5,5′-Dicapsaicin, 4′-O-5-Dicapsaicin Ether, and Dehydrogenation Polymers with High Molecular Weights Are the Main Products of the Oxidation of Capsaicin by Peroxidase from Hot Pepper

Article abstract of DOI:10.1021/jf950826y

The nature of the products of capsaicin oxidation by hot pepper peroxidase was studied by gel permeation chromatography. Three main types of oxidation products were identified. Peak 1, with an average molecular weight of 603 ± 12 and corresponding to dimers of capsaicin, was resolved by gas chromatography-mass spectrometry into the dimers 5,5′-dicapsaicin (1a) and 4′-O-5-dicapsaicin ether (1b). Peak 2 appears to be a polymeric product with a molecular weight of 4500 and probably corresponds to polymeric products with a mean degree of 15. Capsaicin-protein copolymers (peak 3) also appear to be products of capsaicin oxidation, probably arising from protein and capsaicin copolymerization. These results suggest that Capsicum peroxidase oxidizes capsaicin to yield 5,5′-dicapsaicin and 4′-O-5-dicapsaicin ether, together a mixture of highly polymerized dehydrogenation products, all of them therefore of a lignin-like nature.

Full text of DOI:10.1021/jf950826y

J. Agric. Food Chem. 1996, 44, 3085
−3089
3085
5
,5′-Dica p sa icin , 4′-O-5-Dica p sa icin Eth er , a n d Deh yd r ogen a tion
P olym er s w ith High Molecu la r Weigh ts Ar e th e Ma in P r od u cts of
th e Oxid a tion of Ca p sa icin by P er oxid a se fr om Hot P ep p er
†
,‡
Mar ´ı a A. Bernal and A. Ros Barcel o´ *
Department of Plant Biology, Faculty of Sciences, University of La Coru n˜ a, E-15071 La Coru n˜ a, Spain, and
Department of Plant Biology (Plant Physiology), University of Murcia, E-30100 Murcia, Spain
The nature of the products of capsaicin oxidation by hot pepper peroxidase was studied by gel
permeation chromatography. Three main types of oxidation products were identified. Peak 1, with
an average molecular weight of 603 ( 12 and corresponding to dimers of capsaicin, was resolved by
gas chromatography-mass spectrometry into the dimers 5,5′-dicapsaicin (1a ) and 4′-O-5-dicapsaicin
ether (1b). Peak 2 appears to be a polymeric product with a molecular weight of 4500 and probably
corresponds to polymeric products with a mean degree of 15. Capsaicin-protein copolymers (peak
3
) also appear to be products of capsaicin oxidation, probably arising from protein and capsaicin
copolymerization. These results suggest that Capsicum peroxidase oxidizes capsaicin to yield 5,5′-
dicapsaicin and 4′-O-5-dicapsaicin ether, together a mixture of highly polymerized dehydrogenation
products, all of them therefore of a lignin-like nature.
Keyw or d s: Capsaicin; capsaicin oxidation products; hot pepper; peroxidase
INTRODUCTION
and 4′-O-5-dicapsaicin ether, together with a mixture
of highly polymerized dehydrogenation products, all of
them therefore of a lignin-like nature. A radical-
radical coupling reaction mechanism to explain the
appearance of these oxidation products is proposed.
Capsaicin, the major pungent compound of hot pepper
fruits (Capsicum annuum var. Annuum), is an amide
derivative of vanillylamine and 8-methylnontrans-6-
enoic acid (Leete and Louden, 1968; Bennett and Kirby,
1
968). The vanillylamine moiety of capsaicin is biosyn-
MATERIALS AND METHODS
thetically derived from L-phenylalanine, while the
branched fatty acid moiety is derived from valine (Leete
and Louden, 1968; Bennett and Kirby, 1968; Iwai et al.,
P la n t Ma ter ia l. C. annuum var. Annuum fruits were
obtained from a local market and stored at 4 °C until use.
P er oxid a se F r a ction . Capsicum fruits (40 g formula
weight) were homogenized with a mortar and pestle in the
presence of acetone at -20 °C. The homogenate was im-
mediately filtered through one layer of filter paper at 4 °C in
a Buchner funnel and the residue thoroughly washed with
acetone at -20 °C until all pigments were removed. The
protein precipitate was resuspended in 100 mL of 50 mM Tris
1
979).
