Enzymatic oxidation of gallocatechin and epigallocatechin: Effects of C-ring configuration on the reaction products

Article abstract of DOI:10.1016/j.phytochem.2007.08.007

Tea leaf is rich in pyrogallol-type catechins, and their oxidation is important in the generation of black tea polyphenols. In the present study, the enzymatic oxidation of three pyrogallol-type catechins, (+)- and (-)-gallocatechins and (-)-epigallocatechin, was compared. The reactions yielded unstable quinone products, which were trapped as condensation products with o-phenylenediamine. The oxidation of (+)-gallocatechin proceeded very slowly compared to the reaction of (-)-epigallocatechin, and yielded a proepitheaflagallin-type dimer as the major product, though oxidation of (-)-epigallocatechin gave predominantly dehydrotheasinensin C. The cis-configuration of the C-3 hydroxyl group and the B-ring of (-)-epigallocatechin was apparently crucial for rapid and selective production of dehydrotheasinensin C. Oxidation of (-)-gallocatechin proceeded in a manner similar to that of (+)-gallocatechin, and produced an enantiomer of the (+)-gallocatechin product. The results suggest that enzymes catalyze oxidation of the pyrogallol B-ring to the o-quinone, with subsequent non-enzymatic coupling reactions proceed under highly steric control.

Full text of DOI:10.1016/j.phytochem.2007.08.007

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 3054–3061
www.elsevier.com/locate/phytochem
Enzymatic oxidation of gallocatechin and epigallocatechin: Effects of
C-ring configuration on the reaction products
*
Yosuke Matsuo, Yuko Yamada, Takashi Tanaka , Isao Kouno
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Received 5 April 2007; received in revised form 20 July 2007
Available online 20 September 2007
Dedicated to the memory of Professor Yoshihiro Matsumura
Abstract
Tea leaf is rich in pyrogallol-type catechins, and their oxidation is important in the generation of black tea polyphenols. In the present
study, the enzymatic oxidation of three pyrogallol-type catechins, (+)- and (ꢀ)-gallocatechins and (ꢀ)-epigallocatechin, was compared.
The reactions yielded unstable quinone products, which were trapped as condensation products with o-phenylenediamine. The oxidation
of (+)-gallocatechin proceeded very slowly compared to the reaction of (ꢀ)-epigallocatechin, and yielded a proepitheaflagallin-type
dimer as the major product, though oxidation of (ꢀ)-epigallocatechin gave predominantly dehydrotheasinensin C. The cis-configuration
of the C-3 hydroxyl group and the B-ring of (ꢀ)-epigallocatechin was apparently crucial for rapid and selective production of dehyd-
rotheasinensin C. Oxidation of (ꢀ)-gallocatechin proceeded in a manner similar to that of (+)-gallocatechin, and produced an enantio-
mer of the (+)-gallocatechin product. The results suggest that enzymes catalyze oxidation of the pyrogallol B-ring to the o-quinone, with
subsequent non-enzymatic coupling reactions proceed under highly steric control.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Black tea; Camellia sinensis; Theaceae; Epigallocatechin; Gallocatechin; Oxidation; Polyphenol
1. Introduction
production, tea catechins are enzymatically oxidized to
produce oxidation products characteristic of black tea
(Hashimoto et al., 1992; Haslam, 2003). There are two
major oxidation routes of 1. One is condensation with
coexisting epicatechin and its gallate, yielding theaflavins,
pigments with a benzotropolone moiety (Takino et al.,
1964, 1965). The other is production of some epigallocate-
chin dimers, such as theasinensins and oolongtheanins
(Nonaka et al., 1983; Hashimoto et al., 1988). Our previous
work has indicated that a highly stereoselective reaction of
epigallocatechin quinone (1a) produces unstable quinone
metabolites, dehydrotheasinensin C (2) and proepitheafla-
gallin (3), which are subsequently degraded to give theasi-
nensins A and C, oolongtheanin and epitheaflagallin
(Tanaka et al., 2002c, 2003; Matsuo et al., 2006). The ste-
reoselectivity of the oxidative dimerization is presumed to
be controlled by the configuration at the C-2 carbon, and
the geometrical configuration between the C-2 and C-3 of
Flavan-3-ols and proanthocyanidins are widely distrib-
uted in the plant kingdom (Porter, 1998), and are thought
to be defense substances (Feucht and Treutter, 1999).
These polyphenols are oxidized by coexisting enzymes
when the plants are damaged by predators. This chemistry
is also important in food production, especially in the man-
ufacture of black tea. However, the chemical details of the
reactions and structures of the reaction products are not
yet clearly understood. (ꢀ)-Epigallocatechin (1) and its gal-
loyl ester are the major polyphenols in fresh leaves of the
tea tree Camellia sinensis, and account for 50–76% of the
total tea catechins (Saijo and Takeda, 1999). In black tea
*
Corresponding author. Tel.: +81 95 819 2433; fax: +81 95 819 2477.
