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Abstract
A peroxidase-based bioelectrochemical sensor of hydrogen peroxide (H2 O2 ) and a Clark-type oxygen electrode were applied to
continuous monitoring and kinetic analysis of the autoxidation of catechins. Four major catechins in green tea, (3)-epicatechin, (3)epicatechin gallate, (3)-epigallocatechin, and (3)-epigallocatechin gallate, were used as model compounds. It was found that dioxygen (O2 )
is quantitatively reduced to H2 O2 . The initial rate of autoxidation is suppressed by superoxide dismutase and H , but is independent of
buffer capacity. Based on these results, a mechanism of autoxidation is proposed; the initial step is the one-electron oxidation of the B ring
of catechins by O2 to generate a superoxide anion (O3
2 ) and a semiquinone radical, as supported in part by electron spin resonance
measurements. O3
2 works as a stronger one-electron oxidant than O2 against catechins and is reduced to H2 O2 . The semiquinone radical is
more susceptible to oxidation with O2 than fully reduced catechins. The autoxidation rate increases with pH. This behavior can be
interpreted in terms of the increase in the stability of O3
2 and the semiquinone radical with increasing pH, rather than the acid dissociation
of phenolic groups. Cupric ion enhances autoxidation; most probably it functions as a catalyst of the initial oxidation step of catechins. The
product cuprous ion can trigger a Fenton reaction to generate hydroxyl radical. On the other hand, borate ion suppresses autoxidation
drastically, due to the strong complex formation with catechins. The biological significance of autoxidation and its effectors are also
discussed. 2002 Elsevier Science B.V. All rights reserved.
Keywords : Catechin ; Autoxidation ; Superoxide dismutase; Cupric ion; Borate; H2 O2 sensor
1. Introduction
It has been pointed out that oxygen free radicals such as
superoxide anion radical (O3
2 ) and hydroxyl radical
(OH ) cleave DNA and peroxidize low-density lipoproteins and lipids to cause several kinds of disease [1]. Ascorbic acid and K-tocopherol are known to function as
antioxidants to scavenge those toxic free radicals and to
prevent the related diseases. Recently, increasing attention
has been paid to polyphenols as water- and fat-soluble
radical scavengers [2]. Strong reactivity of polyphenols toward O3
2 has been evidenced with electron spin resonance
(ESR) measurements [3]. Haliwell et al. have revealed high
reactivity of catechins against OH with the deoxyribose
assay method [4]. It was also reported that catechins suppress lipid peroxidation induced by water-soluble radical
generators,
2,2P-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [5], azo-bis(2-amidinopropane)hydrochloride
Abbreviations : SOD, superoxide dismutase ; SHE, standard hydrogen
electrode
* Corresponding authors. Fax: +81-75-753-6456.
E-mail address : kkano@kais.kyoto-u.ac.jp (K. Kano).
0304-4165 / 02 / $ ^ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 2 3 0 - 6
36
erty. In spite of increased interest in these biological activities, quantitative and mechanistic aspects of the autoxidation of catechins remain to be elucidated. Although consumption of O2 to be measured by a Clark-type oxygen
electrode is one of the useful methods for continuous monitoring of autoxidation [17], the autoxidation of catechins
has been monitored so far by the absorbance change of
catechins or by the determination of H2 O2 . The interpretation of spectral change is, however, complicated because
of the generation of several oxidized products including
polymerized ones. H2 O2 can be detected by the spectrophotometric or chemiluminescent method using peroxidase. However, these homogeneous enzymatic methods
cannot be applied to continuous monitoring of H2 O2 during the reaction. Furthermore, catechins must be separated
from H2 O2 -containing samples before the measurement
[10,11], since catechins function as electron donors in the
peroxidase reaction and interfere with the H2 O2 measurement. All these situations make it dicult to study the
autoxidation of catechins from kinetic viewpoints.
In the present study, we attempted to monitor the generation of H2 O2 and the consumption of O2 with a peroxidase-based amperometric sensor [18^21] and a Clarktype oxygen electrode, respectively, during the autoxidation of catechins in order to get insight into the mechanism. Four types of catechins as major polyphenol components of green tea were used as model compounds.
Special attention was paid to epigallocatechin gallate,
which is very susceptible to autoxidation. The eects of
superoxide dismutase (SOD), buer capacity, pH, Cu2 ,
and borate on autoxidation kinetics were investigated. The
result shows that O3
2 and the semiquinone intermediate of
catechins play signicant roles in autoxidation. The structural inuence on autoxidation is also discussed. Furthermore, this work reveals that Cu2 accelerates the autoxidation rate, while borate ion can suppress it drastically.
