Cleavage Products of Lycopene Produced by in Vitro Oxidations: Characterization and Mechanisms of Formation

Article abstract of DOI:10.1021/jf034735+

The aim of this study was to produce in vitro oxidation products of lycopene, which could be possible in vivo metabolites. An oxidation of lycopene with potassium permanganate gave a range of lycopene degradation compounds resulting from the oxidative cleavage of one or two carbon-carbon double bonds. Eleven apo-lycopenals/ones and six apo-carotendials were obtained and tentatively characterized by HPLC-DAD-MS. Apo-11-lycopenal and apo-8,6′-carotendial were isolated and characterized by ¹H NMR for the first time. Lycopene was submitted to an oxidation by atmospheric oxygen catalyzed by a metalloporphyrin, a model system of the active center of cytochrome P450 enzymes. (Z)-Isomers, monoxides, and cleavage compounds of (E)-lycopene were formed. We propose a mechanism of oxidation of lycopene by this system.

Full text of DOI:10.1021/jf034735+

7318 J. Agric. Food Chem. 2003, 51, 7318−7325
Cleavage Products of Lycopene Produced by in Vitro
Oxidations: Characterization and Mechanisms of Formation
CATHERINE CARIS-VEYRAT,*,† ANTJE SCHMID,†,‡ MICHEL CARAIL,† AND
‡
VOLKER BO¨ HM
UMR Safety and Quality of Plant Products, INRA, Domaine Saint Paul, Site Agroparc,
84914 Avignon Cedex 9, France, and Institute of Nutrition, Friedrich-Schiller University Jena,
Dornburger Strasse 29, D-07743 Jena, Germany
The aim of this study was to produce in vitro oxidation products of lycopene, which could be possible
in vivo metabolites. An oxidation of lycopene with potassium permanganate gave a range of lycopene
degradation compounds resulting from the oxidative cleavage of one or two carbon-carbon double
bonds. Eleven apo-lycopenals/ones and six apo-carotendials were obtained and tentatively character-
ized by HPLC-DAD-MS. Apo-11-lycopenal and apo-8,6′-carotendial were isolated and characterized
by 1H NMR for the first time. Lycopene was submitted to an oxidation by atmospheric oxygen catalyzed
by a metalloporphyrin, a model system of the active center of cytochrome P450 enzymes. (Z)-Isomers,
monoxides, and cleavage compounds of (E)-lycopene were formed. We propose a mechanism of
oxidation of lycopene by this system.
KEYWORDS: Lycopene; oxidation; apo-lycopenal/one; apo-carotendial
INTRODUCTION
of lycopene under different conditions of autoxidation, but so
far these compounds have not been found in vivo.
Carotenoids are plant secondary metabolites which present a
potential beneficial effect on human health (1). Except for the
well-known provitamin A activity of some carotenoids, they
could also be involved in protective effects against degenerative
diseases such as cancers or cardiovascular diseases. The
mechanism by which they act in vivo is not fully understood.
Hypotheses are: antioxidant actions (2), influence on the
immune system (3), and interaction with gap junctional inter-
cellular communication (GJIC) (4), but metabolites, and par-
ticularly oxidative metabolites of carotenoids could contribute
in vivo to effects attributed to the native molecule (5-7).
However, only a few metabolites have been detected in vivo,
one reason being their low concentration and the difficulty of
detection in biological fluids.
Lycopene, the red pigment of tomato, is one of the most
interesting carotenoids considering beneficial effects on health
(8, 9). It has been suggested that lycopene could be involved in
protection against chronic diseases (10, 11) such as prostate
cancer (12). It is frequently consumed in the Western world
through fresh or processed tomatoes. Even if lycopene has been
proven to be the most effective carotenoid as singlet oxygen
quencher in vitro (13), no clear mechanisms of action in vivo
have been found. Only one metabolite of lycopene was detected
in vivo, namely, 2,6-cyclolycopene-1,5 diol (14). Recently, Kim
et al. (15) have described the formation of cleavage compounds
This investigation was designed to characterize oxidation
products of lycopene obtained by two different systems of
oxidation, with potassium permanganate and by atmospheric
oxygen catalyzed by a metalloporphyrin.
MATERIALS AND METHODS
Chemicals. KMnO4, cetyltrimethylammonium bromide, carbonyl-
ruthenium(II)-tetraphenylporphyrin (Ru(CO)TPP), 3-chloroperoxyben-
zoic acid, CaCl2, Na2SO4, basic Al2O3, NH4OAc, were purchased from
Sigma-Aldrich (St Quentin Fallavier, France). Methanol was from
Merck (Darmstadt, Germany) and of HPLC grade. All other solvents
were from Carlo Erba (Val de Reuil, France). Ethanol, toluene, and
methylene chloride were of analytical grade. Acetone, n-hexane, tert-
butyl methyl ether (MTBE) and tetrahydrofuran (THF) were of HPLC
grade. Methylene chloride was poured through basic alumina before
use to eliminate traces of acidity. Water was purified through a Millipore
Q-Plus.