E-mail address: t-tanaka@nagasaki-u.ac.jp (T. Tanaka).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.08.007

Y. Matsuo et al. / Phytochemistry 69 (2008) 3054–3061
3055
flavan-3-ols, because the same selectivity was also observed
in a non-enzymatic oxidation with potassium hexacyano-
ferrate(III) as an oxidant (Tanaka et al., 2003). In the
present study, we compared the oxidation of (+)- (4) and
(ꢀ)-gallocatechins (7) with that of (ꢀ)-epigallocatechin
(1), and describe the reason for the stereoselective forma-
tion of dehydrotheasinensins and proepitheaflagallin. In
addition, this study may contribute to the understanding
of the production of minor black tea polyphenols, because
a small amount (<2% of total catechins) of 4 is contained in
fresh tea leaves (Saijo and Takeda, 1999).
ucts similar to those obtained by the reaction with tea leaf
homogenate (Tanaka et al., 2002a,b). Furthermore, on
HPLC analysis, it did not show interfering peaks in the
background arising from the homogenate itself. The initial
oxidation products of 1 were found to be unstable quinones
or their dimers, which were difficult to isolate in their ori-
ginal forms (Tanaka et al., 2002c, 2003; Matsuo et al.,
2006). Therefore, in this experiment, an ethanolic solution
of o-phenylenediamine was added to the reaction mixtures
to trap the unstable quinones as phenazine or quinoxaline
derivatives. The reaction products were analyzed by
reversed-phase HPLC equipped with a photodiode array
detector. When 1 was treated with the pear homogenate,
it was rapidly converted into dehydrotheasinensin C (2),
which was detected as the phenazine derivatives 2a and
2b (Tanaka et al., 2002c; Matsuo et al., 2006) (Fig. 1a).
The phenazine derivative 1b of monomeric epigallocatechin
quinone (1a) was also observed (Matsuo et al., 2006). The
2. Results and discussion
In the course of our study on catechin oxidation, Japa-
nese pear homogenate was used as an enzyme source
because it showed strong activity and gave oxidation prod-
35
30
25
20
15
10
5
35
30
25
20
15
10
5
0
0
0
20
40
60
80
100
120
0
20
40
60
80
100
120
min
min
1
1b
2a+2b
3a
4
4b
5a
6a
Fig. 1. Time course of oxidation of 1 (a) and 4 (b) and production of phenazine derivatives.
OH
OH
OH
OH
OH
major
HO
O
2
HO
O
OH
R
H
OH
3
OH
HO
O
O
OH
R
HO
OH
OH
OH
O
OH
1
-H2
2
-H2
OH
OH
OH
O
OH
O
OH
-CO2
O
OH
O
HO
HO
O
O
HO
HO
O
HO
O
O
O
OH
HOOC
minor
O
H
OH
OH
OH
H
OH
O
O
O
1a
OH
OH
OH
3b
3
Scheme 1.

3056
Y. Matsuo et al. / Phytochemistry 69 (2008) 3054–3061
peaks of these products were identified by comparison of
the retention time and UV absorption with those of
authentic samples. Although dimer 2 was produced from
1 and 1a (Scheme 1) (Li et al., 2007), the peak area of 1b
was much smaller than that of 2a and 2b, indicating that
dimerization proceeded rapidly. Another small peak with
UV absorption at 382 nm was identified as the bisquinoxa-
line derivative 3a, based on the retention time and compar-
ison of the UV spectrum (Matsuo et al., 2006). The
derivative 3a was originally obtained as the major product
on treatment of proepitheaflagallin (3) with o-phenylenedi-
amine. From these results, it was concluded that the oxida-
tion of 1 predominantly yielded 2, and 3 was a minor
product (Scheme 1). The oxidation of 1 with potassium
hexacyanoferrate(III), and subsequent treatment with o-
1b. The product 5a exhibited an [M+H]+ peak at m/z 681
in the FAB-MS spectrum, which coincided with that of
1
2a. The H NMR spectrum showed signals arising from
two sets of flavan A C-rings along with aromatic reso-
nances attributable to a tri-substituted phenazine ring (d
