Properties of chlorogenic acid quinone: Relationship between browning and the formation of hydrogen peroxide from a quinone solution

Article abstract of DOI:10.1271/bbb.66.2525

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7 × 10³ for 325 nm and 9.7 × 10 ² for 400 nm in an acidic aqueous solution. Chlorogenic acid and H₂O₂ were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H₂O₂ spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.

Full text of DOI:10.1271/bbb.66.2525

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Properties of Chlorogenic Acid Quinone: Relationship
between Browning and the Formation of Hydrogen
Peroxide from a Quinone Solution
Masatsune MURATAa, Makie SUGIURAa, Yuko SONOKAWAa, Tomomi SHIMAMURAa & Seiichi
HOMMAa
a Department of Nutrition and Food Science, Ochanomizu University 2-1-1 Otsuka,
Bunkyo-ku, Tokyo 112-8610, Japan
Published online: 22 May 2014.
To cite this article: Masatsune MURATA, Makie SUGIURA, Yuko SONOKAWA, Tomomi SHIMAMURA & Seiichi HOMMA (2002)
Properties of Chlorogenic Acid Quinone: Relationship between Browning and the Formation of Hydrogen Peroxide from a
Quinone Solution, Bioscience, Biotechnology, and Biochemistry, 66:12, 2525-2530, DOI: 10.1271/bbb.66.2525
To link to this article: http://dx.doi.org/10.1271/bbb.66.2525
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Biosci. Biotechnol. Biochem., 66 (12), 2525–2530, 2002
Properties of Chlorogenic Acid Quinone: Relationship between Browning
and the Formation of Hydrogen Peroxide from a Quinone Solution
Masatsune MURATA,† Makie SUGIURA, Yuko SONOKAWA
,
Tomomi SHIMAMURA, and Seiichi HOMMA
Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku,
Tokyo 112-8610, Japan
Received February 20, 2002; Accepted August 9, 2002
ly been making an intensive progress.5,6) Several trials
Chlorogenic acid is the major polyphenol in foods
derived from plants and is a good substrate for poly-
phenol oxidase. Chlorogenic acid quinone (CQA-Q),
which is an oxidative product of chlorogenic acid by
polyphenol oxidase, is an important intermediate com-
pound in enzymatic browning. CQA-Q was prepared,
and its properties and the relationship with browning
were examined. The quinone solution was yellow or
orange, and its molecular absorption coe‹cient was
to regulate enzymatic browning by repressing the
expression of PPO have been conducted.7–10) On the
other hand, clariˆcation of the reactivity of poly-
phenol quinones is also important to understand the
mechanism for enzymatic browning and to regulate
the eŠect. Among the polyphenols, 5-caŠeoylquinic
acid (chlorogenic acid, CQA) is one of the major
polyphenol in such plants as apples, coŠee and
lettuce. However, there is little data on the reactivity
3
2
×
×
estimated to be 1.7 10 for 325 nm and 9.7 10 for
400 nm in an acidic aqueous solution. Chlorogenic acid
and H2O2 were spontaneously generated in the CQA-Q
solution as the yellowish color of the solution gradually
faded. A pale colored polymer was the major product in
the reaction solution. Amino acids such as lysine and
arginine added to CQA-Q solution did not repress the
fading of the yellowish color of the solution. We con-
cluded from these results that CQA-Q itself and a mix-
ture of CQA-Q and amino acids did not form intensive
brown pigments in the acidic aqueous solution. H2O2
spontaneously formed in the CQA-Q solution, and
other polyphenols might have played an important role
in the formation of the brown color by enzymatic
browning.
of isolated
o-quinones or CQA quinone (CQA-Q;
Fig. 1). Cilliers and Singleton have reported the
products from the non-enzymatic auto-oxidation of
caŠeic acid as caŠeicins which are dimers of caŠeic
Key words: chlorogenic acid; chlorogenic acid
quinone;
H2O2
o-quinone; enzymatic browning;
The polyphenols in a variety of plants are oxidized
to their corresponding quinones by the action of
polyphenol oxidase (EC 1.10.3.1, PPO) when plant
tissue is broken or crushed.1,2) These quinones are
considered to be further polymerized with quinones
or amines to form a brown pigment.3,4) This brown-
ing has been commonly observed in fruit and vegeta-
bles such as apples, bananas and lettuce, and the
regulation of the browning is an important aspect for
food processing and preservation.4,5)
Fig. 1. Structures of Chlorogenic Acid (CQA), CQA Quinone
Molecular biological studies on PPOs have recent-
(CQA-Q), and 5-
S
-Cysteinyl-chlorogenic Acid (5-S-Cys-CQA).
