Direct antioxidant activity of purified glucoerucin, the dietary secondary metabolite contained in rocket (Eruca sativa Mill.) seeds and sprouts

Article abstract of DOI:10.1021/jf047945a

Rocket (Eruca sativa Mill, or Eruca vesicaria L.) is widely distributed all over the world and is usually consumed fresh (leafs or sprouts) for its typical spicy taste. Nevertheless, it is mentioned in traditional pharmacopoeia and ancient literature for several therapeutic properties, and it does contain a number of health promoting agents including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates (GLs). The latter phytochemicals have recently gained attention as being the precursors of isothiocyanates (ITCs), which are released by myrosinase hydrolysis during cutting, chewing, or processing of the vegetable. ITCs are recognized as potent inducers of phase II enzymes (e.g., glutathione transferases, NAD(P)H:quinone reductase, epoxide hydrolase, etc.), which are important in the detoxification of electrophiles and protection against oxidative stress. The major GL found in rocket seeds is glucoerucin, GER (108 ±5 μmol g^-1 d.w.) that represents 95% of total GLs. The content is largely conserved in sprouts (79% of total GLs), and GER is still present to some extent in adult leaves. Unlike other GLs (e.g., glucoraphanin, the bio-precursor of sulforaphane), GER possesses good direct as well as Indirect antioxidant activity. GER (and its metabolite erucin, ERN) effectively decomposes hydrogen peroxide and alkyl hydroperoxides with second-order rate constants of k₂ = 6.9 ± 0.1 × 10^-2 M ^-1 s^-1 and 4.5 ± 0.2 × 10^-3 M ^-1 s^-, respectively, in water at 37 °C, thereby acting as a peroxide-scavenging preventive antioxidant. Interestingly, upon removal of H₂O₂ or hydroperoxides, ERN is converted into sulforaphane, the most effective inducer of phase II enzymes among ITCs. On the other hand, ERN (and conceivably GER), like other ITCs, does not possess any chain-breaking antioxidant activity, being unable to protect styrene from its thermally (37 °C) initiated autoxidation in the presence of AMVN. The mechanism and relevance of the antioxidant activity of GER and ERN are discussed.

Full text of DOI:10.1021/jf047945a

J. Agric. Food Chem. 2005, 53, 2475−2482 2475
Direct Antioxidant Activity of Purified Glucoerucin, the Dietary
Secondary Metabolite Contained in Rocket (Eruca sativa Mill.)
Seeds and Sprouts
JESSICA BARILLARI,† DONATELLA CANISTRO, MORENO PAOLINI,
,‡
‡
‡
§
§
,†
FIAMMETTA FERRONI, GIAN FRANCO PEDULLI, RENATO IORI,* AND
,
§
LUCA VALGIMIGLI*
Consiglio per la Ricerca e la Sperimentazione in Agricoltura (C.R.A.), Istituto Sperimentale per le
Colture Industriali, Via di Corticella 133, I-40129 Bologna, Italy, Department of Pharmacology,
Biochemical Toxicology Unit, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy, and
Department of Organic Chemistry “A. Mangini”, University of Bologna, Via San Giacomo 11,
I-40126, Bologna, Italy
Rocket (Eruca sativa Mill. or Eruca vesicaria L.) is widely distributed all over the world and is usually
consumed fresh (leafs or sprouts) for its typical spicy taste. Nevertheless, it is mentioned in traditional
pharmacopoeia and ancient literature for several therapeutic properties, and it does contain a number
of health promoting agents including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates
(GLs). The latter phytochemicals have recently gained attention as being the precursors of
isothiocyanates (ITCs), which are released by myrosinase hydrolysis during cutting, chewing, or
processing of the vegetable. ITCs are recognized as potent inducers of phase II enzymes (e.g.,
glutathione transferases, NAD(P)H:quinone reductase, epoxide hydrolase, etc.), which are important
in the detoxification of electrophiles and protection against oxidative stress. The major GL found in
rocket seeds is glucoerucin, GER (108 ( 5 µmol g- d.w.) that represents 95% of total GLs. The
content is largely conserved in sprouts (79% of total GLs), and GER is still present to some extent
in adult leaves. Unlike other GLs (e.g., glucoraphanin, the bio-precursor of sulforaphane), GER
possesses good direct as well as indirect antioxidant activity. GER (and its metabolite erucin, ERN)
effectively decomposes hydrogen peroxide and alkyl hydroperoxides with second-order rate constants
1
) 6.9 ( 0.1 × 10 M-1 s-1 and 4.5 ( 0.2 × 10 M-1 s- , respectively, in water at 37 °C,
-
2
-3
of k
2
2 2
thereby acting as a peroxide-scavenging preventive antioxidant. Interestingly, upon removal of H O
or hydroperoxides, ERN is converted into sulforaphane, the most effective inducer of phase II enzymes
among ITCs. On the other hand, ERN (and conceivably GER), like other ITCs, does not possess
any chain-breaking antioxidant activity, being unable to protect styrene from its thermally (37 °C)
initiated autoxidation in the presence of AMVN. The mechanism and relevance of the antioxidant
activity of GER and ERN are discussed.
KEYWORDS: Antioxidant; rocket; Eruca sativa (Mill.); Eruca vesicaria (L.); glucosinolate; isothiocyanate;
glucoerucin; erucin
INTRODUCTION
spicy flavor of young leaves. Rocket seeds are also used for
the production of oil and for appreciated pungent taste sprouts.
Rocket comprises a number of species of the Brassicaceae
Mentions of its medical use can be found throughout ancient
literature. Rocket was believed to have aphrodisiac properties;
such a virtue is mentioned by Virgil, and in the first century
AD, Lucius Columella affirms “excitet ut Veneri tardos eruca
maritos”, translated as “the rocket excites as the lovers embrace
the lazy husbands” (De Re Rustica, X: 108). Both Dioscorides
(3) and Galen (4) recommended eating seeds for increasing
semen production; for these properties, rocket was forbidden
in gardens of monasteries (5), despite the fact that both the Bible
(Second Kings 4:39-40) and Pliny (Historia Naturalis) had
(
Cruciferae) family belonging to the Eruca (Miller) and
Diplotaxis (DC.) genera. Eruca satiVa (Mill.) or Eruca Vesicaria
L.) has its origin in the Mediterranean region (1) but is widely
(
distributed all over the world (2). It is mostly harvested from
the wild or cultivated as an edible vegetable for the distinct
*
To whom correspondence should be addressed. E-mail: (L.V.)
luca.valgimigli@unibo.it; (R.I.) r.iori@isci.it.