8.12–8.20, 2H, m; 7.87–7.89, 2H, m; 7.85, 1H, s) and a
penta-substituted pyrogallol ring (d 6.69). These signals
were also related to those of 2a, except for the large J2,3 val-
ues (7.8 and 5.9 Hz) of the C-rings. The 13C NMR spec-
trum also resembled that of 2a, with the difference in
chemical shift of C-2 and C-3 indicating that the product
5a was a 2,3-trans isomer of 2a. The atropisomerism of
the biphenyl bond was deduced to be in the R configura-
tion by comparison of the CD spectrum with that of 2a:
both compounds showed positive Cotton effects at
OH
Table 1
HO
1H and 13C NMR spectroscopic data for bisquinoxaline derivative 6aa
OH
HO
HO
N
N
Position
dC
87.6
dH
HMBC (H–C)
O
OH
H
OH
HO
O
2
3
4
4.21 (d, 8.5)
3, 4, c, d, e
2, d
N
64.4
29.0
4.09 (ddd, 6.0, 8.5, 9.6)
3.01 (dd, 6.0, 15.6)
2.41 (dd, 9.6, 15.8)
OH
H
OH
2, 3, 4a, 5, 8a
2, 3, 4a, 5, 8a
HO
O
N
OH
4a
5
6
7
8
8a
20
30
40
100.5
157.0b
95.7c
157.2b
96.3c
156.2b
81.3
OH
OH
OH
5.97b (d, 2.3)
5.98b (d, 2.3)
4a, 5, 7, 8
1b
2a
4a, 6, 7, 8a
OH
4.27 (d, 9.8)
3.51 (ddd, 6.0, 9.8, 10.3)
2.83 (dd, 6.0, 15.3)
30, 40, e, f,j, k
20, 40, k
HO
OH
68.6
28.1
20, 30, 4a0, 50,
8a0
20, 30, 4a0, 50,
8a0
HO
HO
O
O
OH
N
H
HO
HO
O
O
2.58 (dd, 10.3, 15.3)
N
N
OH
OH
N
4a0
50
60
70
80
8a0
a
99.9
157.8b
95.7c
O
H
H
HO
N
OH
6.00b (d, 2.3)
6.06b (d, 2.3)
4a0, 50, 70, 80
4a0, 60, 70, 8a0
N
O
157.8b
96.8c
OH
OH
OH
156.2b
158.0
153.4d
34.5
2b
3a
b
c
4.63 (dd, 2.6, 15.6)
3.70 (d, 15.6)
a, d, e
2, a, b, d, e
phenylenediamine, also afforded 2a and 2b along with
small amounts of 1a and 3a, indicating the stereoselectivity
observed was non-enzymatic and controlled by configura-
tion of the flavan-3-ol.
d
e
f
131.6
132.6
34.7
6.19 (d, 2.5)
3.65 (d, 19.2)
2.85 (d, 19.2)
2, 20, c, d, j, k
20, g, h, j, k
20, e, g, h, k
g
h
i
j
153.0d
152.8d
95.3
50.9
43.5
On the other hand, with oxidation of 4 under similar
conditions, the decrease in 4 was significantly slower than
that for 1, and three derivatives 4b, 5a and 6a were pro-
duced, which showed either UV absorption characteristic
of phenazine (4b, 370 nm; 5a, 374 nm) or quinoxaline (6a,
320 nm) derivatives (Fig. 1b). The three products were iso-
lated in a preparative scale experiment. Product 4b was
readily identified as the phenazine derivative of the mono-
meric gallocatechin quinone (4a), by comparison of the
spectroscopic data with that of 1b. Except for the large
coupling constant between H-2 and H-3 (J2,3, 7.3 Hz), the
1H NMR spectrum of 4b was closely related to that of
4.83 (s)
k
e
e
e
e
Quinoxaline
CH
130.2, 130.1 6.87 (br d, 8.4)
129.6, 129.5 7.40, 7.59 (each br t, 8.4)
129.4 (3C)
129.1
143.4, 141.8
141.4, 140.9
7.84 (3H, m)
7.97, 8.21 (br d, 8.0)
Quinoxaline
CN
1H NMR (500 MHz), 13C NMR 125 MHz, measured in acetone-d6.
Assignments may be interchanged in each column.
a
b–d
e
Complex cross-peaks in quinoxaline moieties were observed.

Y. Matsuo et al. / Phytochemistry 69 (2008) 3054–3061
3057
275 nm and a negative one at 252 nm. This result indicated
that the atropisomerism reflected the configuration at the
C-ring C-2 position, because 1 and 4 had the same R con-
between H-20 and H-j indicated that these protons are
located on the same side of the molecule. In addition, since
the configuration at C-20 was R, the absolute configuration
of 6a was determined as shown in Fig. 2. Production of this
derivative 6a clearly indicated that compound 6 was pro-
duced as the major product of oxidation of 4 (Scheme 2).
The quinone dimer 6 is an isomer of decarboxyl proepi-
theaflagallin (3b) (Matsuo et al., 2006), which has been
shown to be a precursor of epitheaflagallin (Nonaka
et al., 1986).