While considerable progress has been made in un-
derstanding the biosynthesis of capsaicin, the enzymol-
ogy of the last steps of capsaicin metabolism and
degradation is still not completely known. Feeding
experiments with capsaicin precursors suggest that
capsaicin biosynthesis in hot pepper fruits competes
with an active accumulation of lignin-like substances
in cell walls, which are probably derived from phenyl-
propanoid precursors and from capsaicin itself (Hall et
al., 1987; Sukrasno and Yeoman, 1993). The reactions
driving this competing sink are apparently mediated by
peroxidase (EC 1.11.1.7) (Bernal et al., 1993a, 1995).
Arguments in favor of the participation of peroxidase
in this metabolic pathway are based on the exclusive
localization of peroxidase in placental epidermal cells
from hot pepper fruits (Bernal et al., 1994b), and in the
colocalization of peroxidase and capsaicin in vacuoles
and cell walls of hot pepper cells (Bernal et al., 1993b),
this evidence being further supported by the strong
capsaicin-oxidizing activity of hot pepper peroxidase
[tris(hydroxymethyl)aminomethane]-HCl buffer (pH 7.5), con-
taining 1 M KCl, and incubated with agitation for 1 h at 4 °C.
The pepper protein solution thus obtained was clarified by
centrifugation at 10000g for 30 min. The supernatant was
used for the further purification of pepper peroxidase.
Purification of pepper peroxidase was performed by chro-
matography on phenyl-Sepharose CL-4B (Sottomayor et al.,
1
996). For this, the supernatant of the above centrifugation
was concentrated with Aquacid I. (NH
4
)
2
SO was added up
4
to 1 M, and the protein was applied to a phenyl-Sepharose
CL-4B column (50 × 1.5 cm). Bound proteins were eluted with
a decreasing linear gradient of (NH ) SO [from 1.0 to 0.0 M
4
2
4
-1
4 2 4
(NH ) SO at a concentration change rate of 25 mM min and
-
1
with a flow rate of 2 mL min ] in 50 mM Tris-HCl (pH 7.5).
Fractions of 8 mL were collected. All the fractions with
peroxidase activity were pooled, concentrated with Aquacid I,
and dialyzed overnight against 0.1 M Tris-acetate (pH 6.0).
All procedures were carried out at 4 °C.
(Bernal et al., 1994a).
With this in mind, the present study was undertaken
Sp ectr op h otom etr ic Assa ys. The spectrophotometric
assay of capsaicin oxidation by Capsicum peroxidase was
performed at 25 °C in a reaction medium containing 1 mM
capsaicin (Sigma Chemical Co.) (from a 10 mM stock in
to investigate the products of capsaicin oxidation by
peroxidases from Capsicum fruits. The results shown
below illustrate that these products are 5,5′-dicapsaicin
2 2
methanol of HPLC grade), 0.1 mM H O , and 0.1 M Tris-
acetate buffer (pH 6.0) (Bernal et al., 1993a). The reaction
was initiated by the addition of enzyme. Capsaicin oxidation
was monitored by increases in absorbance at 262 nm in a
Uvikon 940 spectrophotometer (Kontron Instruments, Madrid,
Spain), the oxidation rate being expressed in nanomoles per
*
Author to whom correspondence should be ad-
dressed (fax 34 68 363963).
†
University of La Coru n˜ a.
University of Murcia.
‡
S0021-8561(95)00826-0 CCC: $12.00
© 1996 American Chemical Society

3086 J. Agric. Food Chem., Vol. 44, No. 10, 1996
Bernal and Ros Barcelo´
second, for which a ꢀ262 of 5.3 × 10 M cm-1 was used for the
oxidation products (Bernal et al., 1993a). One nanokatal of
peroxidase was defined as the amount of protein that oxidized
3
-1
1
nmol of substrate per second.
Oxid a tion of Ca p sa icin by P er oxid a se. The oxidation
of capsaicin by peroxidase was performed in a closed vessel
containing 400 nkat of pepper peroxidase (corresponding to a
catalytic activity against capsaicin of 0.4 µmol of capsaicin
oxidized per second) in 25 mL of 0.1 M Tris-acetate buffer (pH
6
2 2
.0). Capsaicin and H O were added slowly up to a final
concentration of 0.6 and 1.0 mM, respectively. After incuba-
tion for 24 h at 25 °C, the reaction was stopped by the addition
of 25 mL of dimethylformamide (DMF). A control was carried
2 2
out in the absence of H O .
