Biology Reference
In-Depth Information
(Huang
et al.
, 2002). Additionally, the assay
has proven its versatility because some
changes have been introduced for measur-
ing the effect of antioxidants on reactive
species of oxygen and nitrogen, giving rise
to variations for the hydroxyl radical
(HORAC), peroxynitrite (NORAC) and
superoxide anion (SORAC) (Ou
et al.
, 2002;
http://www.brunswicklabs.com/).
The use
of ORAC has been employed by the United
States Department of Agriculture (USDA) as
a criterion of antioxidant power. The USDA
has published a series of tables containing
not only the food composition of many
medicinal plants used in North America but
also information about their antioxidant
activity. Moreover, in the Laboratory of
Antioxidants at the Institute of Nutrition
and Food Technology (INTA), University
of Chile, a website was recently created
where ORAC values and polyphenolic
contents of Chilean fruits are presented in
a complete database
(www.portalantioxi-
dantes.com).
This project, led by Dr Hernan
Speisky and funded by INNOVA-Chile,
makes Chile the only country in Latin
America and second in the world to carry
out this type of initiative.
2.5.3
CUPRAC
In recent years an assay called cupric ion
reducing antioxidant capacity (CUPRAC)
has been developed and successfully used
for the determination of antioxidant cap-
acity of samples with polyphenols, ascorbic
acid and thiols (Apak
et al.
, 2004, 2007;
Cekic
et al.
, 2009). In this assay, antioxi-
dants reduce Cu
2+
to Cu
+
. The complex
formed between neocuproine and Cu
+
can
be detected spectrophotometrically, and its
concentration is proportional to the ability
of antioxidants to transfer electrons to cop-
per. Among the main advantages of CUPRAC
assay it is worthy to mention that it can be
performed in a medium with physiological
pH (7.4) and that measurements are regis-
tered at an end point (30 min). This method
has significant advantages over FRAP
because the redox chemistry of copper (II),
as opposed to that of the chemically inert
high-spin ferric ion with half-filled d-orbitals
in its electronic configuration, involves
faster kinetics. For this reason, some phe-
nolic antioxidants continue promoting the
formation of the TPTZ-Fe
3+
complex even
hours after the study has been completed
(typically FRAP readings are performed at
5-10 min). Also it has been established that,
owing to lower redox potential of the
CUPRAC reagent, reducing sugars (fructose)
and citric acid are not oxidized with the
CUPRAC reagent. This assay also is applic-
able to thiol-type antioxidants, which can-
not be measured by the FRAP assay. The
method has also been adapted for the analy-
sis of hydrophilic and lipophilic antioxi-
dants (Çelik
et al.
, 2007) and in the
determination of hydroxyl radical scaveng-
ing capacity (Bektasoglu
et al.
, 2008).
2.5.2
FRAP
The ferric reducing antioxidant power
(FRAP) assay is one of the methods histori-
cally used to evaluate the antioxidant cap-
acity of samples from different origins. In
fact, the method measures the ability of a
sample to reduce Fe
3+
to Fe
2+
. Therefore, the
ability to donate electrons from a molecule
is reflected by the concentration of ferrous
ions in the medium. These ions can be
quantified owing to their ability to form an
intense blue-coloured complex with
2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ).
Despite its simplicity and low cost, this
method has some important limitations
such as a pH under the physiological range
(pH = 3.6), and the arbitrariness of reading
time. Many of these disadvantages are over-
come in the method CUPRAC.
2.6
Cell-based Assays
2.6.1
ERYCA
Red blood cells can be used as a simple and
inexpensive biological system to determine
antioxidant capacity of extracts and pure sub-
stances. In the original assay, erythrocytes
Search WWH ::
Custom Search