Next, we examined the oxidation of (ꢀ)-gallocatechin
(7), which is an enantiomer of 4. After treatment of the
reaction mixture with o-phenylenediamine, HPLC analysis
indicated that the reaction proceeded in a manner similar
to that of 4, and the retention time and UV absorption of
the peaks of three major products coincided with those of
4b, 5a and 6a. Although purification of the products cor-
responding to 5a failed, 7a and 8 were isolated and their
1H NMR spectra were found to be identical to those of
4b and 6a, respectively. However, the optical rotations
had the opposite sign. The result indicated that the oxida-
tion reaction did not discriminate between the enantio-
mers 4 and 7.
1
figuration at that position. The H and 13C NMR spectra
of product 6a showed signals arising from two sets of
2,3-trans-flavan A C-rings, along with aromatic resonances
due to two quinoxaline moieties (Table 1). The remaining
signals were assigned to be two aliphatic methylene (C-c
and C-f), an aliphatic methine (C-j), an aliphatic quater-
nary (C-k), an acetal (C-i), a tri-substituted double bond
(C-d and C-e), and four aromatic carbons connected to
nitrogen atoms (C-a, C-b, C-g and C-h). The appearance
1
of mutual H–1H long-range couplings between the H-e,
1
H-2 and H-c in the H-1H COSY spectrum suggested an
allylic relationship between these protons. This was sup-
ported by the HMBC correlations between these positions
(Table 1). The H-e was also correlated with C-20, and the
H-20 was correlated, in turn, with the C-e, C-f, C-j and
C-k. Among the four nitrogen-bearing carbons, C-a and
C-b were correlated to H-c, and C-g and C-h were coupled
with H-f. Furthermore, correlation of H-j with C-20, C-a,
C-b, C-e, C-f, C-k, C-h, and C-i allowed us to construct
the structure of the B-rings. The molecular formula of 6a
OH
OH
HO
OH
2
OH
N
a
2'
c
HO
HO
N
N
O
OH
O
O
HO
HO
H
OH
H
N
HO
O
k
j
H
e
N
i
OH
OH
f
OH
H
2'
h
HO
O
N
OH
O
N
2
N
OH
OH
OH
OH
4b
5a
6a
was deduced to be C41H32N4O9, based on the 13C NMR
signals and FAB-MS (m/z: 725, [M+H]+) data, and finally
confirmed by HR FAB-MS. The unsaturation index 28 and
up- and low-field shifts of the C-20 and C-30, respectively,
compared to the C-2 and C-3, suggested the formation of
a hemiacetal ring at C-i with a C-30 hydroxyl group. As
for the stereochemistry, the strong NOESY correlations
OH
OH
OH
HO
O
S
OH
R
OH
N
OH
HO
O
N
H
H
7
HO
O
OH
OH
O
N
N
N
N
OH
OH
3
OH
HO
O
N
a
2
b
d
e
O
HO
HO
c
8
OH
N
H
OH
j
H
i
O
2'
OH
k
f
7a
h
N
g
O
3'
N
OH
The difference in the oxidation of 1 and 4 was very interest-
Fig. 2. Selected NOESY correlations for 6a.
ing because it implies that the reactions have a difference in

3058
Y. Matsuo et al. / Phytochemistry 69 (2008) 3054–3061
OH
OH
OH
OH
OH
OH
minor
HO
O
HO
O
R
OH
H
OH
OH
HO
O
O
OH
HO
S
OH
OH
O
4
OH
-H2
5
-H2
OH
OH
O
O
O
OH
HO
HO
O
O
HO
O
H
H
O
OH
major
OH
O
OH
O
O
OH
4a
6
Scheme 2.
enzyme specificity and stereochemical features. As for en-
zyme specificity, it was obvious that enzymes preferentially
oxidized 1 rather than 4. Since the enzyme did not distin-
guish between 4 and 7, it only oxidized the B-ring to o-qui-
none, and the subsequent dimerization reaction was
probably non-enzymatic. Stereoselectivity in the produc-
tion of 2, which has previously been demonstrated (Tanaka
et al., 2002c, 2003), was further confirmed in this experi-
ment. The rapid and highly selective coupling between 1
and 1a may be explained by their hydrophobic association
at their sterically unhindered faces, which were on opposite
sides of the C-3 hydroxyl group and B-rings (Scheme 3). In
contrast, the C-3 hydroxyl group of 4 and 4a probably hin-
dered the intermolecular association; therefore dimeriza-
tion proceeded slowly, despite the presence of monomeric
quinone 4a in the reaction mixture, which is shown as 4b
in Fig. 1. The difference in the position at which the cou-
pling reaction occurred (between B-ring C-2 of 1a and C-
20 of 1 for production of 2; and between B-ring C-1 of 4a
and C-20 of 4 for production of 6) may also have been
caused by the difference in the manner of molecular associ-
ation. In order to evaluate the difference in hydrophobic
OH
H+
1
O
HO
OH
OH
OH
OH
O
hydrophobic
R
R
OH
O
-H2
+H2O
R
OH
O
OH
OH
H
2
HO
H
H
R
H
H+
O
A
OH
HO-
C
O
O
HO
OH
HO
O
O
OH
H+
OH
O
H+
OH
1a
R =
OH
OH
OH
H+
O
O
O
HO
4
R
O
H+
H
R
OH
R
OH
OH
R
O
1) - H2
HO-
- H2
HO2C
OH
O
O
OH
O
OH
O
O
OH
OH
6
R
H
O
R
H
O
HO
2) - CO2
H+
R
R
O
OH
O
O
OH
O
OH
HO
H+
O
H
O
HO
HO
O
OH
4a
Scheme 3.

