Assessing the activity of natural food antioxidants - Oxidation in Foods and Beverages and Antioxidant Applications - page 350

M n+ ROOH ! M (n1) RO · OH ÿ

(R14.4)

M (n1) ROOH ! M n ROO · H (R14.5)

Phenolic compounds may participate in redox reactions with transition metal
ions and/or form coordination complexes. 141 Owing to such interactions, some
phenolic compounds (e.g., flavonoids) may either promote or retard oxidation.
An observed prooxidant activity is postulated to be due to reduction of metal
ions by phenols to more prooxidative species. On the other hand, a protective
effect may be due to metal chelating ability, thus hindering the participation of
metal ions in the abovementioned reactions (R14.3±14.5). Information on pro/
antioxidant activity of phenols is obtained by the following techniques.

Metal reducing assays
The reducing ability of phenols towards iron or copper ions can be estimated
spectrophotometricaly with the ferric reducing antioxidant power (FRAP) and the
cupric ion reducing antioxidant capacity (CUPRAC) assays respectively. In brief,
the former is based on the ability of phenolics to reduce ferric tripyridyltriazine
complex (Fe(III)-TPTZ) to ferrous complex (Fe(II)-TPTZ) at pH 3.6 (in order
to maintain iron solubility), as described by Benzie et al. 142 A second technique is
based on the reduction of Cu(II)-neocuproine complex to Cu(I) at pH 7, as
described by Apak et al. 143 These two assays are often employed in order to
estimate the radical scavenging activity by single electron transfer
reactions. 3,11,143±146 However, this practice is often criticized as compounds that
reduce transition metals do not necessarily reduce free radicals. 147 Nevertheless,
the application of the two methods gives evidence for the possible prooxidant
activity of phenols in lipid systems in the presence of Fe(III) or Cu(II).

Metal chelating studies
A straightforward way to screen compounds for chelating ability is to record
their aqueous spectra in a selected pH prior to, and after, the addition of variable
concentrations of metal ions. The chelating capability is influenced by pH value
as ionization of hydroxyl groups of phenols assist metal binding. 148 Chelation is
evidenced by significant spectral changes as in the case of caffeic acid (Fig.
14.2), wherein a bathochromic shifting of absorption was observed after Cu(II)
addition. 63 EDTA is typically added afterwards to investigate the stability of the
formed complex. The spectra of phenolic solution is restored if the metal ion is
chelated by the added EDTA. 63,149
While the aforementioned assay provides qualitative data, chelating ability
may also be quantitatively assessed. An approach is the one adopted by
Andjelkovic et al., 149 where UV-Vis spectroscopy was used to study the Fe(II)
chelating effect of a series of phenolic acids by calculation of binding constants*

* Binding constants are defined as the ratio intercept/slope, where intercept and slope are
parameters of a linear relationship between 1/(total ferrous ion concentration) and 1/(absorbance
of complexed ion).