Spectrophotometric Apparatus. For purity controls, UV-vis
spectra were recorded on a Cary-1E (Varian). Measurements were made
at room temperature in quartz cells (length 1 cm).
Analytic HPLC-DAD-MS Apparatus and Conditions. HPLC
analyses were carried out with a Hewlett-Packard (HP) model 1050
equipped with a quaternary pump solvent delivery and a diode array
detector (DAD) switched in line with a Micromass Platform LCZ 4000.
The column used was a 250 × 4.6 mm i.d., 5 µm, YMC Pack C30
(YMC Inc., Wilmington, NC) equipped with a 20 × 4.6 mm, 5 µm,
C-30 precolumn. The column was kept at 27 °C. Absorption spectra
were recorded between 220 and 600 nm. Acquisition of the mass data
between m/z 100 and 700 was performed in the positive electrospray
mode. The program used for data analyses was Masslynx version 3.4.
* To whom correspondence should be addressed. E-mail: caris@
avignon.inra.fr.
† UMR Safety and Quality of Plant Products, INRA.
‡ Friedrich-Schiller University, Jena.
10.1021/jf034735+ CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/04/2003

Oxidation Products of Lycopene
J. Agric. Food Chem., Vol. 51, No. 25, 2003 7319
Parameters, and especially cone voltage, were optimized in order to
avoid fragmentation. The following gradient system was used with H2O
containing NH4OAc 25 mM (solvent A), methanol containing NH4OAc
25 mM (solvent B), and MTBE (solvent C): 0 to 2 min, %A-%B-
%C, 40-60-0; 5 min, %A-%B-%C, 20-80-0; 10 min, %A-%B-
%C, 4-81-15; 60 min, %A-%B-%C, 4-11-85; 80 min, %A-
%B-%C, 4-11-85; 80.01 min, %A-%B-%C, 0-100-0. The flow
was 1 mL/min.
Preparative HPLC Apparatus and Conditions. For apo-8,6′-
carotendial, preparative HPLC was carried out with a Waters model
600 equipped with a quaternary pump solvent. The column used was
a 250 × 20 mm i.d., 5 µm, YMC Pack C30 (YMC Inc., Wilmington,
NC) equipped with a 21.2 × 30 mm i.d., 10 µm C-18 precolumn. The
column was kept at 25 °C. The following gradient system was used
with H2O (solvent A), methanol (solvent B), THF (solvent C) and
acetone (solvent D): 0 min, %A-%B-%C-%D, 40-60-0-0; 10
min, %A-%B-%C-%D, 30-65-5-0; 15 min, %A-%B-%C-%D,
25-65-10-0; 20 min, %A-%B-%C-%D, 20-65-15-0, 30 min,
%A-%B-%C-%D, 15-55-15-15; 35 min, %A-%B-%C-%D,
10-45-10-35; 40 min, %A-%B-%C-%D, 5-30-5-60; 50 min,
%A-%B-%C-%D, 0-0-0-100. The flow was 15 mL/min.
Apo-11-lycopenal was isolated after HPLC using an Agilent 1100
analytical system. Column and eluant were the same as those described
above for analytical HPLC-DAD-MS.
Figure 1. HPLC chromatograms obtained by (A) UV−vis detection and
(B) mass spectrometric detection of the compounds resulting from the
oxidation of lycopene by potassium permanganate. Numbers correspond
to compounds listed in Figure 2 and 3. 1′ and 1′′ are (Z)-isomers of
compound 1. 2′ and 2′′ are (Z)-isomers of compound 2.
NMR Apparatus and Conditions. NMR analysis were performed
on a Bruker Avance DRX-500 apparatus equipped with a cryoprobe.
1
1H NMR of apo-8,6′-carotendial was performed in CDCl3. H NMR
Table 1. 1H NMR of Apo-11-lycopenal (8) in CD OH at 500 MHza
3
of apo-11-lycopenal was performed in CDCl3 and CD3OD.
