Novel hydroxyl radical scavenging antioxidant activity assay for
water-soluble antioxidants using a modified CUPRAC method
Burcu Bektas
ß
og
˘
lu, Saliha Esin C¸ elik, Mustafa O
¨
zyu
¨
rek, Kubilay Gu
¨
c¸lu
¨
,Res
ß
at Apak
*
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320
_
Istanbul, Turkey
Received 25 April 2006
Available online 15 May 2006
Abstract
Reactive oxygen species (ROS) such as superoxide anion, hydroxyl (
Å
OH), peroxyl, and alkoxyl radicals may attack biological mac-
romolecules giving rise to oxidative stress-originated diseases. Since
Å
OH is very short-lived, secondary products resulting from
Å
OH
attack to various probes are measured. Although the measurement of aromatic hydroxylation with HPLC/electrochemical detection
is more specific than the low-yield TBARS test, it requires sophisticated instrumentation. As a more convenient and less costly alterna-
tive, we used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent
mixture of Fe(II) + EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble anti-
oxidants in 37 °C-incubated solutions for 2 h. The CUPRAC (i.e., our original method for total antioxidant capacity assay) absorbance
of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of
Å
OH scavengers, the difference being proportional to the scavenging ability of the tested compound. A rate constant for the reaction
of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. The second-order rate constants of the
scavengers were determined with competition kinetics by means of a linear plot of A
0
/A as a function of C
scavenger
/C
probe
, where A
0
and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively, and C is the molar concen-
tration of relevant species. The 2,4- and 3,5-dimethoxybenzoates were the best probes in terms of linearity and sensitivity. Iodide, meta-
bisulfite, hexacyanoferrate(II), thiourea, formate, and dimethyl sulfoxide were shown by the modified CUPRAC assay to be more
effective scavengers than mannitol, glucose, lysine, and simple alcohols, as in the TBARS assay. The developed method is less lengthy,
more specific, and of a higher yield than the classical TBARS assay. The hydroxyl radical scavenging rate constants of ascorbic acid,
formate, and hexacyanoferrate(II) that caused interference in other assays could be easily found with the proposed procedure.
Ó 2006 Elsevier Inc. All rights reserved.
Keywords: Hydroxyl radical scavenging; Antioxidant activity; Water-soluble antioxidants; CUPRAC method; Spectrophotometry
When natural defenses of the organism (of enzymatic,
non-enzymatic, or dietary origin) are overwhelmed by
an excessive generation of reactive oxygen species
(ROS), a situation of ‘oxidative stress’ occurs, in which
cellular and extracellular macromolecules (proteins, lipids,
and nucleic acids) can suffer oxidative damage, causing
tissue injury [1,2]. The hydroxyl radical (
Å
OH) is the most
reactive product of ROS formed by successive 1-electron
reductions of molecular oxygen (O
2
) in cell metabolism,
and is primarily responsible for the cytotoxic effects
observed in aerobic organisms extending from bacteria
to plants and animals [1,3,4].
It is generally assumed that
Å
OH is generated in biolog-
ical systems [3,5] from H
2
O
2
by the Fenton reaction:
Fe
2þ
þ H
2
O
2
! Fe
3þ
þ
Å
OH þ OH
À
whereby Fe
2+
is regenerated by superoxide anion O
2
ÅÀ
, giv-
ing rise to an overall Fe(II,III)-catalyzed Haber–Weiss
reaction producing
Å
OH from H
2
O
2
and O
2
ÅÀ
, potentially
available in aerobic cells [6].
When one needs to simulate Fenton reactions in the lab-
oratory to generate hydroxyl radicals and measure the
Å
OH
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2006.05.038
*
Corresponding author. Fax: +90 212 473 7180.
E-mail address: rapak@istanbul.edu.tr (R. Apak).
www.elsevier.com/locate/ybbrc
Biochemical and Biophysical Research Communications 345 (2006) 1194–1200