antioxidant concentration that causes a
decrease in the initial DPPH · concentration by 50% (EC 50 ). The % RSA is
usually preferred for rapid protocols (e.g., 10±30min) because EC 50 values are
based on kinetic data. The latter can be time-consuming as a plateau is reached in
seconds, minutes, or even many hours. 101 Calculation of reaction rate constants of
phenolic compounds have been also used, 102,107 as well as a parameter proposed
by SÂnchez-MorÂno and co-workers 105 called `antiradical efficiency (AE)', which
is defined as [1/(EC 50 T EC50 )], where T EC50 is the time needed to reach the
steady state when antioxidant concentration equals that of EC 50 . The order of
activity obtained with the DPPH · assay, in general, corresponds to the number
and position of hydroxyl groups in the aromatic ring of the test compounds.
Nevertheless, the stoichiometry of the reaction calculated for phenolic
compounds is higher than the available hydroxyl groups in many cases, 11,101 as
some side reactions have been proposed to occur. 101 Aside from not being a
biologically relevant radical, the assay has been reported to have other
limitations. DPPH · is not water soluble, though a mixture of ethanol and water
(1:1 v/v) can be used. 110 The steric effects imposed by the radical may affect its
reactivity, as small compounds may have better access to the radical's active site
compared with larger compounds. 37 Colored compounds or turbid samples affect
spectrometric measurements. Adaptation of the assay to HPLC 111 or
amperometric reduction of the radical at a glassy carbon electrode 112 eliminates
such sources of interference. Furthermore, reactivity of the radical with phenols is
affected by the solvent, which may determine the magnitude or even the order of
activity of the tested compounds. 113±116 Solvent effect is a parameter that should
always be considered in all radical scavenging assays. Despite these issues, the
DPPH · assay remains a popular method among researchers for obtaining
information on the activity of antioxidants, or for use with advanced
instrumentation for the investigation of complex phenolic mixtures (vide infra).

14.6.2 2,2 0 -Azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation
(ABTS · )
The assay involves monitoring the decay of the radical cation of 2,2 0 -Azinobis-
(3-ethylbenzothiazoline-6-sulphonate) (ABTS) at 734 nm, in the presence of a
test compound. Scavenging has been proposed to take place via electron dona-
tion. 29 The results are expressed as Trolox equivalents, that is, the concentration
of Trolox solution (mM) with an antioxidant capacity equivalent to that
observed for 1 mM of the test compound. For this reason, the assay is also
known as `Trolox equivalent antioxidant capacity (TEAC) assay'. It was
initially introduced by Miller et al., 117 and after several revisions aiming at
preventing the interference of test compounds with radical formation, it was one
of the methods recommended for further standardization in the first international
congress on antioxidant methods. 30 Its selection was based on its simplicity,