†
To whom correspondence should be addressed. Fax: +81-3-5978-5899; E-mail: murata
＠
cc.ocha.ac.jp
Abbreviations: PPO, polyphenol oxidase; CQA, chlorogenic acid; CQA-Q, CQA quinone

2526
M. MURATA et al.
acid.11) Fulcrand et al. have prepared caŠeic acid qui-
none and identiˆed the products from chemical oxi-
dation of caŠeic acid under acidic conditions as two
Identiˆcation of 5-S-cysteinyl-CQA. About 0.8 ml
of a 1 mM L-cysteine aqueous solution was added to
3 ml of the CQA-Q solution, the mixture being left
for 30 min at room temperature and then concentrat-
ed in vacuo. The concentrate was applied to prepara-
tive HPLC, and a peak with a retention time of
12.5 min was collected, concentrated in vacuo and
dried. This procedure was repeated several times.
The residue was dissolved in D2O and analyzed by
isomers of 2,5-(3?,4?-dihydroxyphenyl)tetrahydrofu-
ran 3,4-dicarboxylic acid.12) However, these com-
pounds were colorless. In this present study, we
prepared and isolated CQA-Q by HPLC and then ex-
amined the reactivity. We show that CQA-Q formed
hardly any intense brown pigment by itself or with
amino acids and that part of CQA quinone returned
to CQA with the formation of H2O2.
1
NMR. H-NMR dH (ppm): 2.15 (4H, m), 3.04 (2H,
＝
＝
m), 3.54 (1H, dd,
J
3.9, 8.5), 3.72 (1H, t,
J
4.8),
＝
9.4), 6.25 (1H,
＝
4.16 (1H, t,
J
3.8), 5.18 (1H, t,
J
＝
＝
＝
9.5), 7.13 (1H, d, J
d,
J
16.0), 6.82 (1H, d,
J
Materials and Methods
＝
9.5), 8.12 (1H, d,
J
16.0).
Preparation of CQA-Q. One milliliter of a 100 m
CQA solution in MeOH and 1.0 ml of a 100 m
M
M
Estimation of
e
for CQA-Q. Three methods were
value of CQA-Q. The ˆrst
NaIO4 aqueous solution were mixed and then incu-
bated for about 3 min at room temperature. The mix-
ture was passed through Chromatodisk (0.45 mm;
used for estimating the
e
and second methods involved leaving the CAQ-Q
solution for 10–45 min, before CQA and CQA-Q
were analyzed. With the ˆrst method (the absorbance
method), the absorption spectrum of the quinone
solution was continuously analyzed by a spec-
trophotometer with a photodiode array detector
(MultiSpec-1500, Shimadzu, Kyoto). We assumed
the following equations for this method:
Kurabo, Osaka, Japan) cartridges, before being sub-
jected to analytical or preparative HPLC. The HPLC
systems were as follow: pump, L-6320 (Hitachi,
Tokyo, Japan); column, YMC pack R-ODS
×
(Yamamura, Kyoto, Japan; 4.6 i.d. 250 mm; for
analysis) and YMC pack D-ODS (20 i.d. 250 mm;
×
for preparation); detector, L-4500 photodiode array
(Hitachi, Tokyo; for analysis), L-4200 UV-VIS
(Hitachi; for preparation); wavelength, 250–370 nm
(for analysis), 325 nm (for preparation); eluent,
＝
×
e(325, CQA) [CQA]
A325
＋
×
e
(325, CQA-Q) [CQA-Q]
A400
＝
e
(400, CQA-Q) [CQA-Q]
×
CH3CN and 5
z
aqueous acetic acid (12:88); ‰ow
where [CQA] and [CQA-Q] are the concentrations of
CQA and CQA-Q, respectively, and (325, CQA),
(325, CQA-Q) and (400, CQA-Q) are the values
for CQA at 325 nm, for CQA-Q at 325 nm, and for
CQA-Q at 400 nm, respectively.