†
Istituto Sperimentale per le Colture Industriali.
‡
Biochemical Toxicology Unit, University of Bologna.
§
Department of Organic Chemistry “A. Mangini”, University of Bologna.
1
0.1021/jf047945a CCC: $30.25
© 2005 American Chemical Society
Published on Web 03/08/2005

2476 J. Agric. Food Chem., Vol. 53, No. 7, 2005
Barillari et al.
Scheme 1
reported its benefits in eye treatment. Rocket has also been used
in the traditional pharmacopoeia for various purposes: antiphlo-
gistic, astringent, depurative, diuretic, digestive, emollient, tonic,
stimulant, laxative, and rubefacient (6).
represents 1-2% of dry weight, and it is associated with relevant
amounts of other GLs (e.g., glucoiberin, glucoerucin, 4-hydroxy-
glucobrassicin, progoitrin, etc.); this impairs its isolation and
purification in large quantities. Our previous finding that
glucoerucin (GER) could undergo a high yielding transformation
into GRA by chemoselective oxidation using hydrogen peroxide
(Scheme 1) (17) provided a possible solution to this problem
and boosted our interest for GER and its ITC-derivative, erucin
(ERN, 4-methylthiobutyl ITC).
Although ERN has received up to now poor attention due to
the lesser phase II enzyme inducer activity as compared to SFN
(10), our finding suggests that, unlike SFN and ITC in general
(14), it might possess direct as well as indirect antioxidant
activity.
Like other cruciferous vegetables, rocket contains a range of
health-promoting phytochemicals including carotenoids, vitamin
C, fibers, flavonoids, and glucosinolates (GLs) (7). The latter
phytochemicals have recently garnered great interest for their
potential role in the maintenance of human health. In particular,
many papers associate a highly significant cancer risk reduction
with increasing Brassicaceae consumption (8, 9), and it has been
speculated that the isothiocyanates (ITCs), obtained from
myrosinase hydrolysis of GLs (by chewing, cutting, or process-
ing the vegetable), are in great part responsible for the protective
effects of Brassica vegetables (10, 11). The prevailing mech-
anism identified for the protective activity of ITCs is the
induction of phase II enzymes [e.g., glutathione transferases
With the aim to assess that consumption of vegetables
containing the title GL could afford protection against the
oxidative damage, we report here the determination of GER
content in rocket seeds and sprouts, together with a detailed
kinetic investigation on the reactivity of GER and ERN with
hydrogen peroxide (H2O2), alkyl hydroperoxides (ROOH), and
(
1
GSTs, EC 2.5.1.18), NAD(P)H:quinone reductase (QR, EC
.6.99.2), epoxide hydrolase, and heme oxygenase], which are
important in the detoxification of electrophiles (12). More than
20 different GLs have been identified, and their structures share
1
•
peroxyl radicals (ROO ).
a common core of a â-D-glucopyrano moiety linked via a sulfur
atom to a (Z)-N-hydroximinosulfate ester and a variable aglycon
side chain derived from the R-amino acid biosynthetic precursor,
which allows their classification in three groups: aliphatic,
aromatic, and indole GLs (13). Several methionine-derived GLs,
which constitute the largest group of GLs, bear in their side
chain an additional sulfur atom at different oxidation states
MATERIALS AND METHODS
Materials. Solvents were of the highest grade commercially available
and were used as received. 2,2′-Azobis(2,4-dimethylvaleronitrile),
AMVN (Wako Pure Chemicals Ind. Ltd.), was stored at -20 °C. RRR-
R-Tocopherol (Aldrich) was purified as previously described (18). 2,6-
Di-t-butyl-4-methylphenol (Aldrich) was crystallized from hexane.
Styrene (Aldrich, 99+%) was distilled under reduced pressure and
percolated twice trough silica and once through activated basic alumina.
The other chemicals were commercially available (Sigma-Fluka-
Aldrich) and were used as received.
Seed and Sprout Samples. Rocket (Eruca satiVa Mill.) ripe seeds
and 5 day-old sprouts were supplied by Suba and Unico, Longiano,
Forl `ı (Italy). GER content in the samples was assessed in triplicate by
the EU official method (ISO 9167-1) (19), based on the HPLC analysis
of desulfo-GLs obtained through the removal of the sulfate group of
GLs via sulfatase-catalyzed hydrolysis. Seeds and freeze-dried sprouts
were reduced to a fine powder, and about 400 mg aliquots were
extracted by adding 5 mL of boiling 70% ethanol plus 200 µL of
sinigrin (29.5 mM) as internal standard. The mixture was homogenized
for 5 min at 75 °C using an U-Turrax (IKA T25) homogenizer, and
the extracts were centrifuged. The residue was re-extracted with further
(sulfide, sulfoxide, or sulfone functions), which is retained in
the ITC-derived compounds produced via hydrolysis by myro-
sinase (EC 3.2.3.1) (MYR). Since Zhang et al. (10) reported a
comparative study of QR and GST inductions in mouse tissue
by alkyl thiofunctionalized ITCs, differing in the oxidation state
of sulfur, several hundred papers have focused on sulforaphane
(SFN, 4-methylsulfinylbutyl ITC), which is considered the most
active phase II enzyme inducer among analogues. SFN has also
been attributed indirect antioxidant activity, which would arise
from induction of GSTs, QR, and heme oxygenase, with
increased levels of reduced glutathione (GSH), of reduced
coenzyme Q (QH2), and of bilirubin (14). Broccoli and
particularly broccoli sprouts are the richest easily available
sources of glucoraphanin (GRA), the GL bio-precursor of SFN
(11, 15, 16), and this has significantly increased the interest for
5
mL of boiling 70% ethanol and recentrifuged. Surpernatants were
these vegetables over the last years. Despite the increasing
interest in SFN, the availability of large amounts of pure
compounds for clinical investigation constitutes a major limit.
Indeed, the content of GRA in broccoli seeds or sprouts
combined to give a final volume of 10 mL. Each extract (1 mL) was
loaded onto a minicolumn filled with 0.6 mL of DEAE-Sephadex A-25
anion-exchange resin (Amersham Biosciences, Milano) conditioned with
25 mM acetate buffer, pH 5.6. After being washed with 3 mL of buffer,

Direct Antioxidant Activity of Purified Glucoerucin
50 µL of purified sulfatase (20) was loaded into the minicolumn and
J. Agric. Food Chem., Vol. 53, No. 7, 2005 2477
ITC) was reacted with a 4-fold excess of H or t-butyl hydroperoxide
1
2 2
O
was left overnight at room temperature. The desulfo-GLs was then
eluted with 3 mL of distilled water and finally injected into HPLC.