Preparation of Lycopene. Lycopene was extracted from tomato
oleoresin kindly supplied by Naturex (Avignon, France). Tomato
oleoresin (20.56 g) was mixed with n-hexane (100 mL), the insoluble
fraction was filtered and washed twice with n-hexane (15 mL) and
twice with ethanol (10 mL). The solid was dried overnight under
reduced pressure in a desiccator containing CaCl2. Crude lycopene (1.2
g) was obtained. Purity was checked by UV-vis spectrophotometry
and HPLC-DAD-MS. No other compounds were detected. Lycopene
was kept at -20 °C under an atmosphere of argon.
Oxidation of Lycopene by KMnO4. Lycopene (39.8 mg) and
cetyltrimethylammonium bromide (8.1 mg) were dissolved in methylene
chloride/toluene (20 mL/20 mL). An aqueous solution of KMnO4 (12
mL, 15 mg KMnO4/mL water) was added to the organic solution. The
reaction mixture was stirred on a magnetic stirrer at room temperature
for 1 h and filtered through an Acrodisc, 0.45 µm, 32 mm PVDF filter.
The organic phase was washed with water (30 mL), filtered through a
PVDF filter and dried on Na2SO4. After evaporation under reduced
pressure, the residue was dissolved in 2.2 mL CH3OH/MTBE (80/20,
v/v) for HPLC-DAD-MS analysis.
Oxidation of Lycopene by Atmospheric Oxygen Catalyzed by
Ru(CO)TPP in the Presence of 3-Chloroperoxybenzoic Acid. Ru-
(CO)TPP (4.1 mg) was dissolved in methylene chloride (5 mL),
3-chloroperoxybenzoic acid (1.9 mg) was added, and the reaction
mixture was stirred for 5 min. Then, lycopene (13 mg) was added.
Aliquots of 0.5 mL were taken from the reaction mixture after 1, 5,
24, 48, and 96 h, filtered through PVDF filters, and diluted with 0.5
mL of CH3OH/MTBE (80/20, v/v) for HPLC-DAD-MS analysis.
δ in ppm (in CD OD)
(multiplicity, coupling constant in Hz)
3
proton
CH −C(1) trans
CH −C(1) cis
H−C(2)
H−C(3) and H−C(4)
1.68 (s)
1.62 (s)
5.09 (m) (in CDCl3)
2.17 (m)
1.90 (d, J ) 1.0)
6.05 (d, J ) 11.0)
7.14 (dd, J1 ) 11.0, J2 ) 15.2)
6.34 (d, J ) 15.2)
2.34 (d, J ) 1.0)
5.93 (d, J ) 8.4)
10.05 (d, J ) 8.4)
3
3
CH −C(5)
3
H−C(6)
H−C(7)
H−C(8)
CH −C(9)
H−C(10)
H−C(11)
3
a s ) singlet, d ) doublet, dd ) doublet of doublet, m ) multiplet.
most of them, the UV-vis spectral data, which were compared
with literature values when available. For two compounds, we
were able to confirm their structures, because it was possible
to purify them by preparative HPLC and subsequently analyze
1
them by H NMR.
Analysis of the extracted reaction mixture showed the
presence of two types of oxidation compounds, those resulting
from a single oxidative cleavage, and others derived from a
double oxidative cleavage of lycopene. The first type of
compounds contain on one end an aldehyde or a ketone group
and on the other end of the molecule the ψ-end group of
lycopene. These were called apo-lycopenal and apo-lycopenone,
respectively. The second category of products obtained contain
two aldehyde end groups and were called apo-carotendials,
because their structure could have been obtained from any
carotenoid and not only from lycopene. The chemical structures
of the identified apo-lycopenals/ones and apo-carotendials are
shown in Figure 2 and Figure 3. 1H NMR of apo-11-lycopenal
(8) and apo-8,6′-carotendial (14) are presented for the first time
in Tables 1 and 2.
RESULTS AND DISCUSSION
Oxidation of Lycopene with Potassium Permanganate. The
oxidation of lycopene by potassium permanganate was per-
formed in a biphasic system using cetyltrimethylammonium
bromide as phase transfer catalyst. After 1 h of reaction at room
temperature, an HPLC-DAD-MS analysis showed that all
lycopene was consumed, and a range of products was formed.
The absorbance and total ion chromatograms (TIC) obtained
by UV-vis detection and by mass detection respectively of the
extracted reaction mixture are presented in Figure 1. The
majority of the products were tentatively identified by detecting
the corresponding molecular weight for all of them, and for
When modern analytical techniques were not yet available,
potassium permanganate was used for the oxidative fission of
carotenoids to elucidate their constitution. Large degradation
fragments identified as apo-carotenals were obtained (17). In

7320 J. Agric. Food Chem., Vol. 51, No. 25, 2003
Caris-Veyrat et al.
Table 2. Maximum Wavelengths of Absorption of Apo-carotendials Obtained from the Oxidation of Lycopene by Potassium Permanganate
product
no.
