Assessing the activity of natural food antioxidants - Oxidation in Foods and Beverages and Antioxidant Applications - page 339

A 0 ) at 443 nm for a short period of time (usually 10 min) in the presence (V) or
absence (V 0 ) of antioxidants. Quantitative information expressed as relative rate
constants is derived from the equation:
V 0

V 1 k AH k C [AH]

[C]

where [AH] and [C] are the concentrations of the tested antioxidant and crocin
( 1:3310 5 M ÿ1 cm ÿ1 ) and k C and k AH are rate constants for the reaction of
the radicals with crocin and AH, respectively. By determining the V 0 =V value at
a known ratio [AH]/[C], k AH /k C could then be calculated. Recently, a step by
step examination of CBA led authors 60 to propose a simpler expression of results
in the form of percent inhibition. CBA has been used in SAR studies of
flavonoids 61 as well as simple phenols 5,62 and phenolic acids 5,63 and the derived
order generally obeyed the principles of physical organic chemistry. Neverthe-
less, SARs among compounds belonging to different classes should be inter-
preted with caution. 5 Validation data with regard to probe, test compound
characteristics, conditions for peroxyl radical generation and reaction
monitoring period showed the robustness of the assay. 60 Though the assay is
considered to be applicable to `water-soluble' radical scavengers and related
compounds, a modification of the protocol using either canthaxanthin as a
probe 58 or a lipophilic initiator in organic solvent (e.g., toluene) 59,64 has been
proposed when `lipid-soluble' compounds are to be tested. Automated versions
of the assay using microplates have been reported for plasma testing. 65

Oxygen radical absorbance capacity (ORAC)
Initially introduced by Glazer et al., 66 the oxygen radical absorbance capacity
(ORAC) assay was soon accepted as the `most valuable' alternative to the
DPPH · assay (see below). Peroxyl radicals produced by the decomposition of
AAPH at 37ëC react with a fluorescent probe (fluorescein) at pH 7.0 to form a
non-fluorescent product. 67 Hydrogen atom abstraction from the probe result in a
decrease in intensity of fluorescence, which is recorded until fluorescein is
completely destroyed. In the presence of an antioxidant; however, the probe
decay is inhibited as the antioxidant competes for peroxyl radicals. Evaluation of
the antioxidant capacity is based on the difference of integrated areas under the
decay curves (AUC) obtained for the fluorescein solution in the presence or
absence of the compound of interest. In this way it is stated that lag time, rate,
and total inhibition is taken into consideration in a single value. Therefore, the
assay estimates the `capacity'* and not just the `reactivity'* of the tested
antioxidant. ORAC values are usually reported as Trolox equivalents. Lipophilic
antioxidants can also be evaluated using a solution of acetone/water (1:1, v/v)
containing 7% randomly methylated -cyclodextrin with the same probe. 68
However, fluorescein is not sufficiently lipid soluble, and its fluorescence

* Capacity: estimate of the duration of antioxidative action; reactivity: characterizes the starting
dynamics of an antioxidant at a certain concentration. 31