The second method (the HPLC method) involved
analyzing the quinone solution by HPLC under the
conditions already described. We assumed that the
rate of decrease of CQA-Q was equivalent to the rate
of increase of CQA. The ratio of the peak areas of
CQA and CQA-Q by HPLC at 325 nm enabled the
value of CQA-Q at 325 nm to be estimated. The
rate, 1.0 ml min for analysis and 9.99 ml min for
W
W
e
preparation. The eluate containing CQA-Q with a
retention time of about 12 min by preparative HPLC
was collected and immediately used for the subse-
quent experiments. CQA and CQA-Q were each de-
termined from their peak areas at 325 nm. CQA was
used as the external standard, and the CQA-Q con-
centration was estimated by using a conversion rate
which was determined from the molecular absorption
e
e
e
coe‹cients (e) of CQA and CQA-Q at 325 nm. The e
e
e
value for CQA-Q was determined by the Cys method
described later. The variation among samples was
value of CQA-Q at 400 nm was then calculated by
multiplying of CQA-Q at 325 nm by 1.8, because
less than 10
z
.
e
the ratio of the absorbance of CQA-Q at 325 nm and
that at 400 nm was 1.8.
Identiˆcation of CQA. The isolated CQA-Q solu-
tion was left for 3 h at room temperature and then
concentrated in vacuo, before the concentrate was
applied to preparative HPLC under the conditions
just described. A peak with a retention time of about
17 min, which corresponded to a peak with a reten-
tion time of 7.2 min by analytical HPLC, was collect-
ed, concentrated in vacuo and dried. The residue was
dissolved in CD3OD and analyzed by NMR (Jeol
With the third method (the Cys method), an -Cys
L
solution was added to the CQA-Q solution immedi-
ately after its isolation, and the absorbance at 400 nm
was then measured. The equivalent mole value of
CQA-Q to Cys was estimated from the Cys concen-
tration at which the absorbance at 400 nm was zero.
1
Determination of H2O2. H2O2 was determined by
the iodine titration and the peroxidase methods. The
iodine titration method was conducted with Na2S2O3
and KI in the usual way, while the peroxidase method
was according to the method of Putter and Becker13)
JNM-GSX270). H-NMR dH (ppm): 2.15 (4H, m),
＝
＝
3.70 (1H, dd,
J
3.9, 9.2), 4.14 (1H, t,
J
4.1), 5.35
＝
15.0), 6.75 (1H, d,
＝
8.8), 6.28 (1H, d, J
(1H, t,
J
＝
＝
＝
1.4, 8.0), 7.03 (1H, d, J
J
8.0), 6.93 (1H, dd,
J
1.4), 7.55 (1H, d, 15.0).
J
＝

Chlorogenic Acid Quinone and Formation of Hydrogen Peroxide
2527
Fig. 2. Typical HPLC Trace of a Reaction Mixture of CQA and
NaIO4.
A, Chromatogram; B, UV Spectrum of CQA-Q.
with some modiˆcations. After the reaction mixture
(2.2 ml) containing 1.7 m 2,2 -azinobis(3-ethylben-
M
?
zthiazoline sulfonic acid (ABTS, Sigma, St. Louis,
U.S.A.) and horseradish peroxidase (20 units, Wako
Fig. 3. Typical HPLC Traces of Isolated CQA-Q.
Chemical Co., Osaka), a 67 m
buŠer (pH 6.0) and the sample solution (0.2 ml) had
been incubated 30 C for 2 min, the absorbance at
of H2O2 was
M
K Na phosphate
W
CQA-Q isolated by preparative HPLC was applied to analyti-
cal HPLC after 0.1 min (A) and 60 min (B) of incubation. The
HPLC conditions are described in the Material and Method sec-
tion. C, UV spectra of CQA and CQA-Q from A.
