Isolation, identification, and structure of a potent alkyl-peroxyl radical scavenger in crude canola oil, canolol

Article abstract of DOI:10.1271/bbb.69.1568

Alkylhydroperoxides in oxidized oil are undesirable components because they become alkylperoxyl radicals (ROO^?) in the presence of heme, hemoglobin, or myoglobin in red meat. ROO^? are biochemically reactive and damage nucleic acids and proteins, thereby harming living cells. We isolated a component, a highly potent ROO? scavenger, from crude canola oil (rapeseed), which we designated canolol, and identified its chemical structure, 4-vinyl-2,6-dimethoxyphenol. The canolol content of crude canola oil greatly increased after the seed was roasted as compared with that from unroasted seed, but it decreased in highly purified oil. This anti-ROO? activity was highest in crude oil, deceased after each refining step, and was lowest in highly purified refined oil. Canolol was thus generated during roasting. As shown previously, canolol is one of the most potent anti-ROO? components in crude canola oil and its potency is much greater than that of well-known antioxidants, including α-tocopherol, vitamin C, β-carotene, rutin, and quercetin.

Full text of DOI:10.1271/bbb.69.1568

Biosci. Biotechnol. Biochem., 69 (8), 1568–1574, 2005
Isolation, Identification, and Structure of a Potent Alkyl-Peroxyl
*
Radical Scavenger in Crude Canola Oil, Canolol
3;**
Daisuke WAKAMATSU,1;2 Shigeru MORIMURA,1 Tomohiro SAWA,
y
Kenji KIDA,1 Chiaki NAKAI,2 and Hiroshi MAEDA
3;
1Department of Materials and Life Science, Graduate School of Science and Technology,
Kumamoto University, Kumamoto 860-8555, Japan
2Research and Development Center, Showa Sangyo Co., Ltd., Funabashi 273-0015, Japan
3Laboratory of Microbiology and Oncology, Faculty of Pharmaceutical Sciences, Sojo University,
Kumamoto 860-0082, Japan
Received March 11, 2005; Accepted May 12, 2005
Alkylhydroperoxides in oxidized oil are undesirable
components because they become alkylperoxyl radicals
(ROOꢀ) in the presence of heme, hemoglobin, or
myoglobin in red meat. ROOꢀ are biochemically
reactive and damage nucleic acids and proteins, thereby
harming living cells. We isolated a component, a highly
potent ROOꢀ scavenger, from crude canola oil (rape-
seed), which we designated canolol, and identified its
chemical structure, 4-vinyl-2,6-dimethoxyphenol. The
canolol content of crude canola oil greatly increased
after the seed was roasted as compared with that from
unroasted seed, but it decreased in highly purified oil.
This anti-ROOꢀ activity was highest in crude oil,
deceased after each refining step, and was lowest in
highly purified refined oil. Canolol was thus generated
during roasting. As shown previously, canolol is one of
the most potent anti-ROOꢀ components in crude canola
oil and its potency is much greater than that of well-
known antioxidants, including ꢀ-tocopherol, vitamin C,
ꢁ-carotene, rutin, and quercetin.
as DNA and proteins.3–5) Endogenous lipid peroxidation
caused by oxidative stress is another important source of
LOOH. We previously found that heme iron such as that
in hemoglobin and myoglobin efficiently catalyzes the
formation of lipid peroxyl radicals (LOOꢀ) from LOOH,
whereas nonheme iron catalyzes the formation of carbon
centered radicals (Rꢀ) and alkoxy radicals (ROꢀ).6,7) The
alkyl peroxyl radical ROOꢀ is unique in that it is
relatively stable, has a long half-life, and can travel long
distances in vivo from the site of generation.6,7) We also
showed that intake of a fat-rich diet with hemoglobin,
which may generate LOOꢀ in the digestive tract,
significantly promoted colon carcinogenesis in rats
treated with low levels of a carcinogen.8) LOOꢀ can
induce single-strand breakage of DNA8) and can
efficiently generate abasic sites in DNA.9) These
observations suggest that generation of LOOꢀ in vivo
contributes to carcinogenesis, and hence that compounds
that scavenge LOOꢀ might help to prevent cancer.10–13)
Previously we reported that many natural edible oils
contain high levels of anti-ROOꢀ activity, with extra
virgin olive oil exhibiting the highest ROOꢀ scavenging
activity, followed by crude sesame oil and then canola
oil.8) Such anti-ROOꢀ activity might be attributed to
various components such as sesaminol, lignans, toco-
pherols, flavonoids, and polyphenols. However, canola
oil components have not been studied in detail. We
coined canolol the most active component in canola oil,
and identified its chemical structure after extensive
Key words: antioxidants; antimutagenic; seed oil; oil
refining process; canolol
Polyunsaturated fatty acids are susceptible to oxida-
tion, which results in formation of lipid hydroperoxides
(LOOH). LOOH in oxidized oils are undesirable food
components, because they give rise to reactive meta-
bolites such as lipid-derived radicals and aldehydes
(e.g., malondialdehyde and 4-hydroxy-2-nonenal),
which cause oxidative damage in vital molecules such
*
purification to a single component, as described below.