Desulfo-GLs were analyzed using a Hewlett-Packard Model 1100
HPLC system with an Inertsil ODS3 column (250 × 3 mm, 5 µm).
Chromatography was performed with 1 mL/min flow rate at 30 °C by
eluting with a gradient of water (A) and acetonitrile (B) as follows:
isocratically 1% B for 1 min, linear gradient to 22% B for 21 min, and
linear gradient to 1% B for 3 min. Elution of desulfo-GLs was detected
by a diode array, monitoring the absorbance at 229 nm. The amount
of GER was determined using sinigrin as the internal standard and the
relative response factor (21).
in methanol. Analysis of the reaction mixture was performed by GC-
MS using a Hewlett-Packard 5890 series II gas-chromatograph coupled
with a HP5971 mass selective detector. The most appropriate instru-
mental settings were found to be the following: injection split ratio,
50:1; injection volume, 1.0 µL; column, J&W DB-5MS (30 m × 0.25
mm × 0.25 µm film thickness); carrier gas (H
2
) flow, 1.0 mL/min;
and temperature programming, 40 °C (hold 5 min) to 250 °C (hold 5
min) at 5 °C/min.
Kinetic Measurements with Hydrogen Peroxide and Alkyl
-4
Hydroperoxides. GER (1-5 × 10 M) was incubated with hydrogen
-
2
-2
peroxide (1-5 × 10 M) or t-butyl hydroperoxide (1 × 10 to 0.2
M) in water at 298 or 323 K using a thermostated water bath equipped
with a sealed magnetic microstirrer. Aliquots of the reaction mixture
were sampled at time intervals and analyzed by ESI-MS in a
Micromass ZMD ESI-MS spectrometer, with direct injection in the
electrospray source through a syringe pump (Harvard Apparatus Pump
II) at a flow rate of 15 µL/min. The most appropriate instrumental
settings were determined in a preliminary set of experiments: ESI type,
Extraction and Purification of GER. GER was purified from rocket
(Eruca satiVa Mill.) ripe seeds. Rocket seeds were first ground to a
fine powder and defatted in hexane. The solvent was removed, and
the defatted meal was then treated with boiling 70% ethanol to produce
a quick deactivation of endogenous MYR and to extract the intact GL.
The isolation of GER from the extract was carried out by one-step
anion-exchange chromatography, as previously described (22, 23). The
purity was further improved by gel-filtration removal of contaminants,
which was performed using a XK 26/100 column packed with Sephadex
G10 chromatography media (Amersham Biosciences), connected to an
FPLC System (Pharmacia). The mobile phase was water at a flow rate
2
negative ions; desolvation gas (N ), 60 L/h; cone flow (skimmer), 6
L/h; desolvation temperature, 150 °C; capillary voltage, 3.0 kV; cone
voltage, 40 V; hexapole extractor, 3.0 V; and RF lens, 0.3 V. Calibration
curves were obtained both for GER and for GRA using authentic
samples, under identical instrumental settings. The peak area of signals
at m/z 420 and 436 were used, respectively, for the two glucosinolates
-
1
of 2.0 mL min , and the eluate absorbance was monitored at 254 nm.
Fractions were assayed by HPLC analysis of the desulfo-derivative
according to the ISO 9167-1 method (19), and those containing the
GL (>95%) were collected and freeze-dried. GER was characterized
2
giving A ) 5855 + 743722C (r ) 0.9986) for m/z 420 and A ) -56
2
+ 471691C (r ) 0.9991) for m/z 436. Given the higher response factor
1
13
by H and C NMR spectrometry, and the absolute purity estimated
by HPLC was close to 95%.
of the instrument to m/z 420, the decay of GER was used to measure
the pseudo-first-order kinetic constants. Measurements were repeated
at least with three different starting concentrations of peroxide, and
for each concentration, at each temperature experiments were done in
triplicate.
Preparation of ERN. ERN was produced via MYR catalyzed
hydrolysis of GER, performed in 0.1 M phosphate buffer pH ) 6.5 at
3
7 °C. MYR was isolated from Sinapis alba L. seeds (24, 25), and the
enzymatic solution had a specific activity of 65 units/mg of soluble
protein, where one MYR unit was defined as the amount of enzyme
able to hydrolyze 1 µmol/min sinigrin at pH 6.5 and 37 °C.
The total conversion of 1.5 g of pure GER into ERN by 50 MYR
units was confirmed by HPLC analysis of the desulfo-derivative (19),
which allowed us to monitor the reduction until there was a complete
disappearance of GER in the reaction mixture. The hydrolysis product
was obtained after three consecutive extractions with dichloromethane.
The combined extracts were concentrated to a few milliliters in a rotary
evaporator at 45 °C, and finally the solvent was completely removed
under a stream of nitrogen to afford 465 mg (82% yield) of pure ITC.
The ERN structure was ascertained by GC-MS spectrometry using a
Hewlett-Packard GCD G1800A, equipped with a 30 m × 0.25 mm
capillary column HP-5 (26).
Autoxidation Experiments. Autoxidation experiments were per-
formed as previously described (27, 28, 29) in a two-channels oxygen
uptake apparatus, based on a Validyne DP 15 differential pressure
transducer that has already been described elsewhere (30). The entire
apparatus was immersed in a thermostated bath that ensured a constant
temperature within (0.1 °C. In a typical experiment, an air-saturated
-
5
chlorobenzene solution containing the antioxidant (2.5 × 10 to 5.0
-
4
×
10 M) was equilibrated with the reference solution containing only
-3
-2
an excess of R-tocopherol (1 × 10 to 1 × 10 M) in the same solvent
at 30 °C. After equilibration, a concentrated chlorobenzene solution of
-
2
-3
AMVN (final concentration 5 × 10 to 5 × 10 M) was injected in
both the reference and the sample flasks, and the oxygen consumption
in the sample was measured, after calibration of the apparatus, from
the differential pressure recorded with time between the two channels.
