Oxidation mechanism of black tea pigment theaflavin by peroxidase

Article abstract of DOI:10.1016/j.tetlet.2015.07.037

A large number of black tea polyphenols remain uncharacterized because of the complexity of catechin oxidation reactions that occur during tea fermentation. In the course of our studies on black tea polyphenols, we examined the enzymatic degradation of theaflavins, which are black tea pigments having a benzotropolone chromophore. Oxidation of theaflavin with peroxidase afforded a new product named theacoumarin A together with known pigment theanaphthoquinone. The structure of the new compound was determined by spectroscopic examination and a production mechanism via theanaphthoquinone is proposed.

Full text of DOI:10.1016/j.tetlet.2015.07.037

Tetrahedron Letters 56 (2015) 5099–5102
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Tetrahedron Letters
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Oxidation mechanism of black tea pigment theaflavin by peroxidase
⇑
⇑
Rie Kusano, Yosuke Matsuo , Yoshinori Saito, Takashi Tanaka
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A large number of black tea polyphenols remain uncharacterized because of the complexity of catechin
oxidation reactions that occur during tea fermentation. In the course of our studies on black tea
polyphenols, we examined the enzymatic degradation of theaflavins, which are black tea pigments
having a benzotropolone chromophore. Oxidation of theaflavin with peroxidase afforded a new product
named theacoumarin A together with known pigment theanaphthoquinone. The structure of the
new compound was determined by spectroscopic examination and a production mechanism via
theanaphthoquinone is proposed.
Received 29 May 2015
Revised 6 July 2015
Accepted 13 July 2015
Available online 17 July 2015
Keywords:
Black tea
Peroxidase
Ó 2015 Elsevier Ltd. All rights reserved.
Theaflavin
Theanaphthoquinone
Theacoumarin A
Plant polyphenols have been demonstrated to show a wide
range of biological activities,1 and black tea, one of the most pop-
ular beverages worldwide, is an important source of polyphenols
for humans. Black tea is produced by crushing and kneading the
fresh leaves of Camellia sinensis, which contains epicatechin (1),
epigallocatechin (2), and their galloyl esters as major polyphenols.
During processing, the tea catechins are oxidized by reaction with
oxygen by catalysis with endogenous enzymes, polyphenol oxidase
and peroxidase,2 to afford various oxidation products.3 The most
important products are theaflavins, mainly including theaflavin
(3), theaflavin-3-O-gallate (4), theaflavin-30-O-gallate (5), and
theaflavin-3,30-di-O-gallate (6), which are reddish-yellow
pigments with the benzotropolone chromophore (Fig. 1). The pig-
ments are produced by oxidative coupling between pyrogallol-
type and catechol-type catechins.4 Theaflavins contribute largely
to the quality, taste, and color of black tea, and are shown to have
various biological activities, such as radical scavenging,5
to afford 7;13 however, its degradation reaction has not been exam-
ined in detail.2a,14 In this study, we examined the oxidation
reaction of 3 with peroxidase.
First, we examined the time course of oxidation of a mixture of
epicatechin (1) and epigallocatechin (2) in the presence of
horseradish peroxidase (Fig. 2A).15–17 After 10 min, theaflavin (3)
was observed as the major product. Then, theanaphthoquinone
(7) appeared, along with the disappearance of 3 (t = 30 min).
Subsequently, a new product (8) gradually increased, which was
accompanied by a decrease of 7 (t = 60, 120 min). Therefore, com-
pound 8 was presumed to be an oxidation product of 7. We also
investigated the time course of oxidation of 3 (Fig. 2B); the results
supported the production of 8 from 3 via 7. To elucidate the struc-
ture of 8, we performed the oxidation reaction on a large scale.18
Catechins 1 (1.0 g) and 2 (1.0 g) were dissolved in phosphate buffer
at pH 5.0 and stirred with horseradish peroxidase and H2O2 for 3 h.