Tim e Cou r se of P er oxid a se In a ctiva tion d u r in g Ca p -
sa icin Oxid a tion . The peroxidase activity remaining in the
reaction vessel was determined by removing samples at 0.5,
F igu r e 1. Time course of peroxidase inactivation during the
oxidation of capsaicin by hot pepper peroxidase in the presence
1
.0, 1.5, 2.0, 4.0, 6.0, and 24 h, peroxidase activity being
determined with capsaicin as substrate (see above).
(b) and in the absence (O) of hydrogen peroxide.
F r a ction a tion of Ca p sa icin Oxid a tion P r od u cts by Gel
P er m ea tion Ch r om a togr a p h y. The preliminary charac-
terization of capsaicin oxidation products was performed by
gel permeation chromatography using Sephadex LH-20 (Phar-
macia) and DMF as eluent (Weymouth et al., 1993). For this,
were found in the control carried out in the absence of
H2O2 (Figure 1).
These results are surprising since it is well-known
that the oxidation of coniferyl alcohol (a phenolic
compound structurally anologous to capsaicin) by per-
oxidase is accompanied by enzyme inactivation (Wey-
mouth et al., 1993; Ferrer and Ros Barcel o´ , 1994). In
fact, in the course of coniferyl alcohol oxidation, total
enzyme inactivation takes place in a few hours (Wey-
mouth et al., 1993; Ferrer and Ros Barcel o´ , 1994).
Recently, it has been reported that peroxidase inactiva-
tion during the oxidation of phenols is mainly due to
the inability of phenols, or phenoxy radicals, to reduce
compound III (a highly oxidized species of peroxidase
regarded as a key intermediate in the catalytic cycle of
the enzyme) to the ferric enzyme (Chung and Aust,
1995). If this is so, it may be suspected that capsaicin,
like veratryl alcohol and many other phenols (Chung
and Aust, 1995), is capable of reducing compound III,
thus avoiding peroxidase inactivation.
5
mL of reaction medium, to which previously was added DMF
up to 50% (v/v) (see above), was loaded on a Sephadex LH-20
column (1 × 50 cm) equilibrated with DMF. Fractions of 1.0
mL were collected. The UV absorbance (262 nm) of the eluate
was monitored to detect the presence of capsaicin oxidation
products.
Deter m in a tion of Molecu la r Weigh ts of Ca p sa icin
Oxid a tion P r od u cts. For this, the Sephadex LH-20 column
was calibrated using polystyrene standards with MWs of
1
2 860, 4075, 2500, and 687 (Aldrich Chemical Co.) and
dihydrocapsaicin (MW ) 307.4) (Sigma Chemical Co.). The
apparent MWs of the capsaicin oxidation products were
determined by comparison with the distribution coefficients
(K
d
) calculated for each of the standards.
Ga s Ch r om a togr a p h y-Ma ss Sp ectr om etr y (GC-MS)
of Ca p sa icin Oxid a tion P r od u cts. GC-MS was performed
on a 5993 Hewlett-Packard model coupled to a 5995 gas
chromatograph/mass spectrophotometer and to a 2648A Graph-
ics terminal, using a OW Chrompak 50 m × 0.20 mm inside
diameter column, He pressure of 0.4 MPa, and a GC temper-
Ap p a r en t Molecu la r Weigh ts of Ca p sa icin Oxi-
d a tion P r od u cts. Reaction media, to which was added
DMF up to 50% (v/v), were fractionated by gel perme-
ation chromatography on Sephadex LH-20 using DMF
as eluent. Previously, the column was calibrated with
a series of polystyrene standards (Mw ) 12860-687) and
dihydrocapsaicin (Mw ) 307), and the distribution
coefficients (Kd) for each standard were calculated.