Initiation rates, R
experiments by the inhibitor method using R-tocopherol as reference
antioxidant: R
i
, were determined for each condition in preliminary
Preparation of Reference Compounds GRA and SFN. GRA was
produced from GER by chemoselective oxidation of the sulfide group
into the corresponding sulfoxide, producing a mixture of S-epimers
i
) 2[R-tocopherol]/τ. Blank experiments were performed
by following the autoxidation of styrene in the absence of any
(17). GRA was then isolated from the reaction mixture by anion
antioxidant, while reference experiments were performed using R-to-
exchange chromatography and gel filtration as reported previously for
-
5
1
13
copherol (1.0 × 10 M) or 2,6-di-t-butyl-4-methylphenol (BHT, 5.0
GER and characterized by H and C NMR spectroscopy.
-5
×
10 M) as standard antioxidants.
SFN was prepared in racemic form by MYR catalyzed hydrolysis
of hemi-synthetic GRA. SFN was purified following the procedure
previously used for ERN, and the structure of the compound was
confirmed by GC-MS spectrometry using a Hewlett-Packard GCD
G1800A, equipped with a 30 m × 0.25 mm capillary column HP-5
RESULTS
GER was purified on the gram scale to a high purity degree
with good yields, starting from seeds that contained a strongly
predominant individual GL, using a simple procedure (22, 23).
(26).
Product Studies for Oxidation of GER and ERN. GER (1 mM)
was incubated in water with a 4-fold excess of H or t-butylhydro-
-
1
The starting material used contained 108 ( 5 µmol g d.w.
GER, representing about 95% of the total GLs. Although the
GER content was inferior in rocket freeze-dried sprouts that
also contained its oxidized form GRA, it still retained 79% of
the total GLs content, as shown in Figure 1.
2 2
O
peroxide at room temperature for 2-6 h, and the reaction mixture was
analyzed by HPLC-MS using electrospray ionization (ESI) in a Waters
2
695 separation module (equipped with autosampler) coupled to a
Micromass ZMD ESI-MS spectrometer with the following instru-
mental settings: injection volume 20 µL; column, C18 (Waters X-Terra-
MS, 3 mm × 150 mm, 3.5 µm); eluent 90:10 methanol/water; flow
rate, 0.5 mL/min; splitting ratio, 10:1; ESI type, negative ions;
Identification of purified GER was assessed by NMR
spectroscopy, and the proton chemical shift assignments were
in agreement with previously reported data (31).
Incubation of GER dissolved in water with a 4-fold excess
of H2O2 at room temperature for 6 h resulted in complete
disappearance of the starting material as judged by the HPLC-
MS analysis of the reaction mixture. The compound was
2
desolvation gas (N ), 600 L/h; cone flow (skimmer), 60 L/h; desolvation
temperature, 350 °C; capillary voltage, 3.0 kV; cone voltage, 40 V;
hexapole extractor, 3.0 V; and RF lens, 0.3 V. Calibration curves were
obtained for each analyte using the same instrumental settings with
authentic samples dissolved in water. Similarly, ERN (4-methylthiobutyl

2478 J. Agric. Food Chem., Vol. 53, No. 7, 2005
Barillari et al.
Figure 1. HPLC chromatograms (according to ISO 9167-1) of GLs present in rocket (Eruca sativa Mill.) seeds (A) and freeze-dried sprouts (B): 1,
glucoraphanin and 2, glucoerucin.
Figure 2. ESI−MS spectra of the reaction mixture of 1 mM GER and 4 mM hydrogen peroxide in water (A) immediately after addition and (B) after 6
h incubation at room temperature.
completely (97% by HPLC-MS) converted into a product
SFN (see Scheme 1) with 94% conversion, measured by GC-
MS analysis of the reaction mixture. Clearly, with both starting
materials (the GL or the ITC), regioselective oxidation of the
methylthio moiety occurs to yield quantitatively the correspond-
ing methyl sulfoxide. In contrast, the subsequent oxidation step
(i.e., conversion of the methyl sulfoxide into methyl sulfone)
was not observed under the experimental conditions employed.
Kinetic Studies with Peroxides. The reaction of GER with
hydrogen peroxide and t-butyl hydroperoxide in water was
giving, in ESI-MS spectrometry, a pseudo-molecular ion peak
-
(
[M - H] ) at m/z 436 in negative ionization mode (i.e., at an
m/z value higher by 16 amu than that recorded for the starting
GL (m/z 420, see Figure 2)). The reaction product was identified
as GRA by comparison with a reference sample, previously
1
isolated and characterized by H NMR, in agreement with
previous reports (17). Similarly, the reaction of the correspond-
ing ITC, ERN, in methanol with an excess of H2O2 yielded

Direct Antioxidant Activity of Purified Glucoerucin
J. Agric. Food Chem., Vol. 53, No. 7, 2005 2479
Figure 3. (Left) decay of the signal at m/z 420 (GER,
b
) and growth of the signal at m/z 436 (GRA,
0
) during ESI
C. (Right) first-order plots of typical time-evolution traces of the concentration
C ( ) and 25 C ( ) or with 0.06 M t-butyl hydroperoxide at 37 ) and
−MS analysis of the reaction mixture
-
4
-2
of glucoerucin 1.33
×
10 M with H
2
O
2
(1.17
×
10 M) in water at 37
°
-
4
-2
of GER (1.5
C ( ).
×
10 M) in water incubated with 1.2
×
10 M H
2
O
2
at 37
°
b
°
2
° (0
25
°
O
absorption apparatus, using a differential pressure transducer
(27-30). Control experiments were performed using R-toco-
pherol or BHT as standard antioxidants.
Table 1. Second-Order Rate Constants (k
with t-Butyl Hydroperoxide and Hydrogen Peroxide in Water
2
) for the Reaction of GER
a
(M- s-1)
1
k
2
-5
Autoxidations in the presence of R-tocopherol (1.0 × 10
peroxide
t-BuOOH
298 K
310 K
M) showed a neat inhibition period during which the oxygen
consumption was almost completely suppressed, followed by
an uninhibited autoxidation kinetic when the antioxidant had
been completely consumed. On the other hand, BHT (5.0 ×
-
-
3
2
-
3
2
(2.0
(3.3
±
±
0.3)
0.2)
×
×
10
10
(4.5
(6.9
±
±
0.2)
0.1)
×
×
10
10
-
2
H O
2
a
-5
Errors represent standard deviation.
10 M) only decreased the rate of oxygen consumption, thereby
acting as retardant rather than an inhibitor of the autoxidation.
