Formation mechanism of p-methylacetophenone from citral via a tert-alkoxy radical intermediate

Article abstract of DOI:10.1021/jf035517j

The aim of this study was to clarify the formation mechanism of a potent off-odorant, p-methylacetophenone, from citral under acidic aqueous conditions. An acidic aqueous solution (pH 3.0) containing 10 mg/L of citral was stored at 40 °C for 2 weeks. Among the compounds detected in the stored citral solution, 4-(2-hydroxy-2-propyl)benzaldehyde was identified for the first time as a degradation product from citral. The formation of p-methylacetophenone and 4-(2-hydroxy-2-propyl)-benzaldehyde behaved the same when antioxidants were added to the citral solution. In addition, both compounds were formed by the Fe²⁺-induced decomposition of 8-hydroperoxy-p-cymene, another compound identified in the stored citral solution. These results suggested that both p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde can be formed via the same radical intermediate [p-CH₃C₆H ₄C(CH₃)₂O^.] that can be derived from the O-O bond homolysis of 8-hydroperoxy-p-cymene. On the other hand, the degradation of 8-hydroperoxy-p-cymene without Fe²⁺ under acidic aqueous conditions did not yield p-methylacetophenone and 4-(2-hydroxy-2-propyl) benzaldehyde, but the degradation of citral without Fe²⁺ did. Therefore, other than the decomposition of 8-hydroperoxy-p-cymene, a mechanism to generate the tert-alkoxy radical intermediate was proposed for the formation of p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde in the citral solution.

Full text of DOI:10.1021/jf035517j

J. Agric. Food Chem. 2004, 52, 5677−5684 5677
Formation Mechanism of p-Methylacetophenone from Citral via
a tert-Alkoxy Radical Intermediate
‡
TOSHIO UENO,*,† HIDEKI MASUDA,† AND CHI-TANG HO
Material Research and Development Laboratories, Ogawa & Company, Ltd., 15-7 Chidori,
Urayasushi, Chiba 279-0032, Japan, and Department of Food Science, Rutgers University,
65 Dudley Road, New Brunswick, New Jersey 08901-8520
The aim of this study was to clarify the formation mechanism of a potent off-odorant, p-
methylacetophenone, from citral under acidic aqueous conditions. An acidic aqueous solution (pH
3.0) containing 10 mg/L of citral was stored at 40 °C for 2 weeks. Among the compounds detected
in the stored citral solution, 4-(2-hydroxy-2-propyl)benzaldehyde was identified for the first time as a
degradation product from citral. The formation of p-methylacetophenone and 4-(2-hydroxy-2-propyl)-
benzaldehyde behaved the same when antioxidants were added to the citral solution. In addition,
both compounds were formed by the Fe2+-induced decomposition of 8-hydroperoxy-p-cymene, another
compound identified in the stored citral solution. These results suggested that both p-methylac-
etophenone and 4-(2-hydroxy-2-propyl)benzaldehyde can be formed via the same radical intermediate
[p-CH3C6H4C(CH3)2O•] that can be derived from the O-O bond homolysis of 8-hydroperoxy-p-cymene.
On the other hand, the degradation of 8-hydroperoxy-p-cymene without Fe2+ under acidic aqueous
conditions did not yield p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde, but the
degradation of citral without Fe2+ did. Therefore, other than the decomposition of 8-hydroperoxy-p-
cymene, a mechanism to generate the tert-alkoxy radical intermediate was proposed for the formation
of p-methylacetophenone and 4-(2-hydroxy-2-propyl)benzaldehyde in the citral solution.
KEYWORDS: Citral; degradation; off-odors; p-methylacetophenone; formation mechanism; tert-alkoxy
radical; 4-(2-hydroxy-2-propyl)benzaldehyde
INTRODUCTION
p-Cymen-8-ol and its dehydration product, R,p-dimethylstyrene,
have been proposed to be formed from p-menthadien-8-ols by
their oxidation with dissolved oxygen (5) or disproportionation
reactions (4) producing R-terpineol as the reduction product.
p-Methylacetophenone was then suggested to be formed by the
oxidation of R,p-dimethylstyrene (6). In our previous studies,
on the other hand, 8-hydroperoxy-p-cymene was detected in
the stored acidic solution of citral, and it was demonstrated that
this hydroperoxide decomposes to p-cresol, p-cymen-8-ol, and
R,p-dimethylstyrene, and not to p-methylacetophenone (9) under
the same acidic conditions as the citral solution. The formation
mechanism of p-methylacetophenone from citral under our
experimental conditions thus remains a question.