The methods we used for purification and identifica-
This report is a partial fulfillment of the requirements for the MS degree and MS thesis in the Graduate Division of Kumamoto University.1,2)
To whom correspondence should be addressed. Present address: Sojo University, 22-1, Ikeda 4-chome, Kumamoto 860-0082, Japan; Fax: +81-
96-214-5355; E-mail: msmaedah@gpo.kumamoto-u.ac.jp
*
y
**
Present address: Unit of Endogenous Cancer Risk Factors, International Agency for Research on Cancer (IARC), F-69008, Lyon, France
Abbreviations: TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; MS, mass spectroscopy; NMR, nuclear
magnetic resonance; DTPA, diethylenetriaminepentaacetic acid; IPOX50, inhibitory potential 50% of peroxyl radicals; LOOꢀ, lipid peroxyl radicals;
PMC, 2,2,5,7,8-pentamethy-6-couromanol

Canolol, a New Antioxidant in Canola Oil
1569
tion included differential solvent extraction, thin-layer
chromatography (TLC), high-performance liquid chro-
matography (HPLC), mass spectroscopy (MS), nuclear
magnetic resonance (NMR), and infrared, visible, and
ultraviolet spectroscopy. Purified canolol was found to
have the structure 4-vinyl-2,6-dimethoxyphenol, a de-
carboxyl derivative of sinnapinic acid. Canolol exhibited
the highest ROOꢀ scavenging activity among various
known antioxidants such as ꢀ-tocopherol, vitamin C, ꢁ-
carotene, rutin, and quercetin. Further, we found the
steps used in the oil refining process of canola oil
decreased the final canolol content, while roasting the
canola seeds increased canolol content by as much as
100 times of that before roasting.
TLC analyses. The samples, which were dissolved in
chloroform, were applied to TLC plate silica gel,
0.25 mm thick (Kieselgel 60; Merck, Darmstad, Ger-
many), and then developed in a solvent mixture of
hexane, diethyl ether, and acetic acid (80:30:1, v/v/v).
Neutral lipids were determined in the presence of iodine
vapor, as used for detection of general organic com-
pounds.
After the samples were sprayed with 50 mM ꢁ-carotene
and 1 mM linoleic acid (Wako Pure Chemical Industries,
Osaka) in chloroform solution, antioxidant substances
were detected as yellow spots. Phenolic compounds
were detected as dark blue spots, after samples were
sprayed with phenol reagent (Nacalai Tesque, Kyoto)
diluted two-fold with chloroform.
Materials and Methods
Isolation and purification.
Materials. Canola seeds and degummed canola oil
(expeller and hexane extraction after cooking at 80–
90 ꢁC) were obtained from Showa Sangyo (Tokyo).
Diethylenetriaminepentaacetic acid (DTPA), t-butyl hy-
droperoxide (t-BuOOH), and hemoglobin were obtained
from Sigma-Aldrich (St. Louis, MO). Tocopherols ꢀ-,
ꢁ-, ꢂ-, and ꢃ-, and 2,2,5,7,8-pentamethyl-6-chromanol
(PMC), were obtained from Eisai (Tokyo). Synthetic
canolol was prepared by Junsei Chemical (Tokyo).
Methanol extraction. In brief, methanol (90 ml) was
added to 450 g of crude canola oil (that had been
degummed by pressure after cooking at 80–90 ꢁC), and
the mixture was shaken for extraction for 10 min using a
shaker to obtain a heavy dark biphasic solution. The
mixture was then centrifuged at 1;500 ꢂ g for 30 min.