λ
max (nm)
λ
max (nm)
no. of carbon−carbon
conjugated double bonds
product name
apo-8,12′-carotendial
apo-6,12′-carotendial or apo-10,8′-carotendial
apo-8,8′-carotendial ) crocetinedial
apo-6,10′-carotendial
experimental
literature
12
13
6b
7b
14
396, 414
422, 440
442, 466
444, 464
5
6
7
7
8
410, 433, 459 (EtOH) (23)
apo-8,6′-carotendial
464, 485 (shoulder)
453, 484, 517 (carbon disulfide)
452, 480 (petroleum ether) (24)
15
apo-6,6′-carotendial
484, 505 (shoulder)
9
Figure 3. Structures of apo-carotendials detected by HPLC-DAD-MS
obtained from the oxidation of lycopene by potassium permanganate.
Figure 2. Structures of apo-lycopenals and apo-lycopenones detected
by HPLC-DAD-MS obtained from the oxidation of lycopene by potassium
permanganate.
Table 3. 1H NMR of Apo-8,6′-carotendial (14) in CDCl3 at 500 MHza
1973, Ben-Aziz et al. (18) mentioned that apo-6′- and apo-8′-
lycopenal were obtained by oxidation of lycopene with potas-
sium permanganate. Ukai et al. (19) described a protocol for
the reaction which gave apo-6′-lycopenal as the main product
after 44 h, whereas around 50% of the lycopene was left. We
optimized experimental conditions to completely oxidize lyco-
pene and to obtain mono oxidative cleavage compounds as
major products. A mixture of methylene chloride/toluene (60/
40, v/v) to solubilize lycopene and the use of cetyltrimethyl-
ammonium bromide as phase transfer catalyst were found to
be the best compromise. After the complete disappearance of
lycopene, we detected eight apo-lycopenals, three apo-lyco-
penones (Figure 2), and six apo-carotendials (Figure 3). The
former compounds result from the oxidative cleavage of one
carbon-carbon double bond of lycopene, thus giving a range
of products from the longest apo-6′-lycopenal (1) to the shortest
one detected, apo-5-lycopenone (11). Tentative structure as-
signments were possible with the detection of the molecular
weight and the maximum wavelength of absorption compared
to those found in the literature (15). Mono cleavage compounds
of lycopene have been tentatively identified by Kim et al. (15)
using HPLC analysis with UV-vis detection. Our results were
δ in ppm
(multiplicity, coupling constant in Hz)
proton
H−C(6′)
H−C(7′)
H−C(8′)
9.58 (d, J ) 7.8)
6.19 (dd, J1 ) 15.2, J2 ) 7.8)
7.16 (d, J ) 15.2)
1.97 (s)
CH −C(9′)
3
H−C(10′) H−C(11′) H−C(12′)
CH −C(13′)
6.67 (m)
2.00 (s)
3
H−C(14′)
6.38 (d, J ) 11.0)
H−C(15′) H−C(15)
H−C(14)
6.67 (m)
6.44 (d, J ) 10.8)
2.00 (s)
CH −C(13)
3
H−C(11) H−C(12)
H−C(10)
6.67 (m)
6.93 (d, J ) 9.5)
CH −C(9)
1.89 (s)
9.45 (s)
3
H−C(8)
a s ) singlet, d ) doublet, dd ) doublet of doublet, m ) multiplet.
comparable to what they obtained for the series of compounds
containing 11 (apo-6′-lycopenal (1)) to 3 conjugated double
bonds (3,7,11-trimethyl-2,4,6,10-dodecatetraen ) apo-11-lyco-
penal (8)). We tentatively identified three more compounds
containing lower numbers of conjugated double bonds: 6,10-

Oxidation Products of Lycopene
J. Agric. Food Chem., Vol. 51, No. 25, 2003 7321
can be obtained only when products are characterized by NMR.
We thus confirmed the assignment of apo-11-lycopenal (8) by
1H NMR (Table 1). We did not purify the other mono-cleavage
compounds, as NMR data have been published for apo-6′-
lycopenal (1) (19), apo-8′-lycopenal (2) (20, 21), apo-10′-
lycopenal (3) (20), apo-12′-lycopenal (4), and apo-15-lycopenal
(6a) (22).