Separation of the reaction mixture by Sephadex LH-20 and MCI-gel
CHP20P column chromatography afforded 8 (25.3 mg).
a
-glucosidase inhibition,6 lipase inhibition,7 anti-inflammatory
activity,8 and prevention of mouse type IV allergy.9 However, thea-
flavins are degraded enzymatically in the process of black tea
production, and their degradation is considered to be related to
production of uncharacterized black tea polyphenols.2a,10
Previously, we revealed that theaflavin (3) is oxidized by
polyphenol oxidase in the presence of epicatechin (1) to give
theanaphthoquinone (7) as a major product,11 along with several
minor products.12 Degradation of 3 is also mediated by peroxidase
Compound 819 showed an [M+H]+ peak of m/z 523 by FABMS.
13C NMR and elemental analysis revealed the molecular formula
of 8 to be C27H22O11. Two sets of signals arising from the A-ring
and C-ring of the flavan-3-ol skeleton were observed in the 1H
and 13C NMR spectra, and their signals were assigned by 1H–1H
COSY, HSQC, and HMBC spectra (Table 1). The remaining 11 carbon
signals in the 13C NMR were attributed to the moiety derived from
catechin B-rings. In the HMBC spectrum (Fig. 3), correlations from
C0-ring H-20 (dH 5.08) to C-500 (dC 113.2), C-600 (dC 136.8), C-700 (dC
118.07 or dC 118.10), and from H-30 (dH 4.28) to C-600 were
observed. These correlations indicated the connectivity of
C-500–C-600–C-700, and the connection between C-20 and C-600. In
⇑
Corresponding authors. Tel.: +81 95 819 2434 (Y.M.), +81 95 819 2432 (T.T.).
E-mail addresses: y-matsuo@nagasaki-u.ac.jp (Y. Matsuo), t-tanaka@nagasaki-u.
ac.jp (T. Tanaka).
http://dx.doi.org/10.1016/j.tetlet.2015.07.037
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

5100
R. Kusano et al. / Tetrahedron Letters 56 (2015) 5099–5102
OH
OH
4a'
OH
5'
OR1
OH
OH
OH
6'
4'
C'
O
R
B
3'
2'
A'
7''
OH
8''
8a'O'
OH
OH
OH
6''
O
8a'
HO
O
HO 7'
HO
O
8'
OH
OH
HO
O
C
O
5''
4a''
4''
A
8
O
HO
HO
8a
4a
O
HO
O
2''
OH
O
O
2
3
7
6
3''
C
A
5
OH
4
OR2
OH
OH
OH
1
OH
OH
2
1
2
7
8
: R = H
3
: R = R = H
O
: R = OH
1
2
4
: R = G, R = H
OH
OH
G =
5: R1 = H, R2 = G
6: R1 = R2 = G
OH
Figure 1. Structures of 1–8.
Table 1
t = 120 min
t = 60 min
7
A
1H (500 MHz) and 13C (125 MHz) NMR data for 8 (in acetone-d6 + D2O, d in ppm, J in
8
8
4000000
1
1
Hz)
Position
dH
dC
75.0
64.1
28.7
HMBC (H to C)
7
7
2
3
4
5.35 (br s)
4.35 (m)
2.79 (br d, 16.9)
2.93 (dd, 4.4, 16.9)
4, 300, 400, 4a00
2, 4a
3000000
2000000
2, 3, 4a, 5, 8 (4J), 8a
t = 30 min
t = 10 min
99.40a
157.51b
96.3
1
1
4a
5
6
7
8
8a
20
30
40
3
6.05 (d, 2.3)
5.99 (d, 2.3)
4a, 5, 7, 8
157.47b
95.4
4a, 6, 7, 8a
7
1000000
155.8
78.5
66.5
2
1
5.08 (br s)
4.28 (m)
2.84 (dd, 4.8, 16.5)
2.59 (dd, 4.5, 16.5)
40, 8a0, 500, 600, 700
4a0, 600
t = 0 min
28.2
20, 30, 4a0, 50, 8a0
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
4a0
50
99.36a
157.42b
96.6
Retention Time [min]
60
6.04 (d, 2.3)
5.94 (d, 2.3)
4a0, 50, 70, 80
4a0, 60, 70, 8a0
7
t = 120 min
B
8
70
157.39b
95.2
80
8a0
200
300
400
4a00
500
600
700
800
8a00
156.2
161.0
114.3
153.5
118.10c
113.2
136.8
118.07c
145.3
142.4
7
7
t = 60 min
6.68 (br s)
2, 200, 400, 4a00
8
8
2000000
7.33 (d, 1.5)
7.40 (d, 1.5)
20, 400, 4a00, 600, 700, 800 (4J), 8a00
20, 4a00 (4J), 500, 600, 800, 8a00
t = 30 min
t = 10 min
t = 0 min
7
1000000
3
a–c
Assignments may be interchanged.