The upper methanol layer was collected in an oval flask.
Methanol (90 ml) was added again to the lower phase
that contained the crude canola oil, and the mixture was
shaken again for 10 min for extraction. This procedure
was repeated three times.
Assay of anti-ROOꢀ activity. As described previous-
ly.6–8) ROOꢀ were generated from the reaction between
t-BuOOH and hemoglobin. t-BuOOꢀ can oxidize lumi-
nol to form fluorescent products, and thus the amount of
t-BuOOꢀ can be quantitated by measuring luminol-
derived chemiluminescence. ROOꢀ scavenging activity
can be determined by means of inhibition of chemilu-
minescence. In this assay, 125 ml of 0.01 M phosphate
buffered saline, 25 ml of 10 mM DTPA, 25 ml of 0.3 M t-
BuOOH, and 25 ml of ethanol solution of the test
substance were added to a well of a 96-well microplate.
The plate was shaken and preincubated at 37 ꢁC for
210 s, after which it was placed in a chemiluminescence
microplate reader (Labsystems Luminoskan RS, Dai-
nippon Pharmaceuticals, Osaka). Chemiluminescence
was initiated by the addition of 25 ml of 100 mg/l
hemoglobin, using a dispenser supplied with the instru-
ment. The intensity of maximum chemiluminescence
was immediately recorded. The concentration of the test
substance at which 50% of chemiluminescence was
suppressed relative to the control (no addition of test
substance) was measured, and this value was designated
the 50% inhibitory concentration of peroxyl radicals
(IPOX50). Thus, the smaller the value of IPOX50, the
greater the radical scavenging potency.
Each methanol layer was combined and processed
with a rotary evaporator in vacuo to remove methanol to
dryness.
Liquid-liquid phase partition. The methanol extract
was dissolved in 45 ml of hexane, the mixture was
placed into a separating funnel (no. 1), and 15 ml of
acetonitrile was added to the separating funnel. The
funnel was shaken, and then the lower (acetonitrile)
phase was transferred into another separating funnel
(no. 2) containing 45 ml of new hexane, while 15 ml of
fresh acetonitrile was introduced into separating funnel
no. 1. Separating funnels no. 1 and no. 2 were shaken,
and then the lower phase (acetonitrile) of separating
funnel no. 2 was introduced into an oval flask (no. 3),
and the lower phase of separating funnel no. 1 was
combined with separating funnel no. 2. This procedure
was repeated with 15 ml of acetonitrile two times. The
acetonitrile layer and the remaining hexane layer were
evaporated in vacuo, and the solvents were removed.
Fractionation by silica gel column chromatography.
For column chromatography, 15 g of silica gel (Kanto
Kagaku, Tokyo), with a particle size of <150 mm, was
packed in a glass column ꢄ20 ꢂ 300 mm. The acetoni-
trile fraction (500 mg) was dissolved in 5 ml of chloro-
form and then applied to the column, which was
developed with a mixture of solvents: (hexane/diethyl
ether, 90:10 v/v), 175 ml; chloroform, 225 ml; acetone,
400 ml; and methanol, 200 ml. Elution proceeded at a
rate of approximately 2 ml/min. The eluted fractions
were collected and stored in an oval flask, and the
*
A part of this work was described in the MS thesis of D.
Wakamatsu in 2001,1) and Koski et al. published the antioxidant
activity of this component more recently, during preparation of
this manuscript.14)

1570
D. WAKAMATSU et al.
solvents were evaporated with a rotary evaporator in
vacuo.
1.2% isopropanol/hexane (v/v) mixture. The flow rate
of the mobile phase was 1.2 ml/min, and the injection
volumes were 50 ml of the sample solution.
For detection of canolol in the eluate, the fluorescence
detector was set at 298 nm for excitation and 325 nm for
emission. PMC was detected at about 6 min, and canolol
was detected at about 18 min. These conditions were
also in effect for detection of the tocopherols: ꢀ-, ꢁ-, ꢂ-,
and ꢃ-tocopherol.
Preparative TLC. The chloroform fraction (100 mg)
was subjected to preparative TLC (Kieselgel 60, Merck,
Darmstadt, Germany) with the solvent mixture hexane/
diethyl ether (80:30 v/v). After confirmation of a yellow
band on the plate near Rf ¼ 0:15, the desired substance
together with the silica gel was scraped off with a
spatula. Chloroform (20–30 ml) was added to the
collected silica gel powder. To extract the substance, it
was removed from the silica gel via filtration with a
glass filter, and the solvent was evaporated.