In the case of apo-carotendials issued from the cleavage of
two carbon-carbon double bonds, only the double bonds 5-6
(or 5′-6′), 7-8 (or 7′-8′), 9-10 (or 9′-10′), and 11-12 (or
11′-12′) were affected by the cleavage. We did not detect apo-
carotendials/ones resulting from cleavages of double bonds
closer to the center of the molecule (15-15′ carbon-carbon
double bond). No cleavage was detected on the double bond
which is the farthest from the center of the molecule, i.e., the
1-2 double bond, which is not conjugated and is trisubstituted;
this could explain its non reactivity to the oxidation with
potassium permanganate. Four of the six apo-carotendials
tentatively identified, apo-6,10′-carotendial (7b), apo-8,12′-
carotendial (12), apo-6,12′- or -10,8′-carotendial (13), and apo-
6,6′-carotenendial (15) (Figure 3) were not mentioned in the
literature. The structure of the apo-carotendials were assigned
by detecting their molecular weight and their maximum absorp-
tion in UV-vis spectra, which were consistent either with
their number of conjugated double bonds or with data found
in the literature when available (Table 3) (23, 24). These data
were available only for the apo-8,8′-carotenedial (also called
crocetinedial) (6b) and for apo-8,6′-carotendial (14). The
differences in value of the maximum wavelength can be
accounted for by the different solvents used. It is interesting to
Figure 4. UV−vis spectra of apo-carotendials from the oxidation of
lycopene by potassium permanganate. The number indicates the peak
number given in Figure 3.
dimethyl-3,5,9-undecatriene-2-one (apo-9-lycopenone, 9); 3,7-
dimethyl-2,6-octaene-1-al (apo-7-lycopenal, 10); and 2-methyl-
2-heptene-6-one (apo-5-lycopenone, 11). Compounds 10 and
11 were detected by their mass, but their UV-vis spectra was
not possible to find, probably because these compounds are
present in very small amounts and have a low molecular
extinction coefficient; as a consequence, they are not visible in
Figure 1. However, confirmation of the structure assignment
Figure 5. HPLC chromatograms obtained by UV−vis detection of the compounds issued from the oxidation of (E)-lycopene by oxygen, catalyzed by
Ru(CO)TPP in the presence of 3-chloroperoxybenzoic acid after (A) 1 h, (B) 24 h, and (C) 96 h.

7322 J. Agric. Food Chem., Vol. 51, No. 25, 2003
Caris-Veyrat et al.
Figure 6. HPLC chromatograms obtained by mass detection of the apo-lycopenals at (a) (m/z) + 1 ) 443, 1; (b) (m/z) + 1 ) 417, 2; (c) (m/z) + 1 )
377, 3; (d) (m/z) + 1 ) 351, 4; (e) (m/z) + 1 ) 311, 5; (f) (m/z) + 1 ) 285, 6a; (g) (m/z) + 1 ) 259, 7a; (h) (m/z) + 1 ) 219, 8; (i) (m/z) + 1 ) 193;
9. Number of products correspond to compounds listed in Figure 2.
Figure 7. Evolution with time of the mass peak area of (A) lycopene monoxides + and apo-6′-lycopenal ×; (B) apo-13-lycopenone 0, apo-11-lyco-
penone 9, and apo-9-lycopenal ]; (C) apo-15-lycopenal b and apo-14′-lycopenal O; and (D) apo-12′-lycopenal 4, apo10′-lycopenal 2, and apo-8′-
lycopenal /.

Oxidation Products of Lycopene
J. Agric. Food Chem., Vol. 51, No. 25, 2003 7323
Figure 8. Possible mechanism of formation of apo-lycopenals and apo-lycopenones by oxidation of (E)-lycopene with oxygen from air catalyzed by
Ru(CO)TPP.
note that the UV-vis spectra of apo-lycopenals were bell-
shaped, whereas for the apo-carotendials, a fine structure present
in the UV-vis spectra of lycopene was partly observed (Figure
4). Thus, two maximums of absorption were measured for apo-
carotendials (Table 2). Only the UV-vis data were published
for apo-8,6′-carotendial (14) (24), we purified and characterized
lycopene monoxides, and cleavage compounds assigned to apo-
lycopenals. No products resulting from a double oxidative
cleavage (apo-carotendial) were detected over the 96 h period.
Almost all the apo-lycopenals/ones that were obtained by
oxidation of lycopene with potassium permanganate were
detected, except the short-chain apo-5-lycopenone (11) and apo-
7-lycopenal (10). These compounds are volatile, and even if
they had been formed, they could have evaporated during the
time of the experiment. The analysis of the reaction mixture at
different times showed very complex chromatograms that
allowed the degradation compounds to be distinguished mainly
by their molecular weight, but for most of the compounds it
was not possible to obtain their UV-vis spectra. In Figure 6,
the detection of apo-lycopenals/ones by their molecular weight
is shown. Their retention time is comparable to the same
compounds obtained with oxidation by potassium permanganate
(Figure 1).