When the Kd values were plotted versus the molecular
weights, a strong correlation between Kd values and
molecular weight was apparent (log Mw ) 3.66 - 3.76Kd;
-1
ature program of 90-280 °C at 10 °C min , with a 5 min hold
at 280 °C, as previously described (Bernal et al., 1993a). TMS
derivatives of capsaicin oxidation products for GC were
prepared by reaction with Sigma-Sil-A (Sigma Chemical Co.)
according to the procedure described by the manufacturer.
RESULTS AND DISCUSSION
Oxid a tion of Ca p sa icin by P ep p er P er oxid a se.
The oxidation of capsaicin by a partially purified per-
oxidase from hot pepper was carried out in a closed
vessel containing the enzyme (in excess), to which
capsaicin and H2O2 were added. The reaction mixture
was left for 24 h at 25 °C. This type of peroxidase-
mediated dehydrogenation of phenolics (carried out by
a one-off addition of reactants) is known as “bulk
polymer” and is different from the “end-wise polymer”
type (gradual addition of reactants), in which the degree
of polymer formation is somewhat minor (Lai and
Sarkanen, 1975; Weymouth et al., 1993).
In order to check whether the enzyme activity
was limiting during the oxidative process, aliquots
of the reaction medium were taken at several time
intervals and peroxidase activity was measured. The
results shown in Figure 1 illustrate that about 75%
of the enzyme remains active after 24 h of incuba-
tion. In fact, only about 25% of the enzyme was
inactivated in the course of the reaction, and this was
mainly due to thermal inactivation, since similar results
2
r ) 0.9890) (Figure 2).
Once the column was calibrated, the oxidation prod-
ucts of capsaicin were chromatographed, being detected
in eluates by their absorbance at 262 nm (Figure 3).
Three peaks were detected for capsaicin oxidation
products (peaks 1-3, Figure 3). Peak 1 (K ) 0.234),
d
corresponding to compounds with an average molecular
weight (see Figure 2) of 603 ( 12, apparently corre-
sponds to dimers of capsaicin (M ) 608), while peak 2
w
(K ) 0.005) appears to be a polymeric product with a
d
molecular weight of 4500. This dehydrogenation poly-
mer probably corresponds to polymeric products with a
mean polymerization degree of 15. Peak 2 (Figure 3)
trails a plateau which suggests that other polymeriza-
tion products with a polymerization degree of less than
15 may also be present.
Together with these oxidation products, material
absorbing at 262 nm was also found in the first seven
fractions (0-7 mL, peak 3, Figure 3). However, this

Products of Capsaicin Oxidation by Peroxidase
J. Agric. Food Chem., Vol. 44, No. 10, 1996 3087
Ch a r t 1. Str u ctu r e of 5,5′-Dica p sa icin (1a ) a n d 4′-O-5-
Dica p sa icin Eth er (1b) P r od u cts of th e Oxid a tive
Cou p lin g of Ca p sa icin by Hot P ep p er P er oxid a se
Sch em e 1. Ma ss F r a gm en ta tion P a tter n of th e 4-O,4′-
O-DiTMS Der iva tive of 5,5′-Dica p sa icin
F igu r e 2. Molecular weight determination of capsaicin oxida-
tion products (1, corresponding to peak 1 in Figure 3) from
the plotting of the log M versus K
polystyrene standards of 4075 (S ), 2500 (S
dihydrocapsaicin (S ; 307) as molecular markers.
d
values using a series of
1
2
), and 687 (S ) and
3
4
F igu r e 3. Gel permeation chromatography on Sephadex LH-
0 of reaction media containing capsaicin and hot pepper
peroxidase in the presence (solid line) and in the absence
dashed line) of hydrogen peroxide.
2
Ta ble 1. m /z Ra tio of th e Ma in F r a gm en t a n d th e
Aver a ge Molecu la r Weigh t (a Mw ), Abu n d a n ce (A),
Elem en ta l Com p osition (ec), a n d Th eor etica l Molecu la r
Weigh t (tMw ) of th e Decom p osition F r a gm en ts of th e
TMS Der iva tive of 5,5′-Dica p sa icin
(
material, as can be deduced from its cloudy (colloidal)
nature, appears to represent products of protein and
capsaicin copolymerization, such copolymers frequently
growing in reaction media during peroxidase-mediated
phenolic oxidations (Evans and Himmelsbach, 1991). All
these products (dimers, dehydrogenation polymers, and
capsaicin-protein copolymers) were due to a peroxidase-
mediated capsaicin oxidation since, in the absence of
H2O2, only the peak corresponding to capsaicin (Figure
m/ z
aMw
A (%)
ec
tMw
+
7
3
2
53 (M )
-
-
<2
<2
100
C42H68O6N2Si2
C21H34O3NSi
C13H20O3NSi
753.186
376.593
266.393
+
76 (M /2)
66
266.358
+
m/ z 753 appears to be the molecular ion (M ) of the
4-O,4′-O-diTMS derivative of 5,5′-dicapsaicin (m/ z
753.186). Furthermore, the mass fragment at m/ z 376
appears to result from the symmetric homolytic break-
3
, peak 1′) was found.