The stability of citral (a mixture of neral and geranial) under
acidic aqueous conditions is a critical issue in the field of flavor
chemistry. Under such conditions, citral easily degrades by a
series of cyclization and oxidation reactions to form a variety
of degradation products (1-8). Consequently, not only is the
fresh lemon-like odor of citral lost, but undesirable off-odors
develop. Among the degradation products from citral, p-cresol
and p-methylacetophenone were reported to be the most potent
off-odorants (6, 7). The contribution of these two compounds
to the off-odors formed in the acidic solution of citral was also
confirmed in our experiments (9).
Despite the importance as off-odorants derived from citral,
the formation mechanisms of p-cresol and p-methylacetophe-
none have not yet been fully clarified. Scheme 1 shows the
previously proposed formation pathways of the oxidation
products including p-cresol and p-methylacetophenone from
citral under acidic aqueous conditions. It has been established
that citral in acidic solutions undergoes cyclization reactions
leading to the formation of p-menthadien-8-ols (1-5, 8).
Therefore, the aim of this study was to clarify the formation
mechanism of p-methylacetophenone from citral under acidic
aqueous conditions.
MATERIALS AND METHODS
Chemicals. Citral was purchased from Polarome International (Jersey
City, NJ). p-Cymen-8-ol and R,p-dimethylstyrene were purchased from
Sigma-Aldrich Japan (Tokyo, Japan). (-)-Epicatechin (EC), (-)-
epicatechin gallate (ECg), (-)-epigallocatechin (EGC), (-)-epigallo-
catechin gallate (EGCg), and (+)-catechin were purchased from
Funakoshi (Tokyo, Japan). p-Cymene and quercitrin were purchased
from Wako Pure Chemical Industries (Osaka, Japan). p-Cresol, p-
* Author to whom correspondence should be addressed (fax +81-47-
305-1423; e-mail ueno.toshio@ogawa.net).
† Ogawa & Co., Ltd.
‡ Rutgers University.
10.1021/jf035517j CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/05/2004

5678 J. Agric. Food Chem., Vol. 52, No. 18, 2004
Ueno et al.
Scheme 1. Previously Proposed Formation Pathways of p-Cresol and
p-Methylacetophenone and Other Oxidation Products from Citral under
Acidic Aqueous Conditions (4−6, 9)
Figure 1. Gas chromatographic analysis of the acidic buffer solution (pH
3.0) of citral (10 mg/L) (a) before and (b) after storage at 40 °C for 2
weeks. Numbers correspond to those in Table 1.
in dichloromethane was added to the extract as the internal standard.
The extract was dried over sodium sulfate, concentrated in vacuo to
∼5 mL, and further concentrated under a stream of nitrogen to ∼200
µL.
Gas Chromatography (GC). An Agilent 6890 N gas chromatograph
equipped with a flame ionization detector (FID) and a DB-1 fused silica
capillary column (60 m × 0.25 mm i.d.; film thickness of 0.25 µm;
J&W Scientific) was used. To minimize the decomposition of 8-hy-
droperoxy-p-cymene, an injector temperature of 100 °C was used. The
other operating conditions were as follows: detector temperature, 250
°C; nitrogen carrier gas flow rate, 1 mL/min; oven temperature program,
80 °C, raised at 3 °C/min to 210 °C (15 min); 1 µL of sample was
injected using a split ratio of 1:50. The amounts of the citral degradation
products were estimated by computing the areas versus that of the
internal standard (n-pentadecane). The response factors of all com-
pounds to the FID were assumed to be the same.