(Z)-isomers of lycopene were detected at 1 h and had almost
disappeared after 24 h (Figure 5). The tentative attribution of
the (Z)-isomers was made by detecting their mass, identical to
that of (E)-lycopene, but also with their UV-vis spectrum. The
(Z)-isomers detected are most likely mono-(Z)-isomers due to
the hypsochromic shift of only 4-8 nm. For the isomer with a
retention time close to 52 min ((Z)-1) (Figure 5), the absorbance
in the UV region is low, indicating that the compound is
decentrally isomerized but not completely otherwise the “cis-
peak” would not be visible, it could correspond to the 9-(Z)-
isomer. Peaks eluting at 48 ((Z)-2) and 46.5 min ((Z)-3)
present both a “cis-peak” observed at 360 nm which have a
high relative intensity (%AB/AII as defined in (23), 59 and 71%,
respectively). These results suggest that the isomerized double
bond is in both cases close to the center of the molecule, it is
probably the 13-(Z)- and the 15-(Z)-isomers, respectively.
Moreover, the order of the retention times and the values of
%AB/AII found for the (Z)-isomers we detected are comparable
to the results found by Yeum et al. (25), who used comparable
HPLC conditions. Isolation and NMR studies would be neces-
sary to confirm these tentative attributions. We have already
1
it for the first time by H NMR (Table 3). For one of the
products detected and attributed to an apo-carotendial (13), the
molecular weight and UV-vis spectra were not sufficient to
1
distinguish between two structural possibilities (Figure 3). H
NMR would allow determination of the precise chemical
structure of the compound; however, we did not succeed so far
in purifying the compound.
Oxidation of Lycopene with a Metalloporphyrin. We
studied the catalytic oxidation activity of a metalloporphyrin
on lycopene. A similar system was tested before on â-carotene
(25). These models mimic the active center of cytochrome P450
enzymes, which are involved in the in vivo transformation of
xenobiotics. These enzymes are present in the liver, the organ
in which carotenoids can be stored and eventually metabolized,
thus it was of interest to examine the potential activity of such
a model system on a carotenoid of nutritional importance like
lycopene.
The oxidative catalytic species trans-dioxoruthenium(VI)-
tetraphenylporphyrin Ru(O)2(TPP) is initially formed from
carbonylruthenium(II)-tetraphenylporphyrin Ru(CO)(TPP) by
oxidation with 3-chloroperoxybenzoic acid. After transferring
oxygen to the substrate, Ru(O)2(TPP) is regenerated by atmo-
spheric oxygen (26). The reaction of this catalytic system on
lycopene was followed by HPLC-DAD-MS analysis of the
extracted reaction mixture over a period of 96 h. Lycopene
disappeared after 24 h (Figure 5), but the composition of the
reaction mixture underwent evolution even after the complete
disappearance of the starting material, indicating that the
catalytic system was efficient not only on lycopene, but also
on its degradation products initially formed. Three types of
products were detected: (Z)-isomers, compounds with a mo-
lecular weight of 553 ) Mlycopene + 16 + 1, which we called

7324 J. Agric. Food Chem., Vol. 51, No. 25, 2003
Caris-Veyrat et al.
observed (Z)-isomerization of â-carotene in such an oxidative
system (26). It is known that a (Z)-olefin is at least 10 times
more reactive than the (E)-isomer in a competitive oxidation
by a metalloporphyrin catalytic system similar to the one we
used (27). However, the mechanism by which isomerization
occurs is not known. It could be either metal- (28) or acid-
catalyzed (29).
Further studies are underway to detect oxidative cleavage
compounds of lycopene formed by the action of extracts of cells
containing cytochrome P450 enzymes and in vivo.
LITERATURE CITED
(1) Olson, J. A. Carotenoids and human health. Arch. Latin. Nutr.
1999, 49, 7S-11S.
(2) Krinsky, N. I. Carotenoids as antioxidants. Nutrition 2001, 17,
Compounds having a molecular weight of 553 ) Mlycopene
+
16 + 1 were detected in a positive electrospray mode, suggesting
the presence of epoxide groups on the chain of lycopene, but
no other information on the structure could be obtained, because
UV-vis spectra were not possible to obtain for these peaks. In
a previous study with a similar such catalytic system, we had
been able to detect â-carotene epoxides (26).
815-817.
(3) Hughes, D. A. Effects of carotenoids on human immune function.
Proc. Nutr. Soc. 1999, 58, 713-718.
(4) Stahl, W.; Ale-Agha, N.; Polidori, M. C. Nonantioxidant
properties of carotenoids. Biol. Chem. 2002, 383, 553-558.