GC-MS An a lysis of Ca p sa icin Oxid a tion P r od -
+
down of M at 5,5′ (see Scheme 1, arrow 1, breakdown
u cts w ith Low Molecu la r Weigh ts. From the results
shown in Figure 3, it became apparent that dimers of
capsaicin are the main products of the peroxidase-
mediated capsaicin oxidation. For this reason, and in
order to elucidate their structure, the 18-28th fractions
from the Sephadex LH-20 chromatogram were pooled
and brought to dryness, and the TMS derivatives were
prepared. Analysis of the TMS derivatives by GC-MS
showed the presence of two main compounds. The TMS
derivative of compound 1a eluted with a retention time
of 7.23 min, while the TMS derivative of compound 1b
eluted at 10.57 min.
in a). Theoretically, this symmetric breakdown results
in two fragments with identical molecular weights
(theoretical Mw ) 376.593, Table 1). The abundance of
these fragments is low (<2%, Table 1), as also occurs
+
with the M for the TMS derivative of capsaicin itself
(<2%).
Likewise, the presence of mass fragments at m/ z 266
and 248 supports this structure. Thus, the mass
fragment at m/ z 266 would have its origin in the
â-elimination of the alkyl side chain (breakdown in b,
arrow 2, Scheme 1) of the mass fragment m/ z 376, a
fragmentation pattern typical of alkyl amides. The
mass fragment at m/ z 248 apparently has its origin in
mass fragment m/ z 266 through the loss of H2O (arrow
3, Scheme 1). This pattern of decomposition (â-elimina-
tion of olefin at the carbonyl side followed by the loss of
water) is also typical of the TMS derivative of capsaicin.
Furthermore, the calculation of the average Mw for each
The MS spectrum of the TMS derivative of compound
a showed mass fragments at m/ z 753, 376, 266, and
48, with satellites at (m/ z + 1) and (m/ z + 2). These
1
2
decomposition fragments appear to be conclusive for
assigning to compound 1a the structure of 5,5′-dicap-
saicin (1a in Chart 1, Mw ) 608). In fact, the ion at

3088 J. Agric. Food Chem., Vol. 44, No. 10, 1996
Bernal and Ros Barcelo´
Ta ble 2. m /z Ra tio of th e Ma in F r a gm en t a n d th e
Aver a ge Molecu la r Weigh t (a Mw ), Abu n d a n ce (A),
Elem en ta l Com p osition (ec), a n d Th eor etica l Molecu la r
Weigh t (tMw ) of th e Decom p osition F r a gm en ts of th e
TMS Der iva tive of 4′-O-5-Dica p sa icin Eth er
Sch em e 3. P r op osed Rea ction Sch em e for th e F or -
m a tion of 5,5′-Dica p sa icin (1a ) a n d 4′-O-5-Dica p sa icin
Eth er (1b) th r ou gh
Rea ction d u r in g th e Oxid a tion of Ca p sa icin (1′) by
a
Ra d ica l-Ra d ica l Cou p lin g
Hot P ep p er P er oxid a se
m/z
aMw
A (%)
ec
tMw
6
4
4
3
3
2
2
2
80 (M+)
29
01
55
25
81
67
51
-
<2
100
22
87
12
100
52
35
C39H59O6N2Si
C21H24O6N2Si
C20H24O5N2Si
-
679.996
429.528
401.517
-
429.599
401.498
355.500
325.385
281.485
267.498
251.188
-
-
C13H19O4NSi
C12H17O4NSi
C12H17O3NSi
281.385
267.358
251.358
Sch em e 2. Ma ss F r a gm en ta tion P a tter n of th e 4-O-
TMS Der iva tive of 4′-O-5-Dica p sa icin Eth er
2
51 probably arise from the asymmetric breakdown of
the O-diphenyl ether bond (breakdown in a, arrow 2,
Scheme 2) followed by R-elimination of the side chain
(breakdown in c, arrow 2, Scheme 2). Mass fragments
at m/ z 429 and 401 probably arise by R-elimination of
the side chain as olefin (breakdown in c,c′, arrow 4,
Scheme 2), followed by the loss of CO (arrow 5, Scheme
2
). This fragmentation pattern is typical of capsaicin
and, in general, of amides as well.