The biological signicance of these eectors is also discussed.
2. Materials and methods
2.1. Materials
(3)-Epicatechin, (3)-epicatechin gallate, (3)-epigallocatechin, and (3)-epigallocatechin gallate (see Scheme 1 for
their structures) were purchased from Sigma. H2 O2 , cuprous chloride (CuCl), cupric chloride (CuCl2 ) and glutaraldehyde were purchased form Wako. Horseradish peroxidase, SOD and bovine serum albumin were purchased
from Toyobo, Wako, and Nacalai Tesque, respectively.
Graphite powder was kindly donated by Nippon Kokuen.
Partially saponied (22 mol%) polyvinyl acetate lm
(UMR-150L, 20 Wm thickness) was kindly donated by
Unitika. All other chemicals were of reagent grade. Water
was puried on Nanopure II (Barnstead). The stock solu-
37
[10,11]. This is the rst report on the continuous monitoring of H2 O2 generation during the autoxidation of catechins, and this method allows the kinetic analysis of the
autoxidation. In separate experiments, the reaction between catechins and H2 O2 was also examined under anaerobic conditions. However, the H2 O2 concentration (20
WM, pH 9.0) did not decrease after the addition of 20 WM
of any catechin used here. This indicates that the scavenging activity of catechins toward H2 O2 , if any, is very weak
and that the direct consumption of H2 O2 by catechins can
be ignored under the experimental time windows and at
the experimental concentrations.
We also measured the concentration prole of O2 with
an oxygen electrode during the autoxidation of catechins
(Fig. 1, curve B for epigallocatechin gallate). As compared
to curve A in Fig. 1, the generation of H2 O2 and the
consumption of O2 are almost identical within the experimental error. A similar behavior was observed for other
catechins. This result indicates that O2 is quantitatively
reduced to H2 O2 in the autoxidation. It is expected that
the ortho-di- or trihydroxyl groups of catechins are oxidized with O2 to generate the corresponding quinones
(two-electron transfer), although the quinones are not so
stable and undergo subsequent polymerization. The concentration of the generated H2 O2 (or consumed O2 ) exceeds the initial concentration of catechins (see curves A
and B in Fig. 1 for epigallocatechin gallate). This means
that two-electron oxidation of catechins is followed by
subsequent oxidation, although the subsequent oxidation
is not so fast compared with the rst two-electron oxidation. This is in accord with electrochemical studies of polyphenols, in which more than two-electron oxidation,
often giving two or more oxidation waves, is reported
[9,25].
3.2. Participation of O3
2 in autoxidation
The rst step of the autoxidation of quinols is the gen-
38
eration of O3
2 [26], which is disproportioned rapidly into
O2 and H2 O2 at least under neutral or slightly acidic conditions [27]. The generation of O3
2 has also been reported
for the autoxidation of catechins [10,11]. O3
2 can work as
a strong oxidant (the redox potential at pH 7 being 0.89 V
vs. the standard hydrogen electrode (SHE) [27] and/or a
weak base (pKa of HO2 = 4.68 [27]). Due to these properties, O3
2 itself can participate in the autoxidation of catechins. In order to clarify this point, the eect of SOD on
the autoxidation rate was examined with epigallocatechin
gallate as the model compound. As shown in Fig. 1, curve
C, the generation of H2 O2 (that is, the rate of autoxidation) was suppressed in part by the addition of SOD.
Similar SOD eects have been reported in the literature
[10,11].
Apo-SOD in the enzyme solution has chelating activity
toward metal ions [28]. However, in this experiment, the
addition of EDTA did not aect the autoxidation rate and
the SOD eect. This means that metal ion contaminants
do not exist in the system and that the chelating activity of
apo-SOD, if any, is not responsible for the suppression
eect. Therefore, it can be considered that the O3
2 intermediate plays an important role in the autoxidation. The
suppression eect increased with SOD concentration, but
it was saturated at SOD over 80 units ml31 . Even under
saturated conditions, the autoxidation was not completely
suppressed by SOD. Therefore, O3
is not essential for
2
autoxidation ; rather it works as a catalyst.