Gas Chromatography-Mass Spectrometry (GC-MS). A Hewlett-
Packard 5890 series II gas chromatograph equipped with an HP-5972
mass selective detector and a DB-1 fused silica capillary column (60
m × 0.25 mm i.d.; film thickness of 0.25 µm; J&W Scientific) was
used. The operating conditions were as follows: injector temperature,
250 °C; helium carrier gas flow rate, 1 mL/min; oven temperature
program, 60 °C, raised at 3 °C/min to 210 °C (40 min); 1 µL of sample
was injected, using a split ratio of 1:50; ionization voltage, 70 eV; ion
source temperature, 140 °C.
methylacetophenone, and ascorbic acid were purchased from Nacalai
Tesque (Kyoto, Japan).
Synthesis. 4-(2-Hydroxy-2-propyl)benzaldehyde was synthesized
from p-bromobenzaldehyde dimethylacetal (Sigma-Aldrich Japan)
according to the method described by Creary and Wang (10). A yield
of 13 mg (31%) of 4-(2-hydroxy-2-propyl)benzaldehyde was ob-
tained: 1H MMR (CDCl3) δ 10.00 (s, 1 H), 7.76 (AA′BB′ aromatic
quartet, 4H), 2.16 (br s, 1H), 1.61 (s, 6H) [agreed with the literature
(10)]; MS (EI), m/z (relative intensity) 164 (2), 150 (10), 149 (100),
145 (5), 133 (3), 121 (4), 115 (5), 107 (6), 105 (5), 91 (7), 77 (14), 74
(4), 65 (3), 59 (6), 51 (10), 43 (94).
8-Hydroperoxy-p-cymene was synthesized as follows: A solution
of 150 mg of p-cymen-8-ol in 5 mL of ethanol was added dropwise to
the stirred mixture of 150 mL of 30% hydrogen peroxide and 15 mL
of 2.5% (w/v) sulfuric acid. After stirring for 1 h at room temperature,
the reaction mixture was extracted with 250 mL of dichloromethane,
washed with water (250 mL × 2), dried over sodium sulfate, and
concentrated in vacuo to ∼1 mL. The crude product was purified by
thin-layer chromatography on silica gel plates using a mixture of ethyl
acetate and hexane (1:5, v/v) as the eluent to give 8-hydroperoxy-p-
Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H NMR
spectra were recorded at 400 MHz on a Bruker Avance 400 spectrom-
eter (Bruker, Tsukuba, Japan) in CDCl3 with TMS as internal standard
() 0 ppm).
RESULTS AND DISCUSSION
1
cymene (38 mg, yield 23%). The MS (EI) and H NMR spectra were
Degradation of Citral under Acidic Aqueous Conditions.
During storage under acidic aqueous conditions, citral (neral
and geranial) was completely degraded and almost totally
converted to its cyclization products 1-17 (Figure 1 and Table
1), among which 4-(2-hydroxy-2-propyl)benzaldehyde (17) was
identified for the first time as a degradation product from citral.
The structural estimation based on the MS spectra and subse-
quent synthesis according to the published method (10) led to
the identification of 17. The synthetic sample exhibited an odor
character similar to that of benzaldehyde. Because of the weak
odor, compound 17 was assumed not to contribute to the off-
odors of the stored citral solution.
consistent with the literature values (11, 12).
Model Reactions. A sample solution containing 10 mg/L of citral
in an acidic buffer (0.1 M citric acid/0.2 M sodium hydrogen phosphate,
pH 3.0) with or without 60 mg/L of an added antioxidant (Table 2)
was prepared. One hundred milliliters of the prepared solution was
transferred to a 100-mL glass bottle, and the sample bottle was sealed
with a Teflon liner and a screw cap. The sample was stored in a dark
incubator at 40 °C for 2 weeks. For studies of the metal-induced
decomposition of 8-hydroperoxy-p-cymene, 1 mL of 5 × 10-2
M
ferrous sulfate heptahydrate in water was added to 100 mL of the sample
solution containing 2 mg/L of 8-hydroperoxy-p-cymene in the acidic
buffer. The color of the solution immediately turned yellow. The sample
bottle was shaken and then left at room temperature for 30 min.