Y. Matsuo et al. / Phytochemistry 69 (2008) 3054–3061
3059
Table 2
20P (Mitsubishi Chemical), Chromatorex ODS (Fuji Silysia
Chemical, Japan), and Sephadex LH-20 (Pharmacia Fine
Chemical), respectively. TLC was performed on 0.2 mm
thick precoated Kieselgel 60 F254 plates (Merck) with ben-
zene/HCO2Et/HCO2H (1:7:1, v/v) or CHCl3/MeOH/H2O
(14:6:1, v/v), with spots detected by UV illumination,
sprayed with 2% ethanolic FeCl3 or 10% H2SO4 reagent,
and heated. Analytical HPLC was performed on a Cosmosil
5C18-AR II column (Nacalai Tesque; 250 · 4.6 mm i.d.),
with gradient elution at 10–30% (30 min) and 30–75%
(15 min) of CH3CN in 50 mM H3PO4 (flow rate, 0.8 ml/
min; detection, JASCO photodiode array detector MD-
910). Preparative HPLC was performed on a COSMOSIL
Dd (d10 mg/ml ꢀ d0.5 ros/ml) of 1 and 2 (500 MHz, 2% DMSO-d6-D2O)
1
2
H-2
H-3
H-4
ꢀ0.128
ꢀ0.047
ꢀ0.026
ꢀ0.058
ꢀ0.021
ꢀ0.023
ꢀ0.029
ꢀ0.105
ꢀ0.086
ꢀ0.013
ꢀ0.052
ꢀ0.002
ꢀ0.003
ꢀ0.003
H-6
H-8
B-ring H
1
self-association, the H NMR chemical shift change (Dd =
d10 mg/ml ꢀ d0.5 mg/ml) of 1 and 4 in deuterium oxide, con-
taining 2% DMSO-d6 at different concentrations, was com-
pared (Table 2). Both 1 and 4 showed a large up-field shift
of H-2 at the higher concentration, which was caused by
the anisotropic effect of the aromatic rings of the other
associating molecule. The Dd value of the H-3 of 1 was
smaller than that of 4 because it was located at the equato-
rial position. However, the remaining proton signals of 1
showed larger Dd values compared to those of 4, suggesting
that the self-association of 1 in aqueous solution was stron-
ger than that of 4, and the association mainly occurred
over the C-2 position.
Oxidation of catechins is a very important process dur-
ing black tea production, and it also occurs in other plants
when the tissues are injured. However, the chemical details
are still ambiguous. In this study, we demonstrated some
aspects of the stereochemical nature of the enzymatic oxi-
dation of epigallocatechins. The results showed that the
reaction strongly depended on the geometric configurations
of the C-ring and the stereochemical restriction in the qui-
none–phenol association accounts for the high stereoselec-
tivity in dimerization of epigallocatechin (1) and its gallate
producing the major black tea polyphenol theasinensins
(Tanaka et al., 2002c, 2003; Matsuo et al., 2006). Further
studies on the oxidation mechanism of tea catechins are
now in progress.
5C18-AR-II
column
(Nacalai
Tesque;
10 mm
i.d. · 250 mm) with 20–70% CH3CN in 0.5% TFA (linear
gradient elution). Epigallocatechin was isolated from com-
mercial green tea and recrystallized from H2O.
3.2. Oxidation of 1 and 4 and HPLC analysis
Japanese pear fruits (50 g) were homogenized with H2O
(50 ml) and filtered through four layers of gauze. The fil-
trate was then separately mixed with aqueous solutions
of 1 and 4 (10 mg/2.5 ml) and vigorously stirred. Aliquots
(0.1 ml) of the reaction mixture were taken at 10, 15, 30,
60 and 120 min, and mixed with 0.4 ml ethanolic o-pheny-
lenediamine solution (0.5 mg/ml in 5% AcOH/EtOH). The
mixtures were analyzed by HPLC after filtration by mem-
brane filters (0.45 lm), with peak areas at maximum absor-
bance of each peak obtained by the JASCO-BORWIN
chromatography software.