These findings were in excellent agreement with the known
behavior of such reference antioxidants (28, 32).
investigated at 25 and 37 °C, under pseudo-first-order conditions
-4
by the potassium salt of the glucosinolate (1-5 × 10 M) being
incubated with a 100-fold excess of the peroxide. For conven-
ience, the reaction was followed at time intervals by direct
injection of the reaction mixture in the ISI-MS spectrometer
through a syringe pump. The reaction course was monitored
With these experimental settings, we tested the antioxidant
activity of ERN (4-methylthiobutyl ITC), chosen for conven-
ience due to the higher solubility in organic solvents as
compared to its precursor GER. In none of the autoxidation
experiments performed in the presence of ERN in the concen-
-
from the disappearance of signal at m/z 420 ([M - H] ion of
-
5
-4
-
tration range 1.0 × 10 to 5.0 × 10 M was inhibition or
retarding of the initiated autoxidation observed. Clearly, ERN,
and conceivably GER, do not possess any chain-breaking
antioxidant activity under the experimental conditions employed.
GER) and appearance of the signal at m/z 436 ([M - H] of
GRA) in negative ionization mode. Preliminary studies allowed
adjustment of the optimal instrumental setting for direct analysis
of the reaction mixture under conditions in which the two
pseudo-molecular ions were the only signals due to the
glucosinolates. Calibration of the spectrometer response with
authentic samples was performed using either the MS signal
intensity or the integrated signal peak, which afforded almost
identical results. As can be seen from Figure 3, the decay of
GER and growth of GRA, monitored through the time evolutions
of signals at m/z 420 and 436, respectively, afforded nice first-
order complementary kinetic traces (i.e., the rate of disappear-
ance of the signal due to GER was almost identical to the rate
of formation of GRA). Since decay traces usually showed lower
scattering of the mass signal, for convenience they were chosen
to calculate the second-order rate constants. Measurements were
always performed under pseudo-first-order conditions with
various amounts of starting GL and peroxide. Results are
collected in Table 1.
DISCUSSION
Antioxidants comprise a broad and heterogeneous family of
compounds that share the common task of interfering with (stop,
retard, or prevent) the oxidation (or autoxidation) of an
oxidizable substrate. In a biological environment, such as a
living organism, the most relevant autoxidation process is lipid
peroxidation, a well-known and thoroughly investigated process,
which involves a free radical chain reaction (33).
Initiation
•
•
HOOH (or ROOH) f HO (or RO ) (rate ) Ri) (1)
•
•
•
HO (or RO ) + LH f HOH (or ROH) + L
Propagation
(2)
Kinetic Studies with Peroxyl Radicals. These studies were
performed by the thermally initiated autoxidation of styrene
being investigated either in the absence or in the presence of
the antioxidant. The reaction was carried out in a closed system
under controlled conditions at 30 °C in air-saturated solutions
of a standard oxidizable substrate (styrene) in chlorobenzene.
The autoxidations were initiated with 2,2′-azobis(2,4-dimeth-
ylvaleronitrile) (AMVN) and were followed by the oxygen
consumption being monitored with an automatic recording gas
diff.
•
•
L + O
8 LOO
(3)
(4)
2
kp
•
•
LOO + LH
98 LOOH + L
Termination
kt
9
•
•
LOO + LOO
8 molecular products
(5)

2480 J. Agric. Food Chem., Vol. 53, No. 7, 2005
Barillari et al.
In this simple scheme, LH represents a lipid molecule, and
tively (41)), furthermore yielding an alkyl radical able to
propagate the autoxidation.
•
L is the corresponding carbon-centered radical generated by
abstraction of a hydrogen atom. The first step for lipid
peroxidation is the production of a reactive radical species (e.g.,
by the decomposition of hydrogen peroxide or a hydroperoxide
in alkoxyl or hydroxyl radicals). Immediately upon formation,
in the subsequent propagation step, the lipid radical reacts at a
diffusion-controlled rate with molecular oxygen to form a
peroxyl radical, which is the actual chain-carrying species.
Antioxidants can be classified on the basis of their mechanism
of interference with the peroxidation process as preventive
antioxidants or chain-breaking antioxidants: the former (e.g.,
catalase, metal chelators, UV filters) mainly interferes with the
initiation process (i.e., retard or stop the initial formation of
radical species), while the latter (e.g., R-tocopherol, ascorbate,
flavonoids) actually blocks or slows the autoxidation by
competing with the propagation reactions (i.e., they react with
peroxyl radicals faster than the oxidizable substrate (usually
polyunsaturated fatty acids in bio-membranes) to form species
that do not propagate the oxidation chain (34, 35, 36)). In
addition to these direct antioxidants, other compounds that do
not possess relevant antioxidant activity in vitro, but can
stimulate and increase the efficacy of the physiological anti-
oxidant defenses in vivo, are usually classified as indirect
antioxidants.
Isothiocyanates such as SFN and ERN are known to possess
indirect antioxidant activity through induction of GSTs, QR,
and heme oxygenase (14); however, while this mechanism is
the only to be expected for SFN or its precursor, the present
investigation clearly shows that GER possesses good direct,
albeit preventive, antioxidant activity. This is due to its ability
to decompose hydroperoxides and hydrogen peroxide, which
are most often responsible for the initiation step, as well as being
signaling mediators that lead to activation of redox sensitive
transcription factors and responsive genes (37). Results from
Table 1 can be compared with the reactivity of hydrogen
peroxide with glutathione, phenylaminoethyl sulfide, and fe-
nylaminoethyl selenide (two recently developed synthetic an-
tioxidants), whose rate constants at 37 °C have been reported
Oxidative stress is deeply involved in cancer development,
and oxygen-centered radicals play a role in apoptosis. It is well-
known that higher amounts of the reactive oxygen species, but
particularly hydrogen peroxide, are produced in some cancer
cells (42). Hydrogen peroxide is widely recognized as being
extremely important in signal transduction by acting as a
signaling molecule in the mitogen-activated protein kinase
pathway, which results in the activation of redox-sensitive
transcription factors and a responsive gene that promotes cell
survival, growth, and proliferation (37). However, while the
constitutive high production of hydrogen peroxide in cancer cells
is needed to promote their proliferation, additional amounts of
hydrogen peroxide above a certain threshold cause cell cycle
arrest and/or apoptosis (37). An extensive body of evidence
demonstrates that high concentrations of ITC inhibit cancer cell
proliferation by inducing the formation of intolerable amounts
of reactive oxygen species, thus leading to caspase-mediated
apoptosis, whereas at lower concentrations, these compounds
activate protein kinases mediating the expression of defensive
and survival genes, such as GSTs and QR (43). A recent
investigation has shown that ERN induces apoptosis selectively
in human leukemia cells, but not in nontransformed T lympho-
cytes, thereby being a promising new agent in cancer therapy
(44).