3
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
Retention Time [min]
OH
Figure 2. (A) HPLC-DAD chromatogram (max absorbance) of the reaction mixture
of epicatechin (1) and epigallocatechin (2) by peroxidase. (3: theaflavin; 7:
theanaphthoquinone; 8: theacoumarin A) (B) HPLC chromatogram of the reaction
mixture of theaflavin (3) by peroxidase.
OH
3'
2'
C'
7''
OH
HO
O
6''
4a''
8''
8a''
addition, HMBC correlations from H-500 to C-4a00 (dC 118.10 or dC
118.07), C-600, C-700, and C-8a00 (dC 142.4), and correlations from
H-700 to C-500, C-600, C-800 (dC 145.3), and C-8a00 revealed that the
six carbons (C-4a00, 500, 600, 700, 800, 8a00) formed a benzene ring, and
C-800 and C-8a00 were oxygenated based on their 13C NMR chemical
shifts. Another C-ring H-2 (dH 5.35) was correlated with C-300 (dC
114.3), C-400 (dC 153.5), and C-4a00 in the HMBC spectrum.
Furthermore, the correlations from H-300 (dH 6.68) to C-200 (dC
161.0), C-400, and C-4a00 revealed the connectivity of C-200–C-300–C-
5''
2
O
2''
4''
HO
O
O
3''
C
OH
OH
8
400–C-4a00. This indicated that the
a,b-conjugated carbonyl group
Figure 3. Important HMBC correlations of 8.

R. Kusano et al. / Tetrahedron Letters 56 (2015) 5099–5102
5101
OH
OH
OG
H+
OH
OH
H
O
O
OH
OH
O
HO
O
H2O
OH
OH
HO
O
O
oxidation
oxidation
− CO2
H+
O
O
OH
OH
OH
OH
O
HO
O
HO
O
O
OH
O
OH
OH
OH
OH
7
3
OH
OH
OH
OH
OH
H2O2
OH
O
OH
OH
O
OH
− H2O
− CO2
HO
O
OH
O
+ H2O
− H2O
O
O
O
O
OH
H
H+
H
OH
O
HO
O
O
O
HO
O
O
O
O
O
OH
H+
OH
H2O2
OH
8
Scheme 1. Plausible production mechanism of 8 from 3.
C-200–C-400 is connected to C-2. The IR spectrum also supported the
presence of a conjugated carbonyl group (1695 cmꢀ1). Taking the
molecular formula into account, connection between C-200 and
C-8a00 through an ester bond was deduced; thus, 11 carbons
derived from two B-rings form a coumarin skeleton. Based on these
References and notes
1. For reviews of plant polyphenols, see (a) Crozier, A.; Jaganath, I. B.; Clifford, M.
N. Nat. Prod. Rep. 2009, 26, 1001–1043; (b) Quideau, S.; Deffieux, D.; Douat-
Casassus, C.; Pouysegu, L. Angew. Chem., Int. Ed. 2011, 50, 586–621; (c)
Cheynier, V. Phytochem. Rev. 2012, 11, 153–177.
2. (a) Subramanian, N.; Venkatesh, P.; Ganguli, S.; Sinkar, V. P. J. Agric. Food Chem.
1999, 47, 2571–2578; (b) Finger, A. J. Sci. Food Agric. 1994, 66, 293–305; (c)
Mahanta, P. K.; Boruah, S. K.; Boruah, H. K.; Kalita, J. N. J. Agric. Food Chem. 1993,
41, 272–276; (d) Dix, M. A.; Fairley, C. J.; Millin, D. J.; Swaine, D. J. Sci. Food Agric.
1981, 32, 920–932.