Purification by HPLC. The sample obtained of TLC
was subjected to HPLC with a C18 column (Aquasil
Keystone Scientific, Bellefonte, PA, ꢄ4:6 ꢂ 250 mm)
used at room temperature. An acetonitrile/water mixture
(90:10 v/v, eluent A) followed by acetonitrile
(eluent B) were applied for gradient elution as follows:
isocratic elution A, 0–20 min; linear gradient elution
from 100% A to 100% B, 20–25 min; isocratic elution
with 100% B, 25–40 min. The flow rate of the mobile
phase was 1.0 ml/min, and the injection volumes were
100 ml of the sample solution (5 mg/ml). Eluate absorb-
ance was measured at 300 nm.
Results
Reduced anti-ROOꢀ activity in canola oil during
processing
As shown in Table 1, anti-ROOꢀ activity and canolol
in canola oil decreased significantly as oil refining
proceeded. Processes, such as degumming, alkaline-
washing with 10% NaOH, decoloration with acid clay
adsorption, and exposure to high-temperature (260 ꢁC)
steam led to anti-ROOꢀ activity a significant decrease
whereas the total content of tocopherol remained
constant (Table 1, 2). This result indicates that the
anti-ROOꢀ activity in canola oil can be attributed in
large part to compounds other than tocopherols.
Identification. The purified sample, a single identified
spot by TLC and one peak in HPLC, was subjected to
the following analyses for identification:
i) The elements C, H, and N were analyzed using a
CHN corder (CHN Corder MT-3; Yanaco, Kyoto). The
sulfur content was measured using a sulfur analyzer
(Antec, 7000; Astech, Tokyo).
ii) For liquid chromatography/mass spectroscopy
(LC/MS), canolol was dissolved in methanol and the
mixture was applied to a reverse-phase HPLC column
(Hewlett Packard Hypersil ODS, Palo Alto, CA;
ꢄ40 ꢂ 125 mm) for separation; elution was with meth-
anol. Mass analysis was performed using the JMS–LC
mate mass spectrometer (JEOL, Tokyo) with positive-
mode, atmospheric pressure chemical ionization. The
vaporizer temperature was 500 ꢁC.
Table 1. Change in Anti-ROOꢀ Activity of Canola Oil during
Industrial Processing
IPOX50
Canolol
Total tocopherol
conc ( g/g)
Oil
(mg/ml)
conc (
m
g/g)
m
Degummed crude oil
Deacidified oil
1.46
21.6
220.0
38.0
785
754
749
409
Decolored oil
105
Not detectable
Not detectable
Deodorized (purified) oil >300
Canolol as a potent anti-ROOꢀ component in canola
oil
To identify components with potent anti-ROOꢀ
activity in canola oil, degummed crude oil was subjected
to methanol extraction. This procedure yielded 3,025 mg
with an IPOX50 value of 0.18, that is, activity that was
23.3 times higher than that of the starting material
(Table 3). In the liquid-liquid partition experiment,
strong anti-ROOꢀ activity was recovered from the
acetonitrile phase (Table 3). Further purification was
carried out by means of silica gel column chromatog-
raphy, and we observed the highest anti-ROOꢀ activity
in the chloroform fraction. TLC analysis showed that
this chloroform fraction consisted of three spots having
Rf values of 0.01, 0.15, and 0.3 (Fig. 1). A spot with an
Rf value of 0.3 corresponded to free fatty acid. The spots
with Rf ¼ 0:15 exhibited potent antioxidant activity, as
revealed by ꢁ-carotene. Use of the phenol reagent for
detection of phenolic compounds indicated that fractions
with Rf values of 0.01 and 0.15 were both phenolic
compounds, the yield for each being 13 mg. The anti-
ROOꢀ activity of the fraction with an Rf value of 0.15
iii) For NMR, a sample was dried in a nitrogen
atomosphere, dissolved in chloroform-d (CDCl3), and
introduced into an ampule tube, after which measure-
1
ment was taken at 400 Mz for H NMR; JEOL (Tokyo)
was measured.
iv) Ultraviolet and visible absorption was determined
in water (V-550 UV/VIS spectrophotometer, Jasco,
Tokyo) in the conventional manner.