(5) King, T. J.; Khachik, F.; Bortkewicz, H.; Fukushima, S.;
Morioka, S.; Bertram, J. S. Metabolites of dietary carotenoids
as potential cancer preventive agents. Pure Appl. Chem. 1997,
69, 2135-2140.
(6) Khachik, F.; Bertram, J.; Huang, M.-T.; Fahey, J. W.; Talalay,
P. Dietary carotenoids and their metabolites as potentially useful
chemoprotective agents against cancer. In Antioxidant Food
Supplements in Human Health; Academic Press: London, 1999;
pp 203-229.
(7) Bertram, J. S.; King, T.; Fukishima, L.; Khachik, F.; Sen, C.
K.; Sies, H.; Baeuerle, P. A. Enhanced activity of an
oxidation product of lycopene found in tomato products and
human serum relevant to cancer prevention. In Antioxidant Redox
Regulatory Genes; Academic Press: London, 2000; pp 409-
424.
(8) Agarwal, S.; Rao, A. V. Tomato lycopene and its role in human
health and chronic diseases. Can. Med. Assoc. J. 2000, 163, 739-
744.
(9) Khachik, F.; Carvalho, L.; Bernstein, P. S.; Muir, G. J.; Zhao,
D. Y.; Katz, N. B. Chemistry, distribution, and metabolism of
tomato carotenoids and their impact on human health. Exp. Biol.
Med. 2002, 227, 845-851.
(10) Rao, A. V.; Agarwal, S. Role of lycopene as antioxidant
carotenoid in the prevention of chronic diseases: A review. Nut.
Res. 1999, 19, 305-323.
(11) Rao, A. V. R.; Agarwal, S. Role of antioxidant lycopene in cancer
and heart disease. J. Am. Coll. Nutr. 2000, 19, 563-569.
(12) Hadley, C. W.; Miller, E. C.; Schwartz, S. J.; Clinton, S. K.
Tomatoes, lycopene, and prostate cancer: Progress and promise.
Exp. Biol. Med. 2002, 227, 869-880.
(13) Di Mascio, P.; Kaiser, S.; Sies, H. Lycopene as the most efficient
biological carotenoid singlet oxygen quencher. Arch. Biochem.
Biophys. 1989, 274, 532-538.
(14) Khachik, F.; Spangler, C. J.; Smith, J. C.; Canfield, L.;
Steck, A.; Pfander, H. Identification, quantification, and relative
concentrations of carotenoids and their serum metabolites in
human milk and serum. Anal. Chem. 1997, 69, 1873-1881.
(15) Kim, S. J.; Nara, E.; Kobayashi, H.; Terao, J.; Nagao, A.
Formation of cleavage products by autoxidation of lycopene.
Lipids 2001, 36, 191-199, and references therein.
(16) Jorgensen, K.; Skibsted, L. H. Carotenoid scavenging of radicals,
effect of carotenoid structure, and oxygen partial pressure on
antioxidative activity. Z. Lebensm. Unters. Forsch. 1993, 196,
423-429.
(17) Karrer, P.; Jucker, E. Carotenoids; Elsevier: Paris, 1950.
(18) Ben-Aziz, A.; Britton, G.; Goodwin, T. W. Carotene epoxides
of Lycopersicon esculentum. Phytochemistry 1973, 12, 2759-
2764.
(19) Ukai, N.; Etoh, H.; Yagi, H.; Ina, K.; Oshima, S.; Ojima, F.;
Sakamoto, H.; Ishiguro, Y. Photosensitized oxygenation of
lycopene. Biosci. Biotech. Biochem. 1994, 58, 1718-1719.
(20) Maerki-Fischer, E.; Uebelhart, P.; Eugster, C. H. 10′-Apolyco-
pin-10′-ol und 10′-apolycopin-10′-saure aus Blueten der
Rosenhybride ‘Marechal Niel’ HelV. Chim. Acta 1987, 70, 1994-
2002.