Furthermore, in those cases where the structures
were tentatively identified, the calculation of the aver-
age Mw for each fragment from the abundance of m/ z,
(
m/ z + 1), and (m/ z + 2) is in accordance with the
theoretical Mw calculated from the suspected structure
(Table 2).
A possible reaction scheme for the formation of 1a
5,5′-dicapsaicin) and 1b (4′-O-5-dicapsaicin ether) is
(
proposed in Scheme 3 on the basis of the theory of
radical phenolic coupling (Hapiot et al., 1994). The
proposed reaction mechanism proceeds via an initial
one-electron oxidation of capsaicin (1′) by peroxidase to
form the 4-O-phenoxyl free radical (MO4, in Scheme 3),
which, in turn, produces a series of mesomeric radicals,
the most stable (in the case of capsaicin) being where
the unpaired electron is at C5 (MC5, Scheme 3). These
radicals spontaneously dimerize to form several possible
structures; the coupling of two C5 phenolic radicals
would give 5,5′-dicapsaicin (reaction i, Scheme 3), while
the coupling of C5 phenolic radical with a 4-O-phenoxyl
free radical would give 4′-O-5-dicapsaicin ether (reaction
j, Scheme 3). These dimeric structures can further act
as a nucleus, in which the highly polymerized dehydro-
genation products also formed by the action of Capsicum
peroxidase on capsaicin grow.
In conclusion, these results suggest that Capsicum
peroxidase oxidizes capsaicin to yield 5,5′-dicapsaicin
and 4′-O-5-dicapsaicin ether, together with a mixture
of highly polymerized dehydrogenation products, all of
them therefore of a lignin-like nature. Furthermore,
these results lend weight to the biochemical evidence
for supporting the existence of an oxidative competitive
sink for phenylpropanoid intermediates of capsaicin
fragment from the abundance of m/ z, (m/ z + 1), and
(
m/ z + 2) is in accordance with the theoretical Mw
calculated from its suspected structure (Table 1).
The MS spectrum of the TMS derivative of compound
1
3
b shows mass fragments at m/ z 680, 429, 401, 355,
25, 281, 267, and 251. These decomposition fragments
also appear to be conclusive for assigning to compound
1
1
b the structure of 4′-O-5-dicapsaicin ether (1b in Chart
, Mw ) 608). In fact, the ion at m/ z 680 appears to be
+
the molecular ion (M ) of the 4-O-TMS derivative of 4′-
O-5-dicapsaicin ether (m/ z 679.996, Table 2). Further-
more, the mass fragments at m/ z 325 (325.385, 12%)
and 355 (355.500, 87%) appear to result from the
asymmetric breakdown of (M + H) (325.385 + 355.500
)
+
680.885).
The mass fragment at m/ z 281 probably arises from
the asymmetric breakdown of the O-diphenyl ether bond
(
breakdown in a, arrow 1, Scheme 2) followed by â-elimi-
nation of the side chain (breakdown in b, arrow 1,
Scheme 2), whereas mass fragments at m/ z 267 and

Products of Capsaicin Oxidation by Peroxidase
J. Agric. Food Chem., Vol. 44, No. 10, 1996 3089
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itself to yield lignin-like substances in the cell wall of
C. annuum var. Annuum.
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Received for review December 19, 1995. Revised manuscript
received J uly 30, 1996. Accepted J uly 30, 1996. This work
was supported by Grants CICYT ALI 573/93 and XUGA 10301/
A/95. M.A.B. holds a fellowship from the Xunta de Galicia.
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Abstract published in Advance ACS Abstracts, Sep-
tember 1, 1996.