Preparation of Analytical Samples. The degradation products of
citral or those of 8-hydroperoxy-p-cymene were extracted with dichlo-
romethane (30 mL × 2). One milliliter of 0.01% (w/v) n-pentadecane
It has been accepted that compounds 1, 2, 5, 8, 12, 13, 15,
and 16 are formed from citral by a series of acid-catalyzed
reactions (1-5, 8). In agreement with most published data,

Off-Odorant Formation from Citral
J. Agric. Food Chem., Vol. 52, No. 18, 2004 5679
in the citral solution. Unlike the studies conducted at room
temperature (1, 2, 5), a dehydration product, p-cymene (2), was
also one of the major products in our experiments, probably
due to the relatively high temperature (40 °C) of our experi-
mental conditions.
Table 1. Degradation Products Detected in the Acidic Buffer Solution
(pH 3.0) of Citral (10 mg/L) Stored at 40 °C for 2 Weeks
c
ID
method
b
no.a
compound
RI
1
2
3
4
5
6
7
8
9
10
2,3-dehydro-1,8-cineole
p-cymene
p-cresol
985
1017
1044
1077
1150
1158
1163
1174
1177
1249
B
A
A
A
B
A
A
B
A
C
Oxidation reactions as well as the acid-catalyzed reactions
would be involved in the formation of compounds 3, 4, 6, 7,
9-11, 14, and 17. Compounds 10 and 11 were reported to be
formed via the direct epoxidation of the 6,7-double bond of
citral (13, 14). Kimura et al., on the other hand, demonstrated
the formation of R,p-dimethylstyrene (4), p-cymen-8-ol (7), and
R-terpineol (9) using isolated p-menthadien-8-ols 5 and 8,
respectively, under acidic aqueous conditions (4). It was unclear,
however, whether the other oxidation products, that is, p-cresol
(3), p-methylacetophenone (6), 8-hydroperoxy-p-cymene (14),
and the newly found 17, would be formed via the major acid-
catalyzed degradation products of citral, such as 5 and 8, or
would be directly formed from citral.
R,p-dimethylstyrene
p-mentha-1,5-dien-8-ol
p-methylacetophenone
p-cymen-8-ol
p-mentha-1(7),2-dien-8-ol
R-terpineol
(2R*,5R*)-2-formylmethyl-2-methyl-
5-(1-hydroxy-1-methylethyl)tetrahydro-
furan
11
(2S*,5R*)-2-formylmethyl-2-methyl-
5-(1-hydroxy-1-methylethyl)tetrahydro-
furan
1254
C
12
13
14
15
16
17
trans-p-menth-2-ene-1,8-diol
cis-p-menth-2-ene-1,8-diol
8-hydroperoxy-p-cymene
trans-p-menth-1-ene-3,8-diol
cis-p-menth-1-ene-3,8-diol
4-(2-hydroxy-2-propyl)benzaldehyde
1259
1276
1321
1330
1351
1365
B
B
A
B
B
A
Figure 2 shows the concentration changes of citral (neral
and geranial, Figure 2a), its major acid-catalyzed degradation
products (Figure 2b), and the oxidation products (Figure 2c,d)
in the citral solution during storage. Neral and geranial almost
totally degraded during the first 3 days of storage (Figure 2a).
The concentration of acid-catalyzed degradation products 5, 8,
12, and 13 rapidly increased during this period (Figure 2b) and
then started decreasing with almost the entire loss of citral,
whereas the concentration of their dehydration product 2 kept
increasing during the storage. On the other hand, the concentra-
tion of the oxidation products 3, 6, 7, and 14 (Figure 2c) and
4 and 17 (Figure 2d) also kept increasing after almost the entire
loss of citral, although compound 14 started to decrease after 7
a Numbers correspond to those in Figure 1. b Retention index on DB-1 (60 m).
c Identification methods: A, mass spectrum and retention index agree with those
of authentic compounds; B and C, compounds were tentatively identified on the
basis of the following criteria: (B) mass spectrum agrees with that of the Wiley
mass spectral database (Agilent Technologies, 2000) and RI agrees with literature
value (5) or (C) mass spectrum agrees with literature spectrum (13).
p-menthadien-8-ols (5 and 8) and p-menth-2-ene-1,8-diols (12
and 13) were among the major acid-catalyzed reaction products
Figure 2. Concentration changes of (a) neral (0) and geranial (2), of (b) acid-catalyzed degradation products 2 (2), 5 (9), 8 (b), 12 (O), and 13 (0),
of (c) oxidation products 3 (O), 6 (2), 7 (9), and 14 (0), and of (d) oxidation products 4 (4) and 17 (9) during the storage of citral (10 mg/L) in an
acidic buffer solution (pH 3.0) at 40 °C. Each point is the mean value of three experiments. Coefficient of variation for each point was <10%.