Content of canolol and tocopherol. Canolol shows a
fluorescence emission at 325 nm when excited at
298 nm. Hence canolol elution of content was monitored
by means of an HPLC system with a fluorescence
detector (FP-1520, Jasco, Tokyo). Separation was
achieved via
a
column from Kanto Kagaku,
LiChroCART 250-4 cartridge, LiChrospher 100 NH2;
column ꢄ4 ꢂ 244 mm, particle size of 5 mm; the internal
standard for HPLC was PMC. HPLC was carried out at
temperature below 25 ꢁC. Elution was isocratic, with a

Canolol, a New Antioxidant in Canola Oil
1571
Table 3. Anti-ROOꢀ Activity in Canola Oil at Each Step during
Canolol Purificationa
Purification step
Degummed crude oil
IPOX50, mg/mlb Recovery yield
4.2 (1)b
450 g
#
Methanol extraction
#
0.18 (23.3)
3,025 mg
Liquid-liquid partition
Hexane phase
2.5 (1.68)
1,533 mg
862 mg
Acetonitrile phase
#
0.044 (95.4)
Silicagel column chromatography
Hexane/ether fraction
Chloroform fraction
Acetone fraction
Methanol fraction
# (100 mg of chloroform frn)
TLC
2.6 (1.6)
163 mg
267 mg
379 mg
78 mg
0.022 (190)
0.12 (35)
0.22 (19)
Upper layer (Rf ¼ 0:15)
Lower layer (Rf ¼ 0:01)
# (use frn of Rf 0.015 for HPLC)
HPLC
0.008 (530)
0.016 (260)
13 mg
13 mg
0.004 (1,050)
1 mg
a
Numbers in parentheses indicate relative anti-ROOꢀ activity compared with
degummed crude oil (starting material).
Anti-ROOꢀ activity relative to crude degummed oil is 1. Values in
parentheses are ROOꢀ-scavenging potential relative to the starting material.
b
Fig. 1. TLC Analysis of Canola Oil Constituents Obtained from
Different Purification Steps.
was two times higher than that of the Rf ¼ 0:01 fraction.
Finally, a TLC fraction with an Rf value of 0.15 was
applied to reverse-phase HPLC purification. A single
peak at a retention time of 12 min was collected and

1572
D. WAKAMATSU et al.
Fig. 2. 1H-NMR Spectrum of the Purified Component from Canola Oil in CDCl3 (A) and Its Proposed Chemical Structure of Canolol (B).
TMS, tetramethylsilanes were used as the standard.
Fig. 3. HPLC Chromatogram of Crude Canola Oil, and References (Peak 1, BHT [antioxidant reagent]; peak 2, ꢀ-tocopherol; peak 3, PMC
(internal standard); peak 4, unknown; peak 5, ꢂ-tocopherol; peak 6, ꢃ-tocopherol; peak 7, canolol).
See text for details.
found to have an IPOX50 value of 0.004. This compound
had anti-ROOꢀ activity 1,050 times higher than that of
the starting material (Table 3).
found that canolol had a fluorescence emission max-
imum at 325 nm (data not shown) which was used for
quantifying upon HPLC.
Identification of the chemical structure of canolol
We determined the chemical structure of this com-
pound. Elemental analysis yielded neither nitrogen nor
sulfur. High-resolution MS of atmospheric pressure
chemical ionization (APCI) plus LC/MS showed a
molecular mass of 180, and the structure C10H12O3 was
proposed. On the basis of these results plus the 1H-NMR
data, we concluded that this active component is 4-
vinyl-2,6-dimethoxyphenol (Fig. 2B), and we designat-
ed it canolol. Synthesis via decarboxlation of sinnapinic
acid yielded the same compound as canolol on TLC and
HPLC; identity was also confirmed by NMR spectrosco-
py (data not shown). Therefore the assigned chemical
formula is consistent with sinapinic acid.
HPLC chromatography
Figure 3 shows a typical pattern of HPLC; the
representative components are shown. The conditions
are designated in ‘‘Methods’’. Canolol shows a distinct
separation.