9
405 nm was measured. Ten to 500
m
M
used to produce the calibration curve.
positions 8 and 9 was observed. The two components
were therefore connected at position 5. This adduct
Results and Discussion
Preparation and properties of CQA-Q
was thus identiˆed as 5-
acid (5- -Cys-CQA, Fig. 1). These results enabled the
quinone to be conˆrmed as CQA-Q. Richard et al
have reported 5- -Cys-CQA as a single-addition
S-cysteinyl-5?-caŠeoylquinic
CQA-Q was prepared by the oxidation of CQA by
NaIO4 and isolated by HPLC. Figure 2 shows a typi-
cal chromatogram of the reaction mixture and the
UV spectrum of CQA-Q. The quinone had absorp-
tion maxima at 305–310 nm and 390–400 nm. To
ascertain that this quinone was CQA-Q, the fraction
S
.
S
compound in the reaction mixture of CQA and apple
PPO.14)
CQA-Q was very unstable, Fig. 3 showing a typical
chromatogram of the isolated quinone by HPLC. In
addition to the peak of quinone (a retention time of
5.7 min), a peak with a retention time of 7.2 min ap-
peared. This new peak increased and the peak of
CQA-Q decreased when the isolated quinone solu-
tion was incubated at room temperature. The reten-
tion time and the absorption spectrum of this peak
corresponded with those of CQA. To ascertain that
this peak was of CQA, the peak was isolated by
was collected and -Cys was immediately added. The
L
yellowish color of the quinone instantly disappeared.
This mixture was then applied to HPLC, before the
major adduct of the quinone and
L
-Cys was isolated.
The H-NMR spectrum showed that this compound
was the adduct of CQA and -Cys. The signals at
1
L
3.72 ppm and 5.18 ppm were derived from the Cys
residue, and the other signals were from CQA moie-
ty. The signal at position 5 of CQA had disappeared
in this adduct, and coupling between the protons at
1
preparative HPLC and analyzed by H-NMR. The

2528
M. MURATA et al.
Fig. 4. Decrease in Color Intensity of the CQA-Q Solution in the Absence (A) and Presence (B) of Amino Acids.
CQA-Q and the amino acids were dissolved in a mixture of MeCN and 5
z AcOH (15:85).
shows that CQA-Q was partly reduced to CQA and
the yellowish color faded during incubation. Ful-
crand et al. have also reported that an aqueous solu-
tion of caŠeic acid quinone was unstable and that
caŠeic acid was the major product in the aqueous so-
lution of the quinone.12)
We next tried to determine the molecular absorp-
tion coe‹cient (e) of CQA-Q for a qualitative deter-
mination of the quinone by HPLC. We could not iso-
late the quinone as a solid; therefore, the coe‹cient
was estimated by three methods. The absorbance
method and the HPLC method enabled the e value to
be estimated from the proportional decrease in the
quinone and increase in CQA. We assumed for these
two methods that the proportional decrease in CQA-
Q and increase in CQA were the same, because these
two compounds were the major ones by HPLC. With
Fig. 5. Estimated
e Value of CQA-Q by the Cys Method.
NMR data were identical with those of CQA, con-
ˆrming this peak to be CQA. This result shows that
part of CQA-Q had been reduced to CQA in the
acidic aqueous solution.
the absorbance method,
e was estimated from the
decrease in the absorbance at 400 nm, which was the
absorption maximum for CQA-Q, and the increase
in the absorbance 325 nm, which was the absorbance
We used the isolated solution of CQA-Q without
further concentration, because the quinone was very
unstable. An aqueous acetic acid solution containing
acetonitrile was used as the eluent. As the pH value
of such fruits as apple and pear is acidic, this pH con-
dition was suitable for identifying the change in CQA
in fruit and fruit juice. The isolated solution was yel-
low or orange; however, the color intensity gradually
reduced with the decrease in quinone and increase in
CQA. Figure 4 shows the change in the absorbance at
400 nm of the CQA quinone solution. After 60 min,
the absorbance had reduced by about 30
starting level.