5680 J. Agric. Food Chem., Vol. 52, No. 18, 2004
Ueno et al.
Table 2. Effects of Antioxidant Addition on the Formation of the
Oxidation Products (3, 4, 6, 7, 14, and 17) from Citral under Acidic
Aqueous Conditions
concn of compounda (µg/L)
antioxidantb
3
4
6
7
14
677 171 29
23c 1589 134 trd
17
control
(+)-catechin
197
24 209
170c 68
(−)-epicatechin (EC)
(−)-epigallocatechin (EGC)
(−)-epicatechin gallate (ECg)
344
299
592
42
65
28
30
45
29
21
28c
1018 278
1402 263
805 618
835 565
3.2
tr
tr
(−)-epigallocatechin gallate (EGCg) 556
tr
quercitrin
ascorbic acid
514
132
22 106
23 137
624 625 11
654 152 19
a Concentration was determined after the storage of the citral solution at 40 °C
for 2 weeks. Each value is the mean of five experiments. Coefficient of variation
for each value was <10% unless otherwise specified. b Sixty milligrams per liter of
an antioxidant was added to an acidic buffer solution (0.1 M citric acid/0.2 M sodium
hydrogen phosphate, pH 3.0) containing 10 mg/L of citral. c Coefficients of variation
ranged from 10 to 20%. d Trace.
days of storage, probably due to its degradation to 3, 4, and 7
under the acidic aqueous conditions (9). These results indicate
that the oxidation products 3, 6, 14, and 17 as well as 4 and 7
would be formed not directly from citral but via the major acid-
catalyzed cyclization products from citral.
Antioxidant Additions and the Resulting Formation Cor-
relations among Oxidation Products from Citral. The use
of antioxidants to inhibit the formation of off-odorants from citral
in acidic solutions has already been attempted (4, 5, 9). It was
reported (5) that the addition of isoascorbic acid to an acidic
solution containing citral strongly inhibited the formation of R,p-
dimethylstyrene (4) and p-cymen-8-ol (7). We previously repor-
ted that catechins from green tea showed strong inhibitory effects
on the formation of p-methylacetophenone (6) from citral under
acidic aqueous conditions (9). In the present study, informa-
tion regarding such antioxidant additions was examined to inves-
tigate the formation pathways of oxidation products from citral.
Table 2 shows the effects of the added antioxidants (60 mg/
L) including the catechins, quercitrin, and ascorbic acid on the
formation of 3, 4, 6, 7, 14, and 17 from citral under acidic
aqueous conditions. Among these results, three pairs of oxidation
products from citral, that is, 3 and 14, 4 and 7, and 6 and 17,
showed linear relationships between their concentrations (Figure
3). This means that the formation of the two oxidation products
in each pair was influenced in the same manner by this series
of antioxidant additions, reflecting the formation pathways of
the oxidation products from citral. Therefore, it is not surprising
that good correlations were obtained between the concentrations
of 3 and 14 (correlation coefficient, R ) 0.975) and between
those of 4 and 7 (R ) 0.993). These can be explained by their
reported formation pathways, in which 3 is formed from 14
and 4 is formed from 7 by acid-catalyzed reactions (Scheme
1). It is more interesting to see a good correlation between the
concentrations of 6 and 17 (R ) 0.995, a trace amount of 17
was assumed to be zero), because this might suggest a possible
connection between the potent off-odorant 6 and newly found
product 17 during their formation from citral.
Figure 3. Linear relationships between the concentrations of (a)
compounds 3 and 14, (b) 4 and 7, and (c) 6 and 17 in the citral solution
stored with antioxidants (data from Table 2).