In light of the previous information, it is possible, although
speculative, that the ability of ERN to induce apoptosis
selectively in cancer cells, unlike SFN (45), may be at least
partially related to its unusual capability to act as a hydroper-
oxide-scavenging preventive antioxidant. Further investigation
is in progress to disclose possible relationships between the fine
antioxidant/prooxidant balance of ERN and its selective pro-
apoptotic behavior.
Our current investigation boosts the interest on the role in
the human diet of rocket, GER, and ERN. In a recent study,
Gill et al. (46) found that the extract of cruciferous and legume
sprouts not only exerted significant in vitro antigenotoxic activity
against H O damage in HT29 human colon cells but also
2
2
-
4
-2
-1 -1
as 1.01, 4.2 × 10 , and 4.7 × 10
M
s , respectively (38).
2 2
protected against H O insult of peripheral blood lymphocytes
As the active moiety of GER in this preventive antioxidant
activity is the methylthio group, which undergoes oxidation into
methyl sulfoxide, similar antioxidant activity is expected to be
possessed by its biologically active metabolite ERN. Interest-
ingly, the byproduct of this hydroperoxide-scavenging process
is GRA, (i.e., the bioprecursor of SFN (see Scheme 1), the most
effective chemopreventive ITC of the family (10)).
of volunteers, after they consumed about 100 g of sprouts for
2 weeks. As a matter of fact, specific dietary approaches based
on broccoli sprouts have been recently investigated and proved
effective in reducing oxidative stress and cardiovascular prob-
lems (47). Similar or even higher benefits to human health are
to be expected from the consumption of rocket or rocket sprouts.
Indeed, one additional argument in favor of rocket is that this
vegetable is more often consumed fresh rather than boiled like
other vegetables such as broccoli. This leaves MYR activity at
its best and significantly increases the content of ITCs, which
have been found six times more bioavailable than GLs (48).
On the other hand, it is worth noting that ERN and
conceivably any other ITC of the family (as well as their GL
precursors) do not possess any chain-breaking antioxidant
activity. This finding is not entirely unexpected when consider-
ing the structure of these compounds. Isothiocyanates have been
shown to react with silyl or stannyl radicals (39) and with alkyl
radicals (40) to yield a very reactive carbon centered radical.
To be useful as chain-breaking antioxidants, this reaction should,
however, compete with the propagation process and yield a new
radical that is unable to propagate the chain: this is obviously
not the case. Furthermore, it should be noted that, in oxygen
rich environments, peroxyl radicals, rather than alkyl radicals,
are the chain-propagating species (vide supra), and no reaction
of peroxyl radicals with isothiocyanates has been so far reported.
The other moiety in the ERN structure that would be potentially
reactive toward peroxyl radicalssCH2 hydrogens alpha to the
sulfidesis expected to react too slowly (e.g., the rate constants
for the reaction of benzylphenyl thioether or tetrahydrothiophene
CONCLUSION
It has recently been reported that leaves of E. satiVa contain
-mercaptobutyl GL as the major GL among nine, while GER
4
is present only in low amounts (49). In the present study, we
have shown that rocket seeds and sprouts contain GER as main
GL, in large amounts in comparison to leaves (mature plants),
thereby paralleling previous findings on other Brassicaeae GLs
content (11). When eaten raw, rocket sprouts that are actually
available on the market release large amounts of ERN through
endogenous MYR activity.
GER and ERN possess good direct antioxidant activity as
hydroperoxide-scavenging preventive antioxidants. Upon reac-
tion with hydroperoxides, they produce GRA and SFN, respec-
-
1
-1
with t-butylperoxyl radicals are 0.16 and 0.1 M s , respec-

Direct Antioxidant Activity of Purified Glucoerucin
J. Agric. Food Chem., Vol. 53, No. 7, 2005 2481
tively. Therefore, ERN is potentially capable of protection from
oxidative stress by three mechanisms: (i) through induction of
phase II enzymes, (ii) by scavenging hydrogen peroxide and
alkyl hydroperoxides accumulated in cells and peripheral blood,
and (iii) by acting as a precursor of SFN, a potent inducer of
detoxifying enzymes. The lack of activity as a chain-breaking
antioxidant suggests its use in combination with other health-
promoting nutrients such as R-tocopherol or ascorbate. This
maximization of the protection toward oxidative stress can be
easily obtained through ordinary consumption of fresh uncooked
rocket, an ancient underutilized vegetable crop (50).
(13) Fahey, J. D.; Zalcmann, A. T.; Talalay, P. The chemical diversity
and distribution of glucosinolates and isothiocyanates among
plants. Phytochemistry 2001, 56, 5-51.
(
14) Fahey, J. W.; Talalay, P. Antioxidant Functions of Sul-
foraphane: a Potent Inducer of Phase II Detoxication Enzymes.
Food Chem. Toxicol. 1999, 37, 973-979.
(
15) Vallejo, F.; Garcia-Viguera, C.; Tomas-Barberan, F. A. Changes
in broccoli (Brassica oleracea L. Var. italica) health-promoting
compounds with inflorescence development. J. Agric. Food
Chem. 2003, 51, 3776-3782.
(
16) West, L. G.; Meyer, K. A.; Balch, B. A.; Rossi, F. J.; Schultz,
M. R.; Hass, G. W. Glucoraphanin and 4-Hydroxyglucobrasicin
Contents in Seeds of 59 Cultivars of Broccoli, Raab, Kolrabi,
Radish, Cauliflower, Brussels Sprouts, Kale, and Cabbage. J.
Agric. Food Chem. 2004, 52, 916-926.
ACKNOWLEDGMENT
Financial support from the University of Bologna, MIUR
(
17) Iori, R.; Bernardi, R.; Gueyrard, D.; Rollin, P.; Palmieri, S.
Formation of glucoraphanin by chemoselective oxidation of
natural glucoerucin: a chemoenzymatic route to sulforaphane.
Bioorg. Med. Chem. Lett. 1999, 9, 1047-1048.
