A novel black tea pigment and two new oxidation products of epigallocatechin-3-O-gallate

Article abstract of DOI:10.1021/jf0512656

During tea fermentation, oxidation-reduction dismutation of a number of quinone metabolites of tea catechins yields numerous minor products, which make it difficult to separate and purify black tea polyphenols. In this study, epigallocatechin-3-O-gallate

Full text of DOI:10.1021/jf0512656

J. Agric. Food Chem. 2005, 53, 7571−7578 7571
A Novel Black Tea Pigment and Two New Oxidation Products
of Epigallocatechin-3-O-gallate
TAKASHI TANAKA,* YOSUKE MATSUO, AND ISAO KOUNO
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi,
Nagasaki 852-8521, Japan
During tea fermentation, oxidation-reduction dismutation of a number of quinone metabolites of tea
catechins yields numerous minor products, which make it difficult to separate and purify black tea
polyphenols. In this study, epigallocatechin-3-O-gallate was enzymatically oxidized and then the
unstable quinone metabolites in the oxidation mixture were hydrogenated with 2-mercaptoethanol to
reduce production of inseparable minor dismutation products. As a result, three new oxidation products
including a new black tea pigment were isolated, and their structures were determined based on
chemical and spectroscopic data. Dehydrotheasinensin AQ is a new reddish-orange pigment with a
1,2-diketone structure, and its presence in commercial black tea was confirmed. In addition, a new
quinone dimer with a complex caged structure and a trimer of epigallocatechin-3-O-gallate were also
isolated and their production mechanisms are proposed. The presence of this trimer suggested
participation of galloyl quinones in production of minor polyphenols in black tea.
KEYWORDS: Black tea; dehydrotheasinensin AQ; quinone dimer; polyphenol; oxidation
INTRODUCTION
In addition to theaflavins and thearubigins, many unidentified
colorless oxidation products are produced during tea fermenta-
tion (13, 14). Recently, we demonstrated a new oxidation
mechanism of (-)-epigallocatechin-3-O-gallate (1), the most
abundant tea catechin in fresh tea leaves, during tea fermentation
(15-17). In this mechanism, dehydrotheasinensin A (4) and
EGCg quinone dimer A (5) (18) are produced as major products
by stereoselective dimerization of the o-quinone (1a) (Figure
2). Compound 4 is unstable on heating to yield theasinensins
A (6) and D (7), a pair of atropisomers of B- and B′-linked
dimers of 1. In addition to these reduction products, a number
of oxidation products, including oolongtheanin (10) and 11, are
generated simultaneously; therefore, the decomposition of 4
involves oxidation-reduction dismutation (15). Because th-
easinensins are major black tea polyphenols comparable to
theaflavins, this mechanism is important in the formation of
black tea polyphenols (14). However, the yield of 4 and 5 from
1 in a previous in vitro enzymatic oxidation experiment did not
exceed 40%, suggesting the presence of different oxidation
mechanisms. Our lack of understanding of the oxidation of 1 is
mainly due to the presence of numerous minor inseparable
products, some of which probably originate from the dismutation
of 4 or related unknown quinone metabolites (15). We previ-
ously demonstrated that the dismutation of 4 occurs in the final
stage of tea fermentation, when the fermented leaves are heated
and dried (16). In addition, we also showed that the unstable
quinone 4 is easily hydrogenated by treatment with a moderate
reducing agent such as ascorbic acid or thiol compounds,
yielding theasinensin A (6) stereoselectively (15).
Manufactured and consumed worldwide, black tea is an
important polyphenol-rich beverage with a higher polyphenol
content than coffee, cocoa, and green tea (1-3). During its
fermentation, four major tea catechins originally contained in
fresh leaves are enzymatically oxidized and converted to various
oxidation products comprising black tea polyphenols. Of these
oxidation products, characteristic pigments have been particu-
larly studied since the end of the 1950s (4). Black tea pigments
are usually classified into two major groups, theaflavins and
thearubigins. Theaflavin and its galloyl esters (3) (Figure 1)
are well-characterized catechin dimers with a characteristic
benzotropolone unit produced by oxidative coupling between
pyrogallol type catechins [(-)-epigallocatechin and (-)-epi-
gallocatechin-3-O-gallate (1)] and catechol type catechins [(-)-
epicatechin and (-)-epicatechin-3-O-gallate (2)] (Figure 1) (5).
The theaflavin content of black tea leaves is usually 0.8-2.8%
depending on the conditions of fermentation (6-9). On the other
hand, thearubigins constitute up to 60% of the solids in black
tea infusions (6) and are therefore much more important with
respect to coloration and taste. However, the definition of
thearubigins remains ambiguous and little is known about their
chemical structures despite substantial efforts by many research
groups (10, 11). Recently, structures with benzotropolone units
derived by oxidative coupling between galloyl groups and
catechol type catechin B-rings have been proposed as nonpolar
thearubigin components (12).
In the present study, on the basis of the above findings, we
chemically reduced 4 and related unstable quinones produced
* To whom correspondence should be addressed. Tel: 81(0)95 819 2433.
Fax: 81(0)95 819 2477. E-mail: t-tanaka@net.nagasaki-u.ac.jp.
10.1021/jf0512656 CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/27/2005