The evolution in time of these compounds was followed in
a relative quantitative manner by using the total ion chromato-
grams (TIC) obtained by mass detection. As for UV-vis
detection, we assumed that the area of the peaks of a mass
chromatogram obtained at a precise mass (see for example
Figure 6) is proportional to the quantity of the compound. Thus,
by following the evolution in time of the peak areas at a precise
molecular weight, we obtained information on the evolution of
the relative quantity of the compound considered. When several
peaks with close retention time were obtained at the same
molecular weight, indicating the presence of (Z)-isomers, we
added the areas of all peaks. Thus, for the lycopene monoxides,
their global evolution in time is shown in Figure 7A. Their
amount was at the maximum after 1 h of reaction and then
decreased regularly until 96 h. For the longest-chain apo-
lycopenal (i.e., apo-6′-lycopenal (1)), its amount increased until
5 h and then decreased continuously until complete disappear-
ance at 96 h (Figure 7A). For the apo-13-lycopenone (7a), apo-
11-lycopenal (8) and apo-9-lycopenone (9), their amount
increased quickly during the first 5 h (Figure 7B) and then
more slowly but continuously until 96 h. For apo-15-lycopenal
(6a) and apo-14′-lycopenal (5), their evolution is comparable
to the short-chain apo-lycopenals until 48 h, but then their
amount decreased until 96 h (Figure 7C). For the longer-chain
apo-12′-lycopenal (4), apo-10′-lycopenal (3), and apo-8′-lyco-
penal (2), their amount increased until 5 h, but then their
evolution was not regular between 5 and 48 h (Figure 7D).
It was stable and increased for the apo-12′-lycopenal (4),
decreased and increased for the apo-10′-lycopenal (3) and
apo-8′-lycopenal (2), but after 48 h, for all of them, their
amount drastically decreased until 96 h to almost complete
disappearance in the case of (2) and (3). To summarize the
evolution of apo-lycopenals, we observed that the amount of
the shortest ones (apo-9-lycopenone (9), apo-13-lycopenone
(7a), and apo-11-lycopenal (8)) increased continuously until
96 h (Figure 7B), whereas the concentration of the longer ones
(apo-6′-lycopenal (1), apo-8′-lycopenal (2), apo-10′-lyco-
penal (3), apo-12′- lycopenal (4), apo-14′- lycopenal (5), and
apo-15-lycopenal (6a)) decreased after some time (5 or 48 h)
(Figure 7, parts A, C, and D). The evolution of different
products resulting from lycopene suggests a possible mechanism
of formation of these compounds (Figure 8). (Z)-Isomers are
first formed and then oxidized into epoxides forming the
lycopene monoxides. The epoxide groups undergo an oxidative
cleavage, giving rise to apo-lycopenals. Apo-lycopenals of all
sizes are formed simultaneously from the beginning of the
reaction, then the longer-chain apo-lycopenals (apo-15- to apo-
6′-lycopenal, 6a to 1) are oxidatively cleaved and thus trans-
formed into shorter apo-lycopenals/ones (apo-9- to apo-13-
lycopenal/ones, 9 to 7a).

Oxidation Products of Lycopene
J. Agric. Food Chem., Vol. 51, No. 25, 2003 7325
(21) Kjoesen, H.; Liaaen-Jensen, S. Major carotenoids of Shepherdia
canadensis. Isolation and synthesis of methyl apo-6′-lyco-
penaoate. Phytochemistry 1969, 8, 483-491.
(22) Tsujimo, K.; Aoki, M.; Ohashi, M. Stereoselective photoisom-
erisation of ψ-retinal. J. Photochem. Photobiol. A 1992, 65, 73-
78.
(23) Carotenoids Volume 1B: Spectroscopy; Britton, G., Liaaen-
Jensen, S., Pfander, H., Eds.; 1995.
(24) Karrer, P.; Jaffe´, W. Stufenweiser Abbau des Lycopins. HelV.
Chim. Acta 1939, 22, 69-71.
(25) Yeum, K.-J.; Booth, S. L.; Sadowski, J. A.; Liu, C.; Tang, G.;
Krinsky, N. I.; Russel, R. Human plasma carotenoid response
to the ingestion of controlled diets high in fruits and vegetables.
Am. J. Clin. Nutr. 1996, 64, 594-602
(26) Caris-Veyrat, C.; Amiot, M. J.; Ramasseul, R.; Marchon, J. C.
Mild oxidative cleavage of beta,beta-carotene by dioxygen
induced by a ruthenium porphyrin catalyst: Characterization of
products and of some possible intermediates. New J. Chem. 2001,
25, 203-206.
(27) Groves, J. T.; Quinn, R. Aerobic epoxidation of olefins with
ruthenium porphyrin catalysts. J. Am. Chem. Soc. 1985, 107,
5790-5792.
(28) McGrath, D. V.; Grubbs, R. H. The mechanism of aqueous
ruthenium(II)-catalyzed olefin isomerization. Organometallics
1994, 13, 224-235.
(29) Konovalov, V. V.; Kispert, L. D. AM1, INDO/S and optical
studies of carbocations of carotenoid molecules. Acid-induced
isomerization. J. Chem. Soc., Perkin Trans. 2 1999, 901-909.
Received for review July 7, 2003. Revised manuscript received
September 26, 2003. Accepted October 1, 2003.
JF034735+