If O3
works as a base to enhance autoxidation, as
2
suggested by Nanni et al. [29], it is expected that the suppression eect by SOD decreases with an increase in buer
capacity, since the conjugate acid component of the buer
enhances the protonation of O3
2 and the subsequent dis3
proportionation of O2 into O2 and H2 O2 [27]. However,
no signicant dierence was observed within the buer
1
The rate of this reaction would be slow due to the low
redox potential of the O2 /O3
2 couple (30.16 V vs. SHE at
pH 7 [27]) and spin restriction between the reactants. The
product O3
2 would also oxidize catechins, as evidenced by
2O3
2 2H ! O2 H2 O2
39
40
fects might be determined by the relative rates of the autoxidation of catechins and active oxygen species
scavenging catalyzed by SOD and catalase. The local concentration of O2 would also be an important factor.
3.5. Enhancement of autoxidation by Cu2+
It has been reported that addition of Cu2 accelerates
the autoxidation of ascorbate [33] and quinols [17,34].
Therefore, we investigated the eects of Cu2 on the autoxidation rate of epigallocatechin gallate with an oxygen
electrode. The result is given in Fig. 4. The reaction
reached a steady state after a short time lag, and the O2
consumption tended to reach a saturated value. Thereafter
slow O2 consumption followed (data not shown). The (initial) steady-state rate of the autoxidation increased with an
increase in the CuCl2 concentration. The CuCl2 concentration dependence showed curved characteristics, as depicted in the inset of Fig. 4.
In order to clarify the mechanism, we compared the
spectral change of the catechin on the addition of CuCl2
under anaerobic conditions with that observed during autoxidation (Fig. 5). The spectral change after the addition
of Cu2 was very similar to that observed on autoxidation.
Therefore, we can conclude that Cu2 works as a oneelectron oxidant in place of O2 .
6
A similar mechanism has been proposed for the reaction
between p-hydroquinones and Cu2 [17]. The generated
semiquinone is quickly oxidized by O2 [26], as described
above (Eq. 4). Therefore, the one-electron oxidation of the
fully reduced state (catechins) by Cu2 enhances the over-
Eq. 8 was monitored with the peroxidase-based H2 O2 sensor. On the addition of an aliquot of a CuCl acetonitrile
solution to the H2 O2 solution (20 WM, at pH 9.0), the
H2 O2 concentration decreased immediately. A rough evaluation of the bimolecular rate constant was approx.
2.8U102 M31 s31 . This result also supports the proposal
of Hayakawa et al. In conclusion, Cu2 accelerates the
autoxidation of catechins and the generated H2 O2 and
Cu are responsible for the generation of OH . The rate
constant of Eq. 8 is larger than that of the Fe2 -derived
Fenton reaction (68 M31 s31 [36]), but in the same order
as that of Eq. 7. Therefore, the catechins and the Cu2 derived generation of OH would become very serious
41
9
The dissociation constant of the complex was evaluated
as 4.5U1038 M2 from the absorbance change at 322 nm
(Fig. 6, ) by assuming 2:1 complex formation (Eq. 9)
using non-liner regression analysis. The calculated values
can well reproduce the experimental data, as shown by the
solid line in Fig. 6.
As expected, the rate of autoxidation was drastically
suppressed in the presence of borate. The initial rate of
autoxidation decreased with an increase in borate concentration, as indicated by the open circles in Fig. 6. The
concentration dependence is closely related with the anaerobic absorbance change (Fig. 6, ) reecting the complex
formation. Almost all epigallocatechin gallate in solution
is complexed with borate at 20 mM of borate, where the
autoxidation is almost completely suppressed. Similar suppression eects were observed for the other catechins used.
Therefore, we can conclude that the borate complex of
catechins has strong resistance against autoxidation.
The eect of borate ion complex formation on the redox
property of catechins was also examined by cyclic voltammetry. As shown in Fig. 7, epigallocatechin gallate gave
complicated irreversible oxidation waves (curve A). In the
presence of borate, the oxidation wave shifted to the direction of the positive potential (curve B). Although thermodynamic analysis could not be performed due to the
irreversible characteristics, this result qualitatively supports the idea that the complex formation of the catechin
42
with borate causes the positive shift of the oxidation potential (that is, the decrease in reduction power) due to the
stabilization of the reduced form. This would be the reason why the autoxidation of the catechin is suppressed in
the presence of borate.
Although the autoxidation of the catechin was completely suppressed in the presence of appropriate amounts
of borate, it was found that the addition of CuCl2 causes
generation of H2 O2 even in the presence of borate (data
not shown). This also supports that Cu2 can work as an
initiator (or catalyst) of autoxidation. However, this may
suggest that Cu2 can coordinate catechins more strongly
than borate before oxidation, although the possibility that
Cu2 can oxidize the catechin^borate complex directly is
not ruled out.