7572 J. Agric. Food Chem., Vol. 53, No. 19, 2005
Tanaka et al.
Figure 1. Structures of compounds 1−3.
Figure 2. Oxidation of epigallocatechin-3-O-gallate.
HMBC (nJCH optimized for 8 Hz), and NOESY (mixing time 0.50 s)
experiments were performed using standard Varian pulse sequences.
Mass spectra (MS) were recorded on a JEOL JMS-700N spectrometer,
and glycerol or m-nitrobenzyl alcohol was used as the matrix for fast
atom bombardment mass spectroscopy (FAB-MS) measurements.
in the initial enzymatic oxidation reaction mixture in order to
prevent the dismutation reaction. This procedure was expected
to allow us to isolate unknown and unstable quinone metabolites
related to 4 as reduction products. In addition, the composition
of the reaction mixture was expected to become simplified
owing to the absence of dismutation products; therefore,
chromatographic separations were also expected to become
easier. On the basis of the structure elucidation and biogenesis
of the catechin oxidation products, a new oxidation mechanism
for production of one component of black tea polyphenols is
proposed.
Column chromatography was performed with Diaion HP20SS, MCI-
gel CHP 20P (75-150 µm) (Mitsubishi Chemical Co., Japan), Sephadex
LH-20 (25-100 µm) (Pharmacia Fine Chemical Co., Ltd.), and
Chromatorex ODS (Fuji Silysia Chemical Ltd., Japan). Thin-layer
chromatography (TLC) was performed on 0.2 mm precoated Kieselgel
60 F254 plates (Merck) with benzene-ethyl formate-formic acid
(1:7:1, v/v) or chloroform-methanol-water (14:6:1, v/v). Spots were
detected by UV illumination and by spraying with 2% ethanolic FeCl3
or 10% sulfuric acid reagent followed by heating. Analytical high-
performance liquid chromatography (HPLC) was performed on a 250
mm × 4.6 mm i.d. Cosmosil 5C18-AR II column (Nacalai Tesque Inc.)
with gradient elutions of CH3CN in 50 mM H3PO4 from 10 to 30% in
30 min and 30-75% in 15 min at a flow rate of 0.8 mL/min and
detection with a Jasco MD-910 photodiode array detector. Epigallo-
catechin-3-O-gallate (1) was isolated from commercial green tea and
recrystallized from water.
MATERIALS AND METHODS
General Procedures. Ultraviolet (UV) spectra were obtained with
a Jasco V-560 UV/vis spectrophotometer, and optical rotations were
measured with a Jasco DIP-370 digital polarimeter. 1H, 13C NMR, 1H-
1H correlation spectroscopy (COSY), 1H NMR nuclear Overhauser and
exchange spectroscopy (NOESY), heteronuclear single quantum coher-
ence (HSQC), and heteronuclear multiple bond connectivity (HMBC)
spectra were recorded in a mixture of acetone-d6 and D2O (19:1, v/v)
at 27 °C with a Varian Unity plus 500 spectrometer operating at 500
Enzymatic Oxidation and Treatment with 2-Mercaptoethanol.
Japanese pear fruits (562 g) were homogenized with 500 mL of H2O
and filtered through four layers of gauze (19). The filtrate was then
mixed with an aqueous solution of 1 (10 g/300 mL) (Figure 3A) and
1
MHz for H and 125 MHz for 13C. Coupling constants are expressed
in Hz, and chemical shifts are given on a δ (ppm) scale with
tetramethylsilane as an internal standard. HMQC (JCH ) 140 Hz),