3. For reviews of the chemistry of black tea polyphenols, see (a) Haslam, E.
Phytochemistry 2003, 64, 61–73; (b) Drynan, J. W.; Clifford, M. N.; Obuchowicz,
J.; Kuhnert, N. Nat. Prod. Rep. 2010, 27, 417–462; (c) Li, S.; Lo, C.-Y.; Pan, M.-H.;
Lai, C.-S.; Ho, C.-T. Food Funct. 2013, 4, 10–18; (d) Tanaka, T.; Matsuo, Y.; Kouno,
I. Int. J. Mol. Sci. 2010, 11, 14–40.
4. (a) Takino, Y.; Imagawa, H.; Horikawa, H.; Tanaka, A. Agric. Biol. Chem. 1964, 28,
64–71; (b) Yanase, E.; Sawaki, K.; Nakatsuka, S. Synlett 2005, 2661–2663.
5. Yang, Z.; Jie, G.; Dong, F.; Xu, Y.; Watanabe, N.; Tu, Y. Toxicol. In Vitro 2008, 22,
1250–1256.
results, the structure of
8 was determined as shown in
Figure 1, and 8 was named as theacoumarin A.
A plausible mechanism for the production of 8 is shown in
Scheme 1. After oxidation of the benzotropolone ring of 3, a
benzylic acid-type rearrangement, decarboxylation, and oxidation
afford 7.11 Subsequent Baeyer–Villiger oxidation, which includes
the addition of H2O2 to the dicarbonyl moiety and dehydration
via rearrangement, affords a lactone intermediate. Finally, ring
opening of the lactone by hydration, addition of H2O2 with decar-
boxylation, followed by formation of the lactone ring yield 8.
During black tea production, H2O2 is produced by reduction of O2
in the course of enzymatic oxidation of catechins.20
6. Matsui, T.; Tanaka, T.; Tamura, S.; Toshima, A.; Tamaya, K.; Miyata, Y.; Tanaka,
K.; Matsumoto, K. J. Agric. Food Chem. 2007, 55, 99–105.
In summary, theacoumarin A (8) was identified as a major
product of the oxidation of theaflavin (3) by peroxidase, and its
structure was determined on the basis of spectroscopic data. Our
previous study showed that theanaphthoquinone (7) is produced
from 3 by polyphenol oxidase in the presence of epicatechin;
however, the oxidation reaction of 7 has not been observed.12,21
This study revealed that 7 is oxidized by peroxidase to afford 8.
The production of 8 from 3 via 7 by peroxidase is assumed to occur
during the process of black tea production. The oxidation of theafla-
vin (3) and its galloyl esters (4–6) by peroxidase, including the
production of 8 and related compounds, is expected to contribute
to the generation of uncharacterized black tea polyphenols. In
addition, degradation of theaflavins is considered to affect the
quality of black tea.
7. Kusano, R.; Andou, H.; Fujieda, M.; Tanaka, T.; Matsuo, Y.; Kouno, I. Chem.
Pharm. Bull. 2008, 56, 266–272.
8. Aneja, R.; Odoms, K.; Denenberg, A. G.; Wong, H. R. Crit. Care Med. 2004, 32,
2097–2103.
9. Yoshino, K.; Yamazaki, K.; Sano, M. J. Sci. Food Agric. 2010, 90, 1983–1987.
10. Davies, A. P.; Goodsall, C.; Cai, Y.; Davis, A. L.; Lewis, J. R.; Wilkins, J.; Wan, X.;
Clifford, M. N.; Powell, C.; Parry, A.; Thiru, A.; Safford, R.; Nursten, H. E. In Plant
Polyphenols 2: Chemistry, Biology, Pharmacology, Ecology; Gross, G. G.,
Hemingway, R. W., Yoshida, T., Branham, S. J., Eds.; Basic Life Sciences, Vol.
66; Kluwer Academic/Plenum: New York, 1999; pp 697–724.
11. Tanaka, T.; Betsumiya, Y.; Mine, C.; Kouno, I. Chem. Commun. 2000,
1365–1366.
12. (a) Tanaka, T.; Inoue, K.; Betsumiya, Y.; Mine, C.; Kouno, I. J. Agric. Food Chem.