It is considered that borate plays an important role in
plants, for example as a promoter of sugar transport
Fig. 6. The absorbance at 322 nm (b, left axis) and the autoxidation
rate (a, right axis) of epigallocatechin gallate as a function of borate
concentration. The spectroscopic measurements were performed in 0.1
M Tris buer solution (pH 9.0) under anaerobic conditions. The autoxidation rate was measured using an oxygen electrode or a peroxidasebased H2 O2 sensor at 28C. The inset shows the spectra of the catechin
in the presence of (a) 0, (b) 1.0, (c) 2.0, (d) 4.0, (e) 10, (f) 14, (g) 26, (h)
38, and (i) 100 mM borate.
References
[1] B. Halliwell, J.M. Guttenridge, Role of free radicals and catalytic
metal ions in human disease : an overview, Methods Enzymol. 186
(1987) 1^85.
[2] C.A. Rice-Evans, N.J. Miller, G. Paganga, Structure-antioxidant activity relationships of avonoids and phenolic acids, Free Radic. Biol.
Med. 20 (1996) 933^956.
[3] Y. Tsujimoto, H. Hashizume, M. Yamazaki, Superoxide radical scavenging activity of phenolic compounds, Int. J. Biochem. 25 (1993)
491^494.
[4] B. Halliwell, J.M.C. Gutteridge, O.I. Aruoma, The deoxyribose
method : a simple `test-tube' assay for determination of rate constants
for reactions of hydroxyl radicals, Anal. Biochem. 165 (1987) 215^
219.
[5] N. Salah, N.J. Miller, G. Paganga, L. Tijburg, G.P. Bolwell, C. RiceEvans, Polyphenolic avenols as scavengers of aqueous phase radicals and as chain-breaking antioxidants, Arch. Biochem. Biophys.
322 (1995) 339^346.
[6] J. Terao, M. Piskula, Q. Yao, Protective eect of epicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid
bilayers, Arch. Biochem. Biophys. 308 (1994) 278^284.
[7] A.S. Pannala, C.A. Rice-Evans, B. Halliwell, S. Singh, Inhibition of
peroxynitrite-mediated tyrosine nitration by catechin polyphenols,
Biochem. Biophys. Res. Commun. 232 (1997) 164^168.
[8] S.A.B.E. van Acker, D.J. van den Berg, M.N.J.L. Tromp, D.H.
Grioen, W.P. van Bennekom, W.J.F. van der Vijgh, A. Bast, Structural aspects of antioxidant activity of avonoids, Free Radic. Biol.
Med. 20 (1996) 331^342.
43
[9] B. Yang, K. Arai, F. Kusu, Oxidation potentials of avonoids determined by ow-through column electrolysis, Electrochemistry 69
(2001) 519^525.
[10] T. Nakayama, Y. Enoki, K. Hashimoto, Hydrogen peroxide formation during catechin oxidation is inhibited by superoxide dismutase,
Food Sci. Technol. Int. 1 (1995) 65^69.
[11] Y.H. Miura, I. Tomita, T. Watanabe, T. Hirayama, S. Fukui, Active
oxygen generation by avonoids, Biol. Pharm. Bull. 21 (1998) 93^96.
[12] F. Hayakawa, T. Kimura, T. Maeda, M. Fujita, H. Sohmiya, M.
Fujii, T. Ando, DNA cleavage reaction and linoleic acid peroxidation
induced by tea catechins in the presence of cupric ion, Biochim. Biophys. Acta 1336 (1997) 123^131.
[13] F. Hayakawa, T. Kimura, N. Hoshino, T. Ando, DNA cleavage
activities of (3)-epigallocatechin, (3)-epicatechin, (+)-catechin, and
(3)-epigallocatechin gallate with various kind of metal ions, Biosci.
Biotechnol. Biochem. 63 (1999) 1654^1656.
[14] I. Cakmak, V. Romheld, Boron deciency-induced impairments of
cellular functions in plants, Plant Soil 193 (1997) 71^83.
[15] T.L. Mason, B.P. Wasserman, Inactivation of red beet L-glucan synthase by native and oxidized phenolic compounds, Phytochemistry 26
(1987) 2197^2202.
[16] J.A. Rajaratnam, J.B. Lowry, P.N. Avadhani, R.H.V. Corley, Boron: possible role in plant metabolism, Science 172 (1971) 1142^1143.
[17] Y. Li, M.A. Trush, Oxidation of hydroquinone by copper: chemical
mechanism and biological eects, Arch. Biochem. Biophys. 300
(1993) 346^355.
