Antioxidants from Vegetal Sources: New Research Avenues - Natural Antioxidants and Biocides from Wild Medicinal Plants - page 20

group of compounds. One of the radicals
most used as a probe in cell-free systems is
DPPH because it is very easy to use, fast and
inexpensive and can be used in any labora-
tory with minimal equipment. Because the
chemistry behind the reactions of DPPH
with different antioxidants still requires
some understanding, the results should be
interpreted with some care. Additionally, it
should be considered what type of solvent
is applied in the assay, and the effect of pH,
temperature, light, reading time and the
concentration of the radical. The mathemat-
ical treatment of results deserves special
mention. Traditionally, the percentage of
inhibition is calculated at an arbitrary end
time. Recent studies indicate that measur-
ing time should not be less than 20 min; it is
recommended to use the area under the
curve of DPPH bleaching for the calculation
of this parameter (Cheng et al. , 2006). The
use of Trolox as a calibration substance is
desirable to calculate Trolox equivalent
antioxidant capacity (TEAC) values, which
may be useful for comparison purposes.
Other antioxidants such as vitamin C, gallic
acid, catechin and quercetin can also be
used for calibration purposes. Considering
the DPPH bleaching rate in the first few sec-
onds (fast kinetic) or after 5 min (slow
kinetic), as in the ABTS assay (see below), it
is possible to calculate two TEAC indexes
(Campos and Lissi, 1995, 1997). Detailed
analysis of both portions of these curves can
provide interesting information about the
quality and reactivity of the tested antioxi-
dants. In general, the method can be used as
a preliminary test in the detection of anti-
oxidant extracts and for monitoring the
chromatographic fractions. DPPH can even
be used as post-column reagent for high-
performance liquid chromatography (HPLC)
or to stain thin-layer chromatography (TLC)
plates. The case of ABTS radical is analo-
gous to that of DPPH; it provides similar
information but must be generated in addi-
tional steps. Many ways have been proposed
to do this, for example using 2,2´ azo-bis
(2-amidinopropane) hydrochloride (AAPH)
previously thermo-activated (Henriquez et al. ,
2002), potassium persulfate (Re et al. , 1999)
and recently in situ by mixing peroxidase/

H 2 O 2 /ABTS (Munoz-Munoz et al. , 2010).
In the latter work it is worth noting the
introduction of a new parameter called the
apparent kinetics antioxidant efficiency
(KAE app ), which would be useful for com-
paring different antioxidants. Furthermore,
the authors introduce the concepts of a pri-
mary and secondary antioxidant on the
basis of similar observations with DPPH.

2.5.1 ORAC

Although there is no methodology that can
be considered a 'gold standard', many stud-
ies find that certain tests such as oxygen
radical absorbance capacity (ORAC) have
comparative advantages over the rest of the
assays (Ou et al. , 2001). The ORAC index
combines in a single parameter information
on the kinetics of oxidation using the area
under the curve of fluorescence or absorb-
ance decay of a probe such as fluorescein
(ORAC-FL) or pyrogallol red (ORAC-PGR),
which is challenged with peroxyl radicals
(AAPH). In the ORAC-FL assay, the times of
induction are strongly influenced by the
number of phenolic groups present in the
sample, whereas in the ORAC-PGR assay
such times are not practically observed and
the decay of the absorbance is influenced
mainly by the reactivity of the phenols of
the sample. Recently, it has been suggested
that both ORAC indexes (FL and PGR) are
complementary and their ratio is a better
indicator of the average quality of the anti-
oxidants contained in a sample (Poblete
et al. , 2009). The same research group has
proposed ORAC-PRG as a quick way to
determine the specific content of vitamin C
in extracts and biological fluids, because
this substance is one of the few that pro-
duces induction times in a concentration-
dependent manner (Torres et al. , 2008; Atala
et al. , 2009). In the search for the 'methodo-
logical Holy Grail' and to expand its range
of applications, several refinements have
been introduced to this assay. For example,
the use of methylated cyclodextrin allows
obtaining the lipophilic ORAC index in dif-
ferent plant samples or biological fluids