3.2.1. Non-enzymatic oxidation of 1
An aqueous solution (0.4 ml) containing potassium hex-
acyanoferrate(III) (60 mg) and NaHCO3 (16 mg) as added
dropwise at 0 ꢁC to an aqueous solution of 1 (10 mg/1 ml)
with stirring for 10 min. The reaction was stopped by addi-
tion of 0.1 M HCl (0.6 ml). The mixture was passed
through a Sep-Pak C18 cartridge with H2O, and the pheno-
lic compounds were eluted out with MeOH (2 ml). To the
MeOH solution, o-phenylenediamine (2 mg) and AcOH
(0.1 ml) were added, and the mixture was stirred for 1.5 h
at 35 ꢁC. The HPLC analysis of the mixture showed the
peaks of 1 (tR 17.51 min), 2b (28.02 min), 2a (30.51 min),
1a (46.63 min), and 3a (49.61 min).
3. Experimental
3.1. General
Optical rotations were measured with a JASCO DIP-370
digital polarimeter. CD spectra were measured with a JAS-
CO J-720w apparatus. The 1H and 13C NMR, 1H–1H-
COSY, NOESY, HSQC and HMBC spectra were recorded
with a Unity plus 500 spectrometer (Varian, Palo Alto,
CA, USA) operating at 500 MHz for 1H and 125 MHz for
13C NMR spectroscopy. The 1H and 13C NMR spectra were
also measured using a JEOL JMN-AL400 (JEOL, Japan)
operating at 400 MHz for 1H and 100 MHz for 13C NMR.
FAB and EIMS were recorded on a JMS DX-303 spectrom-
eter (JEOL), and m-nitrobenzyl alcohol or glycerol was used
as the matrix for FAB-MS. Elemental analysis was con-
ducted with a Perkin–Elmer 2400 II analyzer (Perkin–Elmer,
Waltham, MA, USA). CC was conducted on MCI-gel CHP
3.3. Oxidation of 4 and isolation of phenazine derivatives
An aqueous solution of 4 (0.5 g/75 ml) was vigorously
stirred with a Japanese pear homogenate (125 ml) for
180 min. To the mixture, a solution of o-phenylenediamine
(250 mg) in 5% AcOH/EtOH (800 ml) was added and
gently stirred for 1 h. After filtration, the filtrate was con-
centrated and applied to a column of MCI-gel CHP20P
(3.0 cm i.d. · 25 cm), with 10% step-wise elution of H2O
containing increasing proportions of MeOH (0–100%, each
200 ml). Elution of the column with 10–30% MeOH in H2O

3060
Y. Matsuo et al. / Phytochemistry 69 (2008) 3054–3061
(v/v) gave a fraction containing 4, which was further puri-
fied by Sephadex LH-20 CC (2.0 cm i.d. · 20 cm) with
1627, 1606, 1517, 1491, 1468; UV (MeOH) kmax nm (log e):
235(4.76), 320(4.20); CD (3.12 · 10ꢀ5 mol/l in methanol)
1
MeOH–H2O (4:6, v/v) to afford
a
recovery of
4
De (nm): +0.78 (318), ꢀ0.23 (268), +0.17 (255); H and
(171.5 mg). Further elution of the initial column with
MeOH–H2O (4:6–9:1) yielded three fractions containing
phenazine derivatives: Fractions 1 (51.8 mg), 2 (42.7 mg)
and 3 (44.9 mg), respectively. Fractions 1 and 2 were sepa-
rately purified by Sephadex LH-20 CC (2.0 cm i.d. · 20 cm)
eluted with MeOH–H2O (4:1, v/v) to give 5a (12.7 mg)
from fraction 1, and 4b (23.4 mg) from fraction 2. Fraction
3 was subjected to Sephadex LH-20 CC eluted with EtOH
to yield 6a (33.7 mg).
13C NMR see Table 1; HR FAB-MS 725.2247,
C41H33N4O9 requires 725.2247.
3.4. Oxidation of 7 and isolation of phenazine derivatives
(ꢀ)-Gallocatechin (7) was prepared by isomerization of
1. An aqueous solution of 1 (2 g/50 ml) was divided into
five aliquots. Each aliquot was sealed in a screw-capped
vial and heated in a microwave oven (600 W) for 2 min.
