Antioxidants and Bioactive Compounds in Fruits - Fruit Ripening: Physiology, Signalling and Genomics - page 108

biomarker and is determined using an
array of protocols. Niederlander et al.
(2008) divided the antioxidant assays into
three main categories:
• assays involving actual ROS-oxidizable
substrate interactions;
• assays involving a relatively stable
single oxidizing reagent; and
• assays relating antioxidant activity to
electrochemical behaviour.
In the fi rst category of assays, the
oxidizing agents are those that are also
active in biological systems and play a role
in consumer product deterioration. These
are applied to oxidize a substrate of which
the concentration can easily be deter-
mined. The antioxidant activity of a
phytochemical introduced into the system
is related to the decrease in substrate
conversion due to competition between the
substrate and the phytochemical. In this
group, assays use hydrogen peroxide and/
or superoxide anion as the active ROS.
In the second category of assays, the
oxidizing agent is a relatively stable reagent
that represents the oxidizing pressure
exercised on a potential reductant, e.g. a
substrate or an antioxidant. Upon reaction
with a phytochemical introduced into the
system, conversion of the reagent can be
monitored through a change in a specifi c
characteristic (e.g. its UV/Vis absorbance).
In the absence of phytochemicals with
antioxidant activity, no reagent conversion
will take place. Antioxidant activity is
related to the rate and extent of reagent
conversion. The most common assays of
this group involve DPPH radical scavenging
activity, 2,2-azinobis(3-ethylbenzothiazo-
line 6-sulfonic acid) (ABTS + ), ferric
reducing/antioxidant power (FRAP), trolox
equivalent antioxidant capacity (TEAC),
phosphomolybdenum and E -carotene
bleaching inhibition. Measurement of the
antioxidant activity of each assay is based
on different mechanisms. The activity of
antioxidants depends mainly on substrates,
solvents, pH, conditions and stages of
oxidation, and localization of antioxidants
in different phases (Frankel and Meyer,
2000). For example, the evaluation of

extract antioxidant activity using the
E -carotene bleaching inhibition method
permits the determination of antioxidants
in an emulsion, whilst the FRAP method
measures the hydrophilic antioxidants and
the DPPH assay detects antioxidants
soluble in organic solvents. The antioxidant
activity of citrus extracts, determined with
four in vitro assays, showed a higher cor-
relation coeffi cient between the phos-
phomolybdenum assay and the E -carotene
bleaching inhibition assay. On the other
hand, correlations between the DPPH
method and the phosphomolybdenum and
E -carotene bleaching inhibition assays were
substantially lower (Goulas and Man-
ganaris, 2011a). Thus, an approach with
multiple assays for determination of
antioxidant activity is highly recommended
(Sacchetti et al. , 2005; Goulas and
Manganaris, 2011b).
Further to the commonly used methods
for assessing antioxidant activity, an
antioxidant effect can be determined over
time using bulk oil and emulsions (Goulas
and Manganaris, 2011b). Such assays have
a series of advantages: they do not require
expensive instrumentation, they measure
both hydrophilic and lipophilic anti-
oxidants, and they determine the anti-
oxidant effect on substrates with different
physicochemical properties of interest to
the food industry. However, the dis-
advantages include the overall duration of
the assays, the oxidation conditions that
infl uence the estimated activity and the
effect on the antioxidant activity by inter-
facial and phase distribution properties,
not to mention the complexity of
interpreting the results.
It should be noted that assays of
antioxidant capacity are based on discrete
mechanisms using different means to
generate radicals or oxidants used to test
compounds suspected of having anti-
oxidant potential. As a result, each
individual assay generates a unique value
that belies direct comparison. There is no
direct evidence that the benefi cial effects of
polyphenol-rich foods can be attributed to
the measured antioxidant properties of
these foods. It is impossible to extrapolate