(research project “Free Radical Processes in Chemistry and
Biology: Fundamental Aspects and Applications in Environ-
ment and Material Sciences”) and Indena S.p.A. (research
project “Studi su Isotiocianati Provenienti da Glucosinolati di
Origine Naturale”) is gratefully acknowledged. We thank Prof.
Patrick Rollin for helpful discussions and Dr. Guido Fracasso
for assistance with the NMR spectroscopy.
(
18) Valgimigli, L.; Banks, J. T.; Ingold, K. U.; Lusztyk, J. Kinetic
Solvent Effects on Hydroxylic Hydrogen Atom Abstraction Are
Independent of the Nature of the Abstracting Radical. Two
Extreme Tests Using Vitamin E and Phenol. J. Am. Chem. Soc.
1
995, 117, 9966-9971.
(
19) EEC Regulation No. 1864/90. Enclosure VIII. Office of European
LITERATURE CITED
Communication. 1990, L170: pp 27-34.
(
20) Leoni, O.; Iori, R.; Haddoum, T.; Marlier, M.; Wathelet, J.-P.;
Rollin, P.; Palmieri, S. Approach to the use of immobilized
sulphatase for analytical purposes and for the production of
desulfo-glucosinolates. Ind. Crops Prod. 1998, 7, 335-343.
21) Wathelet, J.-P.; Iori, R.; Mabon, N.; Palmieri, S.; Leoni, O.;
Rollin, P.; Marlier, M. Determination of the response factors of
several desulfo-glucosinolates used for quantitative analysis of
Brassicaceae; GCIRC Technical Meetings, June 5-7, 2001,
Poznan, Poland.
(
1) Zeven, A. C.; de Wet, J. M. J. Dictionary of cultiVated plants
and their regions of diVersity, 2nd ed.; Centre for Agricultural
Publishing and Documentation, Wageningen, 1982; p 107.
2) Warwick, S. I. Guide to the wild germplasm of Brassica and
allied crops. Part V. Life History and Geographical Data for wild
species in the tribe Brassicaceae (Cruciferae). Agric. Can. Tech.
Bull. 1994, 2E, 61.
(
(
(
3) Gunther, R. T. The Greek herbal of Dioscoridessde materia
medica. II/170; Hafner Publishing Co.: New York, 1968.
4) K u¨ hn, C. G. Claudi Galeni opera omnia, XII. Leipzig, 1826.
5) Mascagno, V. Coltivata o selvatica la rucola e` ottima in insalata.
Vita Campagna 1987, 5, 42-43.
(
(
(22) Visentin, M.; Tava, A.; Iori, R.; Palmieri, S. Isolation and
Identification of trans-4-(Methylthio)-3-butenyl-glucosinolate
from Radish Roots (Raphanus satiVus L.). J. Agric. Food Chem.
1992, 40, 1687-1681.
(23) Barillari, J.; Gueyrard, D.; Rollin, P.; Iori, R. Barbarea Verna
as a source of 2-phenylethyl glucosinolate, precursor of cancer
chemopreventive phenylethyl isothiocyanate. Fitoterapia 2001,
72, 760-764.
(
6) (a) Arietti, N. Flora medica ed erboristica del territorio
bresciano, 1st ed.; Commentari Ateneo di Brescia, Tip. Fratelli
Geroldi: Brescia, 1965; p 460. (b) Uphof, J. C. T. Dictionary
of economic plants, 2nd ed.; Verlag Von J. Cramer Publ.: New
York, 1968; p 591. (c) Balm e´ , F. Plantas Medicinais; Hemus
Livraria Editora Limitada: S a˜ o Paulo, 1978; p 398. (d) Yaniv,
Z.; Schafferman, D.; Amar, Z. Tradition, Uses, and Biodiversity
of Rocket (Eruca satiVa) in Israel. Econ. Botany 1998, 52, 394-
(24) Palmieri, S.; Iori, R.; Leoni, O. Myrosinase from Sinapis alba
L.: A New Method of Purification for Glucosinolate Analyses.
J. Agric. Food Chem. 1986, 34, 138-140.
4
00. (e) Perry, L. M. Medicinal plants of east and southeast
(
25) Pessina, A.; Thomas, R. M.; Palmieri, S.; Luisi, P. L. An
improved method for the purification of myrosinase and its
physicochemical characterization. Arch. Biochem. Biophys. 1990,
Asia: attributed properties and uses; MIT Press: Cambridge,
MA, 1978.
(
7) Steinmetz, K. A.; Potter, J. D. Vegetables, fruit, and cancer. II.
Mechanisms. Cancer Causes Control 1991, 2, 427-442.
8) Jeffery, E. H.; Jarrell, V. Cruciferous vegetables and cancer
prevention. In Handbook of Nutraceuticals and Functional
Foods; Wildman, R. E. C., Ed.; CRC Press LLC: Boca Raton,
FL, 2001; pp 169-192.
2
80, 383-389.
(
26) Leoni, O.; Iori, R.; Palmieri, S.; Esposito, E.; Menegatti, E.;
Cortesi, R.; Nastruzzi, C. Myrosinase-generated isothiocyanate
from glucosinolates: isolation, characterization, and in vitro
anitproliferative studies. Bioorg. Med. Chem. 1997, 5, 1799-
(
1
806.
(
9) van Poppel, G.; Verhoeven, D. T.; Verhagen, H.; Goldbohm, R.
A. Brassica vegetables and cancer prevention. Epidemiology and
mechanisms. AdV. Exp. Med. Biol. 1999, 472, 159-168.
(
27) Amorati, R.; Ferroni, F.; Lucarini, M.; Pedulli, G. F.; Valgimigli,
L. A. Quantitative Approach to the Recycling of R-Tocopherol
by Coantioxidants. J. Org. Chem. 2002, 67, 9295-9303.
(
(
(
10) Zhang, Y.; Talalay, P.; Cho, C. G.; Posner, G. H. A major inducer
of anticarcinogenic protective enzymes from broccoli: isolation
and elucidation of structure. Proc. Natl. Acad. Sci. U.S.A. 1992,
(
28) Amorati, R.; Ferroni, F.; Pedulli, G. F.; Valgimigli, L. Modeling
the Coantioxidant Behavior of Monofunctional Phenols. Ap-
plications to Some Relevant Compounds. J. Org. Chem. 2003,
8
9, 2399-2403.
6
8, 9654-9658.