Increase of canolol after roasting
We examined the levels of canolol and tocopherols in
five lots of crude oil from canola seeds before and after
roasting (Table 2). The amounts of ꢀ, ꢂ, and ꢃ-toco-
pherol were unchanged during roasting (165 ꢁC, 5 min),
whereas ꢁ-tocopherol remained zero throughout. In
contrast, the canolol content of each lot was increased
by about 10–100 times after roasting, as shown in
Table 2. This finding was consistent with antioxidant
potential.
Canolol had absorption peaks at 218 nm and 269 nm.
The molecular extinction coefficient in water was 29,000
at 218 nm and 13,000 at 269 nm ("mol/l; 1 cm). We also

Canolol, a New Antioxidant in Canola Oil
1573
Discussion
diseases.26–29) We found that ONOOꢃ has, in addition,
strong mutagenic effects on the Salmonella typhimurium
TA102 strain2) and the influenza and Sendai viruses.18,30)
We also found that canolol strongly suppressed bacterial
mutations in Salmonella caused by ONOOꢃ.2) The
antimutagenic potency of canolol was almost equivalent
to or more than that of ebselen, a potent ONOOꢃ
scavenger. All these data together indicate that canolol
might have beneficial effects on both ROOꢀ and
ONOOꢃ mediated diseases. The presence of potent
antioxidant components in the diet is thus clearly
important.
The present results suggest that the oil refining
processes used in the industry today should be revised
so that such important scavengers are retained. It is also
interesting that canolol levels appear to increase during
roasting, which suggests that a precursor of canolol,
such as sinnapinic acid, might exist, in which case
decarboxylation of the precursor would result in for-
mation of canolol. This possibility should be inves-
tigated in a model system.
In the plant seeds from which most edible oils are
prepared, abundant antioxidants have been found.
Highly potent oxygen radical scavengers play a crucial
role in preventing radical-induced damage of DNA,
RNA, and proteins in the seeds. These scavengers are
thus vitally important not only for prevention of genetic
damage but also for various nutritional and enzymatic
functions needed by seeds for germination.
In the gut, where heme becomes available and
accessible to alkyl hydroperoxides (or LOOH) in food,
the peoxyl radical ROOꢀ is generated. Suppression of
such hazardous radical species is of great practical value
for human health and cancer prevention.
In the present study, we isolated a highly potent anti-
ROOꢀ fraction from crude canola oil and identified its
chemical structure as 4-vinyl-2,6-dimethoxyphenol, and
coined this substance canolol. As shown in Fig. 2B,
canolol is a decarboxylated analog of sinapinic acid. It
was detected in five different lots of unroasted canola
seeds (6–58 mg/g) (Table 2). Further, roasting the canola
seeds markedly increased the canolol content in all five
lots of seeds (to 549–1536 mg/g). Our data suggest that
decarboxylation of sinapinic acid during roasting might
produce canolol. This possibility, such as decarboxyla-
tion of sinapinic acid by heat, should be elucidated in a
model experiment. The biosynthetic pathways of canolol
also remain unclear.
Sinapinic acid and other 4-hydroxycinnamic acid
derivatives such as ferulic acid possess radical scaveng-
ing activity.15) It was proposed that these compounds can
readily donate their phenoxyl hydrogen atom to neu-
tralize radical species, with simultaneous formation of
corresponding phenoxyl radicals.16) Unpaired electrons
of phenoxyl radicals, however, can be delocalized, and
hence these phenoxyl radicals show weaker reactivity.
In agreement with this finding, we discovered, via
electron spin resonance spectroscopy, canolol-derived
phenoxyl radicals in a reaction between canolol and t-
BuOOꢀ (data not shown).
In previous studies, we showed that various sources
such as nitroguanosine and heterocyclic amines, as well
as inflammatory insults in hosts, generated superoxide
anion radicals and nitric oxide.17–20) Also, our data
relating peroxyl radical formation by heme plus ROOH
with enhanced colon carcinogenesis8) have been vali-
dated subsequently.21)
Canolol can scavenge not only ROOꢀ but also
peroxynitrite (ONOOꢃ),2) a highly potent oxidizing
and nitrating agent formed during the reaction of
superoxide radicals with nitric oxide.22–24) We showed
that both are formed endogenously in vivo at sites of
inflammation and infection.17,18,25) Because ONOOꢃ can
cause oxidative and nitrative damage to DNA, RNA, and
proteins, overproduction of it might be associated with
various pathogenic states or conditions such as inflam-
mation, cancer, arteriosclerosis, and neurodegenerative
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