The mixture was combined and separated by MCI-gel
CHP20P (3.0 cm i.d. · 25 cm) with H2O–MeOH
(100:0 ! 70:30, v/v, in a 5% step-wise elution) to give 7
3.3.1. Phenazine derivative 4b
25
Brown amorphous powder, ½aꢁD ꢀ27.3 (c = 0.01,
2
MeOH); FAB-MS m/z: 377 [M+H]+; IR mmax cmꢀ1: 3288,
(0.94 g), ½aꢁD5 ꢀ1.2 (c = 1.0, acetone). Oxidation of 7 and
2921, 1631, 1607, 1566, 1519, 1470, 1468; UV (MeOH)
subsequent derivatization and separation were performed
k
max nm (loge): 269 (4.69), 370 (3.83); 1H NMR
in a manner similar to that described for 4, to give 7a
1
(400 MHz, acetone-d6) d: 8.26–8.18 (2H, m, H-90, 120),
7.95–7.88 (2H, m, H-100, 110), 7.79 (1H, br s, H-60), 7.32
(1H, d, J = 1.5, H-20), 6.06, 5.98 (each 1H, d, J = 2.0, H-
6, 8), 4.96 (1H, d, J = 7.3, H-2), 4.19 (1H, m, H-3), 2.94
(1H, dd, J = 5.8, 16.1, H-4), 2.63 (1H, dd, J = 8.8, 16.1,
H-4); 13C NMR (100 MHz, acetone-d6) d: 157.8, 157.2,
156.2 (C-5, 7, 8a), 153.4 (C-30), 144.8, 144.5, 144.3, 142.1
(C-40, 50, 70, 80), 136.0 (C-10), 131.7, 131.2, 130.2, 130.1
(C-90, 100, 110, 120), 118.8 (C-60), 109.7 (C-20), 100.3 (C-
4a), 96.4, 95.2 (C-6, 8), 82.5 (C-2), 68.0 (C-3), 28.7 (C-4);
HR FAB-MS 377.1152, C21H17N2O5 requires 377.1137.
(46.7 mg) and 8 (40.0 mg). The H NMR spectrum of 7a
was identical to that of 4b.
3.4.1. Phenazine derivative 8
25
Brown amorphous powder; ½aꢁD ꢀ89.7 (c = 0.1,
MeOH); FAB-MS m/z: 725 [M+H]+; IR mmax cmꢀ1: 3359,
2919, 1627, 1606, 1517, 1491, 1468; UV (MeOH) kmax nm
1
(loge): 235(4.76), 320(4.20); the H NMR spectrum was
identical to that of 6a; HR FAB-MS 725.2273,
C41H33N4O9 requires 725.2247.
1
3.5. H NMR measurements of 1 and 4 in 2% DMSO-d6/
D2O
3.3.2. Phenazine derivative 5a
25
Brown amorphous powder; ½aꢁD +71.5 (c = 0.05,
MeOH); FAB-MS m/z: 681 [M+H]+; IR mmax cmꢀ1: 3366,
2924, 2853, 1628, 1609, 1517, 1467; UV (MeOH) kmax nm
(log e): 275(4.66), 374(3.75); CD (3.12 · 10ꢀ5 mol/l in
The 1H NMR spectra (500 MHz) of 1 and 4 in 2%
DMSO-d6/D2O at the concentrations of 0.5 mg/ml and
10.0 mg/ml was measured and the chemical shift differences
were compared (Table 2).
1
MeOH) De (nm): +4.7 (275), ꢀ0.9 (252), +0.3 (238); H
NMR (400 MHz, acetone-d6) d: 8.20–8.12 (2H, m, H-90 0,
120 0), 7.89–7.87 (2H, m, H-100 0, 110 0), 7.85 (1H, s, H-60 0),
6.69 (1H, s, H-60 0 0), 6.03, 6.02, 5.92, 5.89 (each 1H, d,
J = 2.4, H-6, 60, 8, 80), 5.18 (1H, d, J = 4.9, H-2), 4.50
(1H, d, J = 6.8, H-20), 4.29 (1H, m, H-3), 4.00 (1H, m, H-
30), 2.73 (1H, dd, J = 5.4, 16.1, H-4 or 40), 2.62 (1H, dd,
J = 4.9, 16.1, H-4 or 40), 2.54 (1H, dd, J = 5.9, 16.1, H-4
or 40), 2.22 (1H, dd, J = 7.8, 16.1, H-4 or 40); 13C NMR
(100 MHz, acetone-d6) d: 157.6, 157.4, 157.3, 157.0,
156.7, 156.4 (C- 5, 7, 8, 50, 70, 8a0), 151.5 (C-30 0), 146.8,
145.7, 144.7, 144.2, 144.1, 142.2 (C-10 0, 50 0, 70 0, 80 0, 30 0 0,
50 0 0), 135.4, 134.4 (C-40 0, 40 0 0), 131.6, 131.2, 130.17,
130.11, 129.5 (C-90 0, 100 0, 110 0, 120 0, 10 0 0), 118.4, 117.8 (C-
20 0, 60 0), 114.9 (C-20 0 0), 106.9 (C-60 0 0), 99.9, 99.5 (C-4a,
4a0), 96.2, 96.0, 95.4 (2C) (C-6, 60, 8, 80), 79.69, 79.67 (C-
2, 20), 67.2, 66.5 (C-3, 30), 27.2, 26.3 (C-4, 40); HR FAB-
MS 681.1711, C36H29N2O12 requires 681.1720.