It is generally believed that CQA-Q is very reactive
and automatically polymerizes by itself or with CQA
and amino acids to form a brown pigment, because
CQA turns brown when it is incubated with PPO in
the presence of oxygen. However, our result clearly
maximum for CQA. With the HPLC method,
estimated from the peak areas of CQA and CQA-Q
by HPLC, this being monitored by the absorbance at
e was
325 nm. The
enabling the
e
value of CQA could be measured,
e
value of CQA-Q to be calculated from
these data. The diŠerence in
e value estimated by
these two methods was less than 15
z
, the mean value
4
3
×
×
of
e
being 1.5 10 for 325 nm and 8.5 10 for
400 nm in this acidic aqueous solution.
The Cys method involved -Cys being added to the
L
z
of the
CQA-Q solution immediately after its isolation.
Figure 5 shows the relationship between added Cys
and the absorbance of CQA-Q at 400 nm. We
assumed that the Cys concentration at the point
where the absorbance of CQA-Q was nearly zero was
equivalent to the original CQA-Q concentration. The
e
value of CQA-Q was estimated to be 1.7
×
103 for

Chlorogenic Acid Quinone and Formation of Hydrogen Peroxide
2529
Fig. 7. Formation of CQA and H2O2 in the CQA-Q Solution.
Fig. 6. Decrease in CQA-Q and Formation of CQA in the Solu-
tion of CQA-Q.
The CQA-Q solution was left for 30 min, before the concen-
trations of CQA (HPLC method) and H2O2 (A, iodine titration
method; B, peroxidase method) were determined.
2
×
325 nm and 9.7 10 for 400 nm. These values seem
to be more reliable than the values estimated by other
two methods, because they are both lower. With the
absorbance and HPLC methods, it was assumed that
the amount of CQA formed was equal to the amount
of CQA-Q lost, any polymerized or decomposed
products of CQA-Q being neglected, because no
clearly deˆned peaks, apart from CQA appeared by
the color of the quinone solution instantly disap-
peared when
tioned, 5-
L
-Cys was added. As already men-
S
-Cys-CQA was identiˆed in the solution.
Sanoner et al. have reported that CQA-Q reacted
with CQA to produce 5-CQA dimers.17) The com-
pound in this solution might have been formed, but
the amount would have been very low. They used
polymer-supported periodate18) for the oxidation of
CQA, and the CQA solution was used without fur-
ther puriˆcation. The diŠerence in methods might
have led to diŠerent results.
HPLC. Therefore, the
e values estimated by these
two methods seem to be much larger than the real
values.
These results suggest that most of CQA-Q was
converted to pale colored polymers with the genera-
tion of H2O2 and CQA. In fact, polymers with a pale
yellowish color were obtained from the faded solu-
Generation of H2O2 in the CQA-Q solution
CQA-Q spontaneously generated CQA in this
acidic solution, so we examined what kind of oxi-
dized product was formed. We could not detect any
clearly deˆned peak of an oxidized product by
HPLC, but did detect H2O2. The amount of H2O2
was measured by both the iodine titration method
and peroxidase method. The amount of H2O2 formed
was almost equivalent to that of CQA formed
(Fig. 7). We don't know the generation mechanism
for H2O2, but the small amount of metal ions con-
taminating this solution might have been linked with
the formation of H2O2. The generation of H2O2 has
been reported during enzymatic browning,19,20) and
our observations might explain this phenomenon.
PPO generally shows some peroxidase activity, and
this peroxidase activity and H2O2 might play an im-
portant role in enzymatic browning. For example,
CQA is the major phenolic in apple21) and is the best
substrate for apple PPO.22) During the incubation of
apple juice, CQA gradually decreases and completely
disappears within 1 h, and its brown color deepens.
