Assessing the activity of natural food antioxidants - Oxidation in Foods and Beverages and Antioxidant Applications - page 341

14.5.2 Superoxide anion radical (O 2 ·ÿ )
Superoxide radical anion (O 2 ·ÿ ) is produced as a result of the donation of one
electron to oxygen. In foods it is formed when xanthine oxidase (XOD) acts on
xanthine or hypoxanthine in the presence of molecular oxygen or by photo-
activation of hematoporphyrin. 77 It is considered to be a rather weak oxidant;
however, it can lead to the formation of more active species such as hydroxyl
radical and peroxynitrite. 77,78 The most common analytical protocol developed
for the determination of O 2 ·ÿ is based on the system XOD/hypoxanthine or
xanthine at pH 7.4 to generate the anion radical. 79 Nevertheless, since phenolic
compounds may inhibit the radical formation by interfering with the enzyme, a
non-enzymatic reaction of phenazine methosulphate (PMS) in the presence of
nicotinamide adenine dinucleotide (NADH) is also reported for assessing only
the radical scavenging ability. 79 In both cases the detection of the radical is
achieved via reduction of nitroblue tetrazolium (NBT) into formazan, which
absorbs at 560 nm. 79 The aforementioned protocols are the most preferable ones,
though other detection methods of the anion radical through reduction of
ferricytochrome C instead of NBT, 80 nitrite formation after reaction with
hydroxylamonnium and detection at 530 nm 81 or by GC determination of
ethylene produced after reaction with -keto--methiolbutyric acid (KMBA)
have been reported. 82 Methods based on electron spin resonance (ESR) spectro-
metry and chemiluminescence have also been reported in the literature. 83,84
However, ESR requires expensive equipment and skills for carrying out such
tests. Superoxide radical anion has been employed in the assessment of activity
of various phenolic antioxidants. 79±81,83±86 The findings, indicate the importance
of the number and position of hydroxyl groups to antioxidant activity. Detection
of the superoxide anion radical using NBT or chemiluminescence have signifi-
cant drawbacks due to solubility problems of colored end product and inter-
ference with the generated radical. 87 To overcome this, a novel high-throughput
assay has been introduced, which is referred to as the superoxide radical
absorbance capacity (SORAC) assay. The particular protocol is based on radical
production using XOD/xanthine but detection is made via fluorescence.
Specifically, a non-fluorescent probe (hydroethidine) is used which is converted
to 2-hydroxyethidium (a strongly fluorescenct compound) when oxidized
( exc 480 nm and emis 567 nm). 87 Unlike other probes, hydroethidine does
not interfere with the system, nor does it yield water-insoluble products. The
method has been validated relative to linearity, precision, accuracy, and
robustness, and then tested using a series of catechin derivatives. 87 Although the
SORAC assay is a promising method for SAR studies, a wider diversity of
compounds should be tested.
14.5.3 Nitric oxide radical (NO · )
Nitric oxide is an important free radical formed in vivo, which may participate in
both physiological and pathological processes. 88 Nevertheless, NO · scavenging is
also of importance from the point of view of food chemistry, as its formation is