Acknowledgements
The authors are grateful to Mr. K. Inada and Mr. N.
Yamaguchi for NMR and MS measurements. This work
was supported by a Grant-in-aid for Scientific Research
No. 18510189 from the Japan Society for the Promotion
of Science.
References
Feucht, W., Treutter, D., 1999. The role of flavan-3-ols and proantho-
cyanidins in plant defense. In: Inderjit, Dakshini, K.M.M., Foy, C.L.
(Eds.), Principles and Practices in Plant Ecology. CRC, Boca Raton,
pp. 307–338.
Hashimoto, F., Nonaka, G., Nishioka, I., 1988. Tannins and related
compounds. LXIX. Isolation and structure elucidation of B, B0-linked
bisflavanoids, theasinensins D-G and oolongtheanin from oolong tea.
(2). Chem. Pharm. Bull. 36, 1676–1684.
3.3.3. Phenazine derivative 6a
25
Brown amorphous powder; ½aꢁD +133 (c = 0.1, MeOH);
Hashimoto, F., Nonaka, G., Nishioka, I., 1992. Tannins and related
compounds. CXIV. Structures of novel fermentation products,
FAB-MS m/z: 725 [M+H]+; IR mmax cmꢀ1: 3359, 2919,

Y. Matsuo et al. / Phytochemistry 69 (2008) 3054–3061
3061
theogallinin, theaflavonin and desgalloyl theaflavonin from black tea,
and changes of tea leaf polyphenols during fermentation. Chem.
Pharm. Bull. 40, 1383–1389.
Takino, Y., Imagawa, H., Horikawa, H., Tanaka, A., 1964. Studies on the
mechanism of the oxidation of tea leaf catechins. Part III. Formation
of a reddish orange pigment and its spectral relationship to some
benzotropolone derivatives. Agric. Biol. Chem. 28, 64–71.
Takino, Y., Ferretti, A., Flanagan, V., Gianturco, M., Vogel, M., 1965.
The structure of theaflavin, a polyphenol of black tea. Tetrahedron
Lett., 4019–4025.
Haslam, E., 2003. Thoughts on thearubigins. Phytochemistry 64, 61–73.
Li, Y., Tanaka, T., Kouno, I., 2007. Oxidative coupling of the pyrogallol
B-ring with a galloyl group during enzymatic oxidation of epigallo-
catechin 3-O-gallate. Phytochemistry 68, 1081–1088.
Matsuo, Y., Tanaka, T., Kouno, I., 2006. A new mechanism for oxidation
of epigallocatechin and production of benzotropolone pigments.
Tetrahedron 62, 4774–4783.
Nonaka, G., Kawahara, O., Nishioka, I., 1983. Tannins and related
compounds. XV. A new class of dimeric flavan-3-ol gallates, theasi-
nensins A and B, and proanthocyanidin gallates from green tea leaf
(1). Chem. Pharm. Bull. 31, 3906–3914.
Tanaka, T., Mine, C., Inoue, K., Matsuda, M., Kouno, I., 2002a.
Synthesis of theaflavin from epicatechin and epigallocatechin by plant
homogenates and role of epicatechin quinone in the synthesis and
degradation of theaflavin. J. Agric. Food Chem. 50, 2142–2148.
Tanaka, T., Mine, C., Kouno, I., 2002b. Structures of two new oxidation
products of green tea polyphenols generated by model tea fermenta-
tion. Tetrahedron 58, 8851–8856.
Nonaka, G., Hashimoto, F., Nishioka, I., 1986. Tannins and related
compounds. XXXVI. Isolation and structures of theaflagallins, new
red pigments from black tea. Chem. Pharm. Bull. 34, 61–65.
Porter, L.J., 1998. Flavans and proanthocyanidins. In: Harborne, J.B.
(Ed.), Flavonoids. Chapman and Hall, London, pp. 21–62.
Saijo, R., Takeda, Y., 1999. HPLC analysis of catechins in various kinds
of green teas produced in Japan and abroad. Nippon Shokuhin
Kagaku Kogaku Kaishi 46, 138–147, CAN 130:351456.
Tanaka, T., Mine, C., Watarumi, S., Fujioka, T., Mihashi, K., Zhang,
Y.J., Kouno, I., 2002c. Accumulation of epigallocatechin quinone
dimers during tea fermentation and formation of theasinensins. J. Nat.
Prod. 65, 1582–1587.
Tanaka, T., Watarumi, S., Matsuo, Y., Kamei, M., Kouno, I., 2003.
Production of theasinensins A and D, epigallocatechin gallate dimers
of black tea, by oxidation-reduction dismutation of dehydrotheasi-
nensin A. Tetrahedron 59, 7939–7947.