Table 3. Quantitative Data for the Fe2+-Induced Decomposition of
8-Hydroperoxy-p-cymene (14) under Acidic Aqueous Conditions
concn of compound (µg/L)
samplea
6
7
14
17
before addition of Fe2+
after addition of Fe2+
9
1378
32
273
2085
nd
ndb
278
a FeSO4 (5 × 10-4 M) was added to the acidic buffer solution (pH 3.0) of 14
(2 mg/L). b Not detected.
further decomposition with the elimination of a methyl radical,
that is, â-fragmentation (16), leading to the formation of
p-methylacetophenone (6). To confirm the formation of 6 and
other products resulting from the O-O bond homolysis of 14,
we conducted the Fe2+-induced decomposition of 14 under
acidic aqueous conditions. As shown in Figure 4, hydroperoxide
14 immediately degraded to 6 along with 7 and 17 by the
addition of a large excess of ferrous sulfate (FeSO4). The
quantitative data for this experiment are given in Table 3.
As shown in Scheme 2, all of the products 6, 7, and 17 were
interpreted to be formed via the tert-alkoxy radical 18 generated
by the reduction of 14 with Fe2+. p-Cymen-8-ol (7) could be
formed by the further reduction of the alkoxy radical 18 by
Fe2+-Induced Decomposition of 8-Hydroperoxy-p-cymene
(14). It is well-known that hydroperoxides can undergo ho-
molytic O-O bond cleavage by accepting one electron from
reducing metal ions such as ferrous ion (Fe2+) to produce alkoxy
radicals (15). In the case of the hydroperoxide 14, the homolytic
O-O bond cleavage would produce the corresponding tert-
alkoxy radical [p-CH3C6H4C(CH3)2O•], which can undergo

Off-Odorant Formation from Citral
J. Agric. Food Chem., Vol. 52, No. 18, 2004 5681
Table 4. Concentration of Degradation Products Obtained from the
Acidic Buffer Solutions (pH 3.0) of 8-Hydroperoxy-p-cymene (14) (2
mg/L), Citral (10 mg/L), and Citral (10 mg/L) with 14 (2 mg/L) Stored
at 40 °C for 2 Weeks
concn of compounda (µg/L)
sample
3
4
6
7
14
17
14
citral
citral with 14
849b
183
1017
25
17
224
317
685
678
1410
127
153
324
ndc
27
41
23d
48
a Concentration was determined after the storage of the sample solutions at 40
°C for 2 weeks. Each value is the mean of three experiments. Coefficient of variation
for each value was <10% unless otherwise specified. b Coefficient of variation was
13%. c Not detected. d Coefficient of variation was 23%.
aryl radical cation B could typically undergo deprotonation at
the benzylic position to form the benzylic radical 19 (18, 19).
Another possibility for the formation of 19 might be H
abstraction at the benzylic position of 7 with free radicals.
However, we did not detect p-formylacetophenone, an oxidation
product that could be obtained from H abstraction at the benzylic
position of 6. This observation suggested that H abstraction from
7 would also not occur. Therefore, we assumed that 19 would
be formed from 18 as mentioned above.
Figure 4. Gas chromatographic analysis of the acidic buffer solution (pH
3.0) of hydroperoxide 14 (2 mg/L) (a) before and (b) after the addition of
Fe2+ (5 × 10-4 M).
Scheme 2. Possible Mechanism for the Fe2+-Induced Decomposition
of Hydroperoxide 14 under Acidic Aqueous Conditions
Degradation of Hydroperoxide 14 under Acidic Aqueous
Conditions without Fe2+. In contrast to the Fe2+-induced
decomposition, there was little or no production of 6 and 17
during the storage of 14 under acidic aqueous conditions in the
absence of Fe2+ (9). This result indicated that 14 without Fe2+
rarely undergoes the O-O bond homolysis under our experi-
mental conditions. In this paper, we also report the degradation
of 14 in the acidic aqueous solution of citral. Table 4 shows
quantitative data for the degradation products from 14 (2 mg/
L), citral (10 mg/L), and citral (10 mg/L) with added 14 (2 mg/
L) stored under acidic aqueous conditions. With the addition
of 14 to the citral solution, the formation of 3 and 7 (and 4)
increased almost as much as expected from the data of the
degradation of 14 by itself. The formation of 6 and 17 in the
citral solution was also increased with the addition of 14, but
the increased amounts were much lower than those of 3 and 7.