11) Fahey, J. W.; Zhang, Y.; Talalay, P. Broccoli sprouts: an
exceptionally rich source of inducers of enzymes that protect
against chemical carcinogens. Proc. Natl. Acad. Sci. U.S.A. 1997,
(
29) Lucarini, M.; Pedulli, G. F.; Valgimigli, L.; Amorati, R.; Minisci,
F. Thermochemical and Kinetic Studies of a Bisphenol Anti-
oxidant. J. Org. Chem. 2001, 66, 5456-5462.
9
4, 10367-10372.
12) Prestera, T.; Zhang, Y.; Spencer, S. R.; Wilezak, C., Talalay, P.
The electrophile counterattack response: protection against
neoplasia and toxicity. AdV. Enzyme Regulation 1993, 33, 281-
(30) Amorati, R.; Pedulli, G. F.; Valgimigli, L.; Attanasi, O. A.;
Filippone, P.; Fiorucci, C.; Saladino, R. Absolute rate constants
for the reaction of peroxyl radicals with cardanol derivatives. J.
Chem. Soc., Perkin Trans. 2 2001, 2142-2146.
2
96.

2482 J. Agric. Food Chem., Vol. 53, No. 7, 2005
Barillari et al.
(
31) Prestera, T.; Fahey, J. W.; Holtzclaw, W. D.; Abeygunawardana,
C.; Kachinski, J. L.; Talalay, P. Comprehensive chromatographic
and spectroscopic methods for the separation and identification
of intact glucosinolates. Anal. Biochem. 1996, 239, 168-179.
32) Burton, G. W.; Ingold, K. U. Autoxidation of Biological
Molecules. 1. The Antioxidant Activity of Vitamin E and Related
Chain-Breaking Antioxidants in Vitro. J. Am. Chem. Soc. 1981,
(44) Fimognari, C.; Nusse, M.; Iori, R.; Cantelli-Forti, G.; Hrelia, P.
The new isothiocyanate 4-(methylthio)butyl isothiocyanate se-
lectively affects cell-cycle progression and apoptosis induction
of human leukemia cells. InVest. New Drugs 2004, 22, 119-
(
1
29.
(
45) Fimognari, C.; Nusse, M.; Berti, F.; Iori, R.; Cantelli-Forti, G.;
Hrelia, P. Isothiocyanates as novel cytotoxic and cytostatic
agents: molecular pathway on human transformed and non-
transformed cells. Biochem. Pharmacol. 2004, 68, 1133-1138.
1
03, 6472-6477.
(
(
33) Ingold, K. U. Peroxyl Radicals. Acc. Chem. Res. 1969, 2, 1-9.
34) Ingold, K. U. Inhibition of the autoxidation of organic substances
in the liquid phase. Chem. ReV. 1961, 61, 563-589.
(46) Gill, C. I.; Haldar, S.; Porter, S.; Matthews, S.; Sullivan, S.;
Coulter, J.; McGlynn, H.; Rowland, I. The effect of cruciferous
and leguminous sprouts on genotoxicity, in vitro and in vivo.
Cancer Epidemiol. Biomarkers PreV. 2004, 13, 1199-1205.
(
35) Burton, G. W.; Ingold, K. U. Vitamin E: Application of the
Principles of Physical Organic Chemistry to the Exploration of
Its Structure and Function. Acc. Chem. Res. 1986, 19, 194-
2
01.
(
47) Wu, L.; Ashraf, M. H.; Facci, M.; Wang, R.; Paterson, P. G.;
Ferrie, A.; Juurlink, B. H. Dietary approach to attenuate oxidative
stress, hypertension, and inflammation in the cardiovascular
system. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7094-7099.
48) Shapiro, T. A.; Fahey, J. W.; Wade, K. L.; Stephenson, K. K.;
Talalay, P. Chemopreventive Glucosinolates and Isothiocyanates
of Broccoli Sprouts: Metabolism and Excretion in Humans.
Cancer Epidemiol. Biomarkers PreV. 2001, 10, 501-508.
(36) Denisov, E. Handbook of Antioxidants; CRC Press: Boca Raton,
FL, 1995; Ch. 1.
(37) Loo, G. Redox-sensitive mechanisms of phytochemical-mediated
inhibition of cancer cell proliferation (review). J. Nutr. Biochem.
(
2
003, 14, 64-73.
(
38) Woznichak, M. M.; Overcast, J. D.; Robertson, K.; Neumann,
H. M.; May, S. W. Reaction of Phenylaminoethyl Selenides with
Peroxynitrite and Hydrogen Peroxide. Arch. Biochem. Biophys.
2
000, 379, 314-320.
(49) Bennett, R. N.; Mellon, F. A.; Botting, N. P.; Eagles, J.; Rosa,
(
39) Bachi, M. D.; Bar-Ner, N.; Melman, A. Stereoselective Synthesis
of (()-R-Kainic Acid Using Free Radical Key Reactions. J. Org.
Chem. 1996, 61, 7116-7124.
E. A.; Williamson, G. Identification of the major glucosinolate
(
(
4-mercaptobutyl glucosinolate) in leaves of Eruca satiVa L.
salad rocket). Phytochemistry 2002, 61, 25-30.
(
40) Barton, D. H. R.; Jaszberenyi, J. C.; Theodorakis, E. A. The
invention of radical reactions. Part XXIII new reactions: Nitrile
and thiocyanate transfer to carbon radicals from sulfonyl cyanides
and sulfonyl isothiocyanates. Tetrahedron 1992, 48, 2613-2626.
41) Howard, J. A. Reaction of organic peroxyl radicals in organic
solvents. In Peroxyl Radicals; Alfassi Z. B., Ed.; John Wiley &
Sons: New York, 1997; pp 283-334.
(
50) Bianco, V. Rocket, an ancient underutilized Vegetable crop and
its potential; Rocket Genetic Resources Network, S. Paludosi,
compiler. IPGRI: Rome 1995; pp 35-58.
(
(
(
42) Szatrowski, T. P.; Nathan, C. F. Production of large amounts of
hydrogen peroxide by human tumor cells. Cancer Res. 1991,
Received for review December 6, 2004. Revised manuscript received
January 26, 2005. Accepted January 27, 2005.
5
1, 794-798.
43) Keum, Y.-S.; Jeong, W.-S.; Kong, A. N. T. Chemoprevention
by isothiocyanates and their underlying molecular signaling
mechanism. Mutat. Res. 2004, 555, 191-202.
JF047945A