Oxidation of Epigallocatechin Gallate
J. Agric. Food Chem., Vol. 53, No. 19, 2005 7573
Figure 3. HPLC profiles of the reaction mixture. (A) Before addition of pear homogenate. (B) After stirring for 3 h with pear homogenate. (C) After
addition of 2-mercaptoethanol (18 h).
vigorously stirred for 3 h. At this stage, HPLC analysis of the reaction
mixture indicated a reduction of 1 and accumulation of 4 and 5 together
with several unknown products (Figure 3B). The mixture was poured
into 8% 2-mercaptoethanol in EtOH (1.3 L) at -20 °C and gently stirred
at room temperature for 18 h. HPLC analysis showed that 4 was
completely converted to 6 (Figure 3C). Insoluble materials in the
mixture were consequently filtered off, and the filtrate was concentrated
until EtOH was removed in vacuo. The resulting aqueous solution was
applied to a 35 cm × 4 cm i.d. MCI-gel CHP20P column with H2O
containing increasing proportions of MeOH (10% stepwise elution from
0 to 80%, each 500 mL) to give two FeCl3 positive fractions, A (eluted
out with 30-60% MeOH) and B (eluted out with 70-80% MeOH).
Fraction A was further fractionated by chromatography over a 27 cm
× 4 cm i.d. Sephadex LH-20 column with H2O containing 0-100%
MeOH (20% stepwise elution, each 500 mL) yielding seven fractions.
The first three fractions eluted out with 0-60% MeOH and contained
2-mercaptoethanol and gallic acid. Fraction A-4 (4.83 g) was further
separated by a 33 cm × 4 cm i.d. Sephadex LH-20 column with EtOH
(1 L) and then with 50% aqueous acetone (1 L) to give two fractions,
A-4-1 and A-4-2. The fraction A-4-1 was applied to a 26 cm × 4 cm
i.d. MCI-gel CHP20P column with 0-50% MeOH (5% stepwise
elution, each 100 mL) to give 1 (519 mg). The fraction A-4-2 was
subjected to chromatography on a 26 cm × 4 cm i.d. MCI-gel CHP20P
column with 0-50% MeOH (5% stepwise elution, each 100 mL) to
give 5 (554 mg), 6 (486 mg), and a crude crop of 9, which was purified
by chromatography on a 26 cm × 4 cm i.d. Chromatorex ODS column
with 0-40% MeOH (5% stepwise elution, each 100 mL) to yield 9
(305 mg). Fraction A-5 (4.42 g) was chromatographed over a 25 cm
× 3 cm i.d. Sephadex LH-20 column with 0-20% H2O in EtOH (10%
stepwise elution, each 300 mL) to give 6 (2.75 g) and a fraction
containing mainly 6, 14, and isomers of 14. This fraction was applied
to a 27 cm × 3 cm i.d. Chromatorex ODS column with 0-40% MeOH
(5% stepwise elution, each 100 mL) and then to a 21 cm × 2.5 cm i.d.
Sephadex LH-20 column with 80-100% MeOH (10% stepwise elution,
each 200 mL) and then MeOH-H2O-acetone (90:5:5, v/v/v, 200 mL)
to give a crude crop of 14. Purification of the compound was achieved
by chromatography on a 21 cm × 2.5 cm i.d. MCI-gel CHP20P column
0-40% MeOH (5% stepwise elution, each 100 mL) to yield 14 (43
mg). Fraction A-6 was subjected to a 21 cm × 2.5 cm i.d. MCI-gel
CHP20P column with 20-60% MeOH (5% stepwise elution, each 100
mL) to give 8 (51.3 mg). Fraction B was separated by chromatography
on a 19 cm × 2 cm i.d. Sephadex LH-20 column with 80-100% MeOH
(10% stepwise elution, each 100 mL) to give epitheaflagallin 3-O-gallate
(12) (60.4 mg).
25
Dehydrotheasinensin AQ (8). Red amorphous powder; [R]D
-603.8° (c 0.12, MeOH). FAB-MS m/z 913 [M + H]+, m/z 935 [M +
Na]+. UV λmax nm (log ꢀ): 447 (4.06), 277 (4.53). IR νmax cm-1: 3355,
1698, 1686, 1604, 1521. Anal. calcd for C44H32O22‚3/2H2O: C, 56.23;
H, 3.75. Found: C, 56.45; H, 4.02. 1H and 13C NMR data: See Table
1.
Preparation of a Quinoxaline Derivative (8a). o-Phenylenediamine
(5 mg) in acetic acid (0.1 mL) was added to a solution of 8 (15 mg) in
0.5 mL of EtOH, and the mixture was stirred at 25 °C for 2 h. The
mixture was directly applied to a 15 cm × 10 cm i.d. Sephadex LH-20
column eluted with MeOH and then MeOH-H2O-acetone (95:5:5,
v/v/v) to give the quinoxaline derivative 8a (7.7 mg).
25
Quinoxaline Derivative (8a). Reddish brown powder; [R]D
-300.7° (c 0.06, methanol). FAB-MS m/z 985 [M + H]+. UV λmax
nm (log ꢀ): 379 (3.80), 272 (4.70). H NMR (400 MHz, in acetone-
1
d6): δ 8.23, 8.01 (each 1H, dd, J ) 1.7, 7.5 Hz, Q-5 and Q-8), 7.73
(2H, m, Q-6 and Q-7), 7.35 (2H, s, galloyl-2, 6), 7.08 (2H, s, galloyl-
2, 6), 7.02 (1H, s, H-6′′′), 6.12 (1H, s, H-2′′), 6.06, 6.01 (each 1H, d,
J ) 2.3 Hz, H-6′′, 8′′), 5.89, 5.85 (each 1H, br d, J ) 3.2 Hz, H-3,
3′′), 5.81 (1H, d, J ) 2.3 Hz, H-6), 4.98 (1H, d, J ) 2.3 Hz, H-8),
4.39, 3.66 (each 1H, d, J ) 15.9 Hz, H-6′), 4.19 (1H, br s, H-2), 3.28
(1H, dd, J ) 4.4, 17.3 Hz, H-4′′), 2.94 (overlapped with HOD signal,
H-4, 4′′), 2.77 (1H, J ) 4.4, 17.6 Hz, H-4).
HPLC Analysis of Black Tea. Commercial black tea (a blended
tea produced in India and Sri Lanka, purchased from Mitsui Norin Co.,
Ltd.) (10 g) was extracted with 70% acetone (2×), and then, the organic
solvent was removed by evaporation. The resulting aqueous solution
was successively partitioned with ether, CHCl3, and EtOAc, and then,
the EtOAc layer was concentrated (0.9 g). The EtOAc extract was
applied to a 10 cm × 2 cm i.d. Sephadex LH-20 column and eluted
with 80-100% MeOH and then with 50% aqueous acetone to give a
fractions mainly containing theaflavins. HPLC analysis of the theaflavin
fraction showed a peak at 34.0 min corresponding to 8, the UV spectrum
of which was also identical with that of 8.
25
EGCg Quinone Dimer B (9). Tan amorphous powder; [R]D
-16.4° (c 0.18, acetone). FAB-MS m/z 931 [M + H]+, 949 [M +
H2O + H]+. MALDI TOF-MS m/z: 953 [M + Na]+, 971 [M + H2O
+ Na]+. UV λmax
nm (log ꢀ): 278 (4.33). IR νmax cm-1: 3405,
EtOH

