Antioxidants from Vegetal Sources: New Research Avenues - Natural Antioxidants and Biocides from Wild Medicinal Plants - page 25

detection in living cells (Qin et al. , 2008;
Sakurada et al. , 1992).

FeTCPP. Although much hope was placed
on this probe, its specific use to detect intra-
cellular superoxide production is contro-
versial. So, Zielonka and Kalyanaraman
critically reviewed the reliability of this
probe, concluding that it must be used with
care and HPLC profiles should be traced in
order to observe all HE oxidation products
(Zielonka and Kalyanaraman, 2010).

Amplex Red

N-Acetyl-3,7-dihydroxyphenoxazine (Amplex
Red) is a highly specific probe for the detec-
tion of hydrogen peroxide produced in neu-
trophils. When this probe is oxidized by
hydrogen peroxide it generates a stable fluor-
escent product resofurin, which can be ana-
lysed using excitation at 520-550 nm and
an emission at 585-595 nm. Although this
compound is not interfered with by the
autofluorescence of biological samples,
some vegetal constituents could interfere
with Amplex Red oxidation (Serrano et al. ,
2009; Mishin et al. , 2010).

2.7.2 New probes for the
detection of ROS

Recently, substantial progress has been
made in the synthesis of new and more spe-
cific probes to detect ROS.

New probes for the hydroxyl radical (OH • )

Tetramethylbenzidine

Traditionally, the hydroxyl radical is
detected by reaction with salicylate or phe-
nylalanine (Althaus et al ., 1993; Halliwell
and Kaur, 1997; Luo and Lehotay, 1997).
However, both strategies do not allow a
realistic estimate of the production of
hydroxyl radical and some new probes have
been introduced.

Tetramethylbenzidine (TMB) is a probe used
to detect HOCl, generating a blue product
when it is oxidized. A disadvantage of this
probe is that it can be a substrate of MPO,
limiting its applications (Freitas, 2009).

Diaminofluoresceins

These compounds (DAF-2 and DAF-FM)
have been used successfully for recognizing
the extracellular production of NO. In an
analogous way to DCHF-DA, their acetylated
derivatives are used for the recognition of
intracellular production of NO. These
probes are highly sensitive and allow moni-
toring of NO production in real time.

t e r e p h t h a l a t e . The product of terephtha-
late hydroxylation (2-hydroxy-terephthalate)
has a higher fluorescence intensity than
2-hydroxy-benzoate (Saran and Summer,
1999). Because of that, this probe has gained
popularity for detecting OH radicals pro-
duced from diverse sources, including liv-
ing systems, in the micromolar range
(Freinbichler et al. , 2008a,b; Page et al. ,
2010). Detection could be done using fluor-
escence readers or, even better, trough
HPLC-FLD (Li et al. , 2004).

Ethanol/a-(4-pyridyl-1oxide)-N-tert-
butylnitrone (4POBN)

This probe is one of the few existing mol-
ecules able to detect specifically the pro-
duction of the hydroxyl radical.

d p p e c . Similarly, the fluorescent probe 1,2-
dipalmitoylglycerophosphorylethanolamine
(DPPEC) has been developed for the detec-
tion of the hydroxyl radical in lipid mem-
branes (Soh et al. , 2008). The probe has high
selectivity for this radical.

MitoSOX Red

This is a fluorescent probe specifically oxi-
dized by mitochondrial superoxide. It can
be used to investigate the production of
ROS in living cells. It should be noted that
their oxidation can be prevented by adding
SOD-mimetic agents such as Tiron and

t e m p o . Recently, Maki and colleagues
developed a new probe from the union of