These results suggested that the degradation of 14 would not
be, at least, the main pathway for the formation of 6 and 17 in
the citral solution.
Formation Mechanisms of p-Methylacetophenone (6) and
Compound 17 from Citral. On the basis of the presented data,
we propose the formation mechanisms of 6 and 17 in the citral
solution as shown in Scheme 3. Our results suggested that both
6 and 17 could be formed via the tert-alkoxy radical 18, yet
the decomposition of 14 would not be the main pathway for
the formation of 6 and 17 in the citral solution. Therefore, it
seems reasonable to assume that, other than the O-O bond
homolysis of 14, a mechanism to generate the radical 18 might
be involved in the formation of 6 and 17 in the citral solution.
In addition, the formation of 14 in the citral solution suggests
the possible formation of the corresponding peroxy radical 20.
Therefore, we assumed that the radical 18 might be generated
from the peroxy radical 20, not via hydroperoxide 14, in the
citral solution.
It is generally accepted that alkoxy radicals can be formed
directly from peroxy radicals, not via hydroperoxides (20-25).
In the case of the formation of 18 from 20, there seem to be
two possible pathways: one is the bimolecular self-coupling
of the peroxy radical 20 followed by the decomposition of the
resulting tetroxide (20-22) (path A), and the other is the addition
Fe2+ (15). Regarding the formation of 17 from 18, the transfor-
mation of 18 to a benzylic radical 19 was postulated to be invol-
ved. A possible mechanism for this transformation is as follows.
The alkoxy radical 18 could undergo 1,3-cyclization to form a
cyclohexadienyl radical A (17), which might convert via the
acid-catalyzed ring opening to an aryl radical cation B. The

5682 J. Agric. Food Chem., Vol. 52, No. 18, 2004
Ueno et al.
Scheme 3.
Possible Mechanism for the Formation of
Scheme 4. Possible Mechanism for the Formation of Peroxy Radical
20 in the Acidic Solution of Citral
p-Methylacetophenone (6) and 4-(2-Hydroxy-2-propyl)benzaldehyde
(17) from Citral under Acidic Aqueous Conditions
tion products including p-menthadien-8-ols 5 and 8 (Figure 2b).
This reaction might involve the acid-catalyzed dehydration of
5 and subsequent isomerization, during which 21 could be
formed as an intermediate. Probably due to the difficulties in
detection, the formation of 21 in the acidic citral solution has
not been elucidated so far, but its possible formation as an
intermediate during the formation of 2 has been postulated (26).
On the other hand, Andemichael et al. reported that 21 was
rapidly oxidized to hydroperoxide 14 upon brief exposure to
air (27). They also pointed out that the facile rate of oxidation
of 21 can be reasonably attributed to the gain in aromaticity
through a free radical chain mechanism, which suggests the
formation of 20 as a radical intermediate. Although the oxidation
of p-cymene (2) could also produce 20, we observed that the
addition of 2 (2 mg/L) to the citral (10 mg/L) solution did not
affect the formation of any of the oxidation products 3, 4, 6, 7,
14, and 17.
Mechanisms of Antioxidant Effects. The observed antioxi-
dant actions (Table 2) were interpreted on the basis of the
proposed mechanism for the formation of oxidation products
from citral. All of the tested catechins strongly inhibited the
formation of both 6 and 17. However, the formation of 3 and
14 was promoted by the catechins except for (+)-catechin, and
the formation of 7 and 4 was also promoted by all of the tested
catechins. Especially, the gallated catechins (ECg and EGCg)
showed strong promoting effects on the formation of 3 and 14,
and the nongallated catechins [(+)-catechin, EC, and EGC]
showed strong promoting effects on the formation of 7 and 4.
These promoting effects of the catechins on the formation of
oxidation products from citral might be accounted for by the
two different radical scavenging mechanisms against the peroxy
radical 20. It is generally accepted that phenolic antioxidants
(ArOH) can scavenge two peroxy radicals (ROO•) by means
of H donation and the subsequent phenoxy-peroxy coupling
reaction (eqs 1 and 2).
reaction of the peroxy radical 20 to olefins, such as p-mentha-
dien-8-ols 5 and 8, followed by the elimination of the alkoxy
radical 18 (23-25) (path B).