[18] H. Kinoshita, M. Torimura, K. Kano, T. Ikeda, Peroxidase-based
amperometric sensor of hydrogen peroxide generated in oxidase reaction: application to creatinine and creatine assay, Electroanalysis 9
(1997) 1234^1238.
[19] H. Kinoshita, M. Torimura, K. Kano, T. Ikeda, Amperometric determination of high-density lipoprotein cholesterol using polyethylene
glycol-modied enzymes and a peroxidase-entrapped electrode, Ann.
Clin. Biochem. 35 (1998) 739^744.
[20] T. Ikeda, T. Shiraichi, M. Senda, An ecient method for entrapping
ionic mediators in the enzyme layer of mediated amperometric biosensors, Agric. Biol. Chem. 52 (1988) 3187^3188.
[21] L. Yang, E. Janle, T. Huang, J. Gitzen, P.T. Kissinger, M. Vreeke, A.
Heller, Applications of `wired' peroxidase electrodes for peroxide
determination in liquid chromatography coupled to oxidase immobilized enzyme reactors, Anal. Chem. 67 (1995) 1326^1331.
[22] A.J. Parker, Hydrometallurgy of copper and silver in solvent mixtures, Search 4 (1973) 426^432.
[23] K. Yamamoto, T. Ohgaru, M. Torimura, H. Kinoshita, K. Kano, T.
Ikeda, Highly-sensitive ow injection determination of hydrogen peroxide with a peroxidase-immobilized electrode and its application to
clinical chemistry, Anal. Chim. Acta 406 (2000) 201^207.
[24] K. Kano, T. Ikeda, Fundamentals and practices of mediated bioelectrocatalysis, Anal. Sci. 16 (2000) 1013^1021.
[25] H. Hotta, H. Sakamoto, S. Nagano, T. Osakai, Y. Tsujino, Unusually large numbers of electrons for the oxidation of polyphenolic
antioxidants, Biochim. Biophys. Acta 1526 (2001) 159^167.
[26] H. Tatsumi, H. Nakase, K. Kano, T. Ikeda, Mechanistic study of the
autoxidation of reduced avin and quinone compounds, J. Electroanal. Chem. 443 (1998) 236^242.
[27] D.T. Sawyer, J.S. Valentine, How super is superoxide ?, Acc. Chem.
Res. 14 (1981) 393^400.
[28] T.D. Rae, A.S. Torres, R.A. Pufahl, T.V. O'Halloran, Mechanism of
Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS, J. Biol. Chem. 276 (2001) 5166^5176.
[29] E.J. Nanni Jr., M.D. Stallings, D.T. Sawyer, Does superoxide ion
oxidize catechol, K-tocopherol, and ascorbic acid by direct electron
transfer?, J. Am. Chem. Soc. 102 (1980) 4481^4485.
[30] J.M. Ricardo da Silva, N. Darmon, Y. Fernandez, S. Mitjavila, Oxygen free radical scavenger capacity in aqueous models of dierent
procyanidins from grape seeds, J. Agric. Food Chem. 39 (1991)
1549^1552.
44
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
Simultaneous determination of catechins in human saliva by highperformance liquid chromatography, J. Chromatogr. B Biomed.
Sci. Appl. 703 (1997) 253^258.
E. Shoji, M.S. Freund, Potentiometric sensors based on the inductive
eect on the pKa of poly(aniline): a nonenzymatic glucose sensor,
J. Am. Chem. Soc. 123 (2001) 3383^3384.
W.M. Dugger, Boron in plant metabolism, in: Encyclopedia of Plant
Physiology, New Series, Vol. 15, Springer-Verlag, Heidelberg 1983,
pp. 626^650.
K.W. Lee, C.M. Whittle, H.J. Dyer, Boron deciency and translocation proles in sunower, Physiol. Plant. 19 (1966) 919^924.
T. Matoh, Boron in plant cell walls, Plant Soil 193 (1997) 59^69.
M.A. O'Neill, D. Warrenfeltz, K. Kates, P. Pellerin, T. Doco, A.G.
Darvill, P. Albersheim, Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cells, forms a dimer that is covalently cross-linked by a borate ester, J. Biol. Chem. 271 (1996) 22923^
22930.
S. Lee, Boron in plants: a biochemical role, Science 158 (1967) 798^
799.
D.H. Lewis, Boron, lignication and the origin of vascular plants ^ a
unied hypothesis, New Phytol. 84 (1980) 209^229.
M. Uchida, M. Ono, Determination of hydrogen peroxide in beer
and its role in beer oxidation, J. Am. Soc. Brew. Chem. 57 (1999)
145^150.