2001, 49, 5785–5789; (b) Li, Y.; Shibahara, A.; Matsuo, Y.; Tanaka, T.; Kouno, I. J.
Nat. Prod. 2010, 73, 33–39.
13. Jhoo, J.-W.; Lo, C.-Y.; Li, S.; Sang, S.; Ang, C. Y. W.; Heinze, T. M.; Ho, C.-T. J. Agric.
Food Chem. 2005, 53, 6146–6150.
14. Sang, S.; Yang, C. S.; Ho, C.-T. Phytochem. Rev. 2004, 3, 229–241.
15. Compounds 1 (2.0 mg) and 2 (2.0 mg) were dissolved in 0.2 M phosphate
buffer at pH 5.0 (2.0 mL), then 100
peroxidase (1.0 mg/mL) (Type II, 150–250 units/mg; Sigma–Aldrich)17 and
10 L of 5% H2O2 were added and stirred. For the first 60 min, 20 L of 5% H2O2
was added with every 10 min. EtOH containing 1% trifluoroacetic acid (100 L)
L) and the resulting mixture was
lL of a buffer solution of horseradish
Acknowledgments
l
l
l
This work was supported in part by JSPS KAKENHI Grant Nos.
25870532 and 26460125. The authors are grateful to Mr. K.
Inada, Mr. N. Yamaguchi, and Mr. N. Tsuda (Center for Industry,
University and Government Cooperation, Nagasaki University) for
measurements of NMR, MS, and elemental analysis.
was poured into the reaction mixture (100
l
filtered through a membrane filter (0.45
lm). The filtered solution (5 lL) was
analyzed by analytical HPLC.16
16. Analytical HPLC was performed on
a
Cosmosil 5C18-ARII column
(250 ꢁ 4.6 mm i.d.; Nacalai Tesque, Kyoto, Japan) with gradient elution from
4% to 30% (39 min) and from 30% to 75% (15 min) of CH3CN in 50 mM H3PO4
(column temperature: 35 °C; flow rate: 0.8 mL/min).
17. In this study, horseradish peroxidase was used instead of tea peroxidase
because the former is commercially available (Sigma–Aldrich). In addition, a
previous study showed that horseradish peroxidase can catalyze the
production of theaflavins from tea catechins,14 which indicated that the
catechin oxidizing capacity of horseradish peroxidase is similar to that of tea
peroxidase.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.07.
037.

5102
R. Kusano et al. / Tetrahedron Letters 56 (2015) 5099–5102
27
18. Compounds 1 (1.0 g) and 2 (1.0 g) were dissolved in 0.2 M phosphate buffer at
pH 5.0 (75 mL), then horseradish peroxidase (5.0 mg) and 30% H2O2 (2 mL)
were added and stirred. For the first 40 min, 2 mL of 30% H2O2 was added with
every 10 min. After 3 h, reaction solution was directly applied to Sephadex LH-
20 (3 ꢁ 28 cm, H2O–MeOH–50% aq acetone) to afford 10 fractions. HPLC
19. Theacoumarin A (8): A brown amorphous powder; [
a
]
D
ꢀ211.4 (c 0.1, MeOH);
FABMS m/z: 523 [M+H]+; Anal. Calcd for C27H22O11ꢂ1.5H2O: C, 59.02, H, 4.59.
Found: C, 58.95, H, 4.61; IR m
max (dry film) cmꢀ1: 3382, 2932, 1695, 1630, 1612,
1591, 1518, 1470; UV kmax (MeOH) nm (log e): 293 (4.07), 256 (4.20).
20. Jiang, Y.; Miles, P. W. Phytochemistry 1993, 33, 29–34.
21. Tanaka, T.; Mine, C.; Inoue, K.; Matsuda, M.; Kouno, I. J. Agric. Food Chem. 2002,
50, 2142–2148.
analysis of each fraction indicated that
Purification of fraction
MeOH) and Sephadex LH-20 (1.5 ꢁ 10 cm, EtOH) afforded 8 (25.3 mg).
8
was contained in fraction 8.16
8
using MCI-gel CHP20P (2 ꢁ 23 cm, 30–100% aq