ROO• + ArOH f ROOH + ArO•
(1)
(2)
Although further studies are needed for the elucidation of
these mechanisms, the postulated formation of 18 from 20 can
give a rational explanation for the formation of 6 and 17 from
citral without Fe2+. Also, on the basis of our proposed
mechanism, the observed increase in 6 and 17 with the addition
of 14 to the citral solution can be rationalized by the formation
of 20 resulting from H abstraction from 14 with free radicals.
This would probably be a minor reaction for 14, which
preferably undergoes acid-catalyzed degradation to 3 and 7 along
with 4 under our experimental conditions.
A possible mechanism for the formation of the peroxy radical
20 in the citral solution is shown in Scheme 4. The oxidation
of p-mentha-1,4(8),5-triene (21) was postulated to be involved
in the mechanism for the following reasons. It appears that
p-cymene (2) is formed from the major acid-catalyzed degrada-
ROO• + ArO• f non-radical products
Relatively stable coupling products were obtained when
phenoxy radicals derived from 2,6-di-tert-butyl-4-methylphenol
(28), R-tocopherol (29), and genistein (30) reacted with peroxy
radicals. However, Valcic et al. (31) reported that phenoxy
radicals derived from EGC and EGCg did not form stable
coupling products with peroxy radicals and the decomposition
of the unstable coupling products afforded the catechin oxidation
products including the seven-membered B-ring anhydrides of
EGC and EGCg (Scheme 5). In these processes, peroxy radicals
(ROO•) might be reduced to the corresponding alcohols (ROH)
as suggested by their proposed mechanism.

Off-Odorant Formation from Citral
J. Agric. Food Chem., Vol. 52, No. 18, 2004 5683
of 7 and 4. Because of the electron delocalization due to the
2,3-double bond of the C-ring of the quercetin moiety (38),
phenoxy radicals derived from quercitrin might be less reactive
to the peroxy radical 20 as compared to those derived from the
catechins.
Scheme 5. Coupling Reaction between the Phenoxy Radical Derived
from EGCg and Peroxy Radicals (ROO•) Followed by the
Decomposition of the Coupling Product (31)
Ascorbic acid also did not inhibit the formation of 6 and 17
as much as the catechins, but the mechanism seems to be
different from that of quercitrin. When we conducted the
degradation of hydroperoxide 14 (2 mg/L) with added EGC,
EGCg, and ascorbic acid (60 mg/L, respectively) under acidic
aqueous conditions, only ascorbic acid induced the decomposi-
tion of 14 to 6 and 17. This reduction of 14 by ascorbic acid
might be accounted for by the low reduction potential of the
ascorbate anion radical (39) compared to those of the catechin
phenoxy radicals (40, 41).
Scheme 6. Possible Radical-Scavenging Mechanisms of Phenolic
Antioxidants (ArOH) on the Formation of Oxidation Products from
Citral
In conclusion, this study provided a specific insight into the
formation mechanism of the potent off-odorant p-methylac-
etophenone (6) from citral under acidic aqueous conditions. Our
data suggested that 6 is formed via the tert-alkoxy radical 18,
which is most likely derived from the corresponding peroxy
radical 20. Further studies including direct detection of the free
radicals by electron spin resonance spectroscopy might provide
unambiguous evidence to support our proposed mechanism for
the formation of 6 from citral.
ACKNOWLEDGMENT
In the present case, as shown in Scheme 6, H donation from
the catechins to the peroxy radical 20 can afford 14, which
subsequently degrades to 3 and 7 along with 4 under acidic
aqueous conditions. On the other hand, coupling reactions
between phenoxy radicals derived from the catechins and the
peroxy radical 20 followed by the decomposition of the coupling
products might produce 7 and 4.
We thank Susumu Kiyohara, Shuichi Muranishi, and Kenji
Kumazawa for helpful discussions and Yuya Sekiguchi for
excellent technical assistance.
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Received for review December 26, 2003. Revised manuscript received
June 16, 2004. Accepted June 20, 2004.
JF035517J