This suggests that regenerated CQA is successively
used by the browning reaction. The second major
polyphenol in apple is catechins. Catechins form an
intense brown color by the action of apple PPO.21)
The properties of catechin quinone and the interac-
tion of CQA-Q in a yield of about 90
z (data not
shown). Figure 6 shows the changes in the concentra-
tions of CQA and CQA-Q estimated by the Cys
method. The CQA-Q concentration decreased and
was partly converted to CQA, the yield of CQA from
CQA-Q being about 10z. These results show that
CQA-Q did not form intense brown pigments, but
pale colored polymers, and partly generated CQA.
Reactivity of CQA-Q with amino acids
The reactivity of CQA-Q with amino acids was ex-
amined. After the quinone solution had been incu-
bated with lysine, glycine, and arginine, the absor-
bance at 400 nm was monitored. The absorbance
decreased with incubation time, and this proˆle was
almost identical with that of the CQA-Q solution
(Fig. 4(B)). This characteristic shows that CQA-Q
formed hardly any intense brown pigment with such
amino acids such lysine and arginine. Yabuta et al
.
have reported the formation of a green pigment from
a mixture of methyl caŠeate and a primary amino
compound under alkaline conditions.15,16) There
seems to have been a deˆnite diŠerence between the
reactivity of CQA-Q in the acidic condition and that
of CQA in the alkaline condition. On the other hand,

2530
M. MURATA et al.
Biochem., 65, 383–388 (2001).
tion between CQA-Q and catechins will be further
examined. Richard-Forget et al. have pointed out the
involvement of the peroxidase in the enzymatic
browning of pear.23) Pear peroxidase did not react
with phenolics, but in the presence of polyphenol oxi-
dase, peroxidase enhanced the phenol degradation.
Considering our results, H2O2, the substrate of
peroxidase, seems to have been generated through
CQA-Q formed in the reaction mixture of pear PPO
and phenolics.
We conclude from these results that CQA-Q itself
and the mixture of CQA-Q and amino acids did not
form the intense brown pigment in the acidic aqueous
solution. H2O2 spontaneously generated from CQA-
Q and polyphenols other than CQA might have
played an important role in forming the intense
brown pigments by enzymatic browning.
11) Cilliers, J. J. L., and Singleton, V. L., Characteriza-
tion of the products of nonenzymic autooxidative
phenolic reactions in a caŠeic acid model system. J.
Agric. Food Chem., 39, 1289–1303 (1991).
12) Fulcrand, H., Cheminat, A., Brouillard, R., and
Cheynier, V., Characterization of compounds ob-
tained by chemical oxidation of caŠeic acid in acidic
conditions. Phytochemistry, 35, 499–505 (1994).
13) Puä tter, J., and Becker, R., Peroxidase. In ``Method
of Enzymatic Analysis, Third Edition, Vol. 3'', ed.
Bergmeyer, H. U., Verlag Chemie, Weihenheim, pp.
286–292 (1983).
14) Richard, F. C., Goupy, P. M., Nicolas, J. J.,
Lacombe, J.-M., and Pavia, A. A., Cysteine as an
inhibitor of enzymatic browning. 1. Isolation and
characterization of addition compounds formed dur-
ing oxidation phenolics by apple polyphenol oxidase.
J. Agric. Food Chem., 39, 841–847 (1991).
15) Yabuta, G., Koizumi, Y., Namiki, K., Hayashi, T.,
and Namiki, M., Novel benzacridine derivative in the
green pigment from methyl caŠeate and butylamine.
Biosci. Biotechnol. Biochem., 60, 1701–1702 (1996).
16) Yabuta, G., Koizumi, Y., Namiki, K., Hida, M., and
Namiki, M., Structure of green pigment formed by
the reaction of caŠeic acid esters (or chlorogenic acid)
with a primary amino compound. Biosci. Biotechnol.
Biochem., 65, 2121–2130 (2001).
17) Sanoner, P., Guyot, S., Marnet, N., and Drilleau,
J.-F., Polyphenol oxidation in cider apple model: ki-
netic and product study. Polyphenol Communication
2000, Freising-Weihenstephan (Germany), September
11–15, pp. 531–532 (2000).
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