7574 J. Agric. Food Chem., Vol. 53, No. 19, 2005
Tanaka et al.
Table 1. 1H and 13C NMR Data for Dehydrotheasinensin AQ (8) and EGCg Quinone Dimer B (9) (500 MHz for 1H NMR and 125 MHz for 13C
NMR)a
8
9
position
δC
δH
HMBC (H to C)
3, 4, 1 , 2
2, 4, 4a, gal-7
166.1)
δC
δH
HMBC (H to C)
3, 4, 1 , 2 , 6
4a, gal-7
167.6)
2, 3, 4a,
5, 8a
NOESY
2
3
76.5
65.9
4.25 (br s)
5.91 (br s)
′
′
77.2
64.6
4.33 (s)
5.83 (br s)
′
′
′
3, 4, 2
2, 4, 2
′
′
, 6
, 6
′
′
(δ
(δ
4
26.7
3.04 (br d, 17.4)
2, 3, 4a, 5, 8a
27.0
2.95 (br d,
2, 3, 4
3, 4
17.5)
2.86 (dd, 4.1,
17.4)
4a, 5, 8a
2.87 (dd, 4.2,
4a, 5, 8a
17.5)
4a
5
6
7
8
98.8
98.8
157.6b
97.3
157.8b
5.98 (d, 2.3)
5.63 (d, 2.3)
4a, 5, 7, 8
96.9
6.06 (d, 2.3)
4a, 5, 7, 8
4a, 6, 7, 8a
157.6b
95.4
154.8
88.6
157.6b
95.7c
155.7
78.3
4a, 6, 7, 8a
5.83 (d, 2.3)
8a
1
2
′
′
136.1
63.6
3.39 (br d,
0.9)
2, 1
′
, 3
′
, 6
′
,
2, 3, 2′′′
3′′′-OH
,
2
′′′, 3′′′, 5′′′
3
4
5
6
′
′
′
′
142.5
178.4
187.1
47.7
200.7
81.9
199.6
69.8
4.02 (d, 15.2)
2, 1
′
′
, 2
′
′
, 4
′
′
, 5
′
3.37 (d, 1.8)
2, 1
′
, 2
′
, 4
′
,
2, 3, 6′′′
5
′
, 4′′′, 5′′′
3.34 (d, 15.2)
6.05 (br s)
2, 1
, 2
, 5
2
3
4
′′
′′
′′
76.4
69.8
26.3
3′′, 4′′, 1′′′
,
76.9
65.1
26.5
4.60 (br s)
5.94 (br d,
4.6)
3.06 (dd, 4.6,
17.5)
2.93 (br d,
17.5)
3, 1′′′, 2′′′, 6′′′
3
2
2
3
′′, 4′′, 2′′′, 6′′′
′′, 4′′, 2′′′
′′, 3′′
2
4
′′′, 6′′′
′′, 4a′′, gal-7
5.78 (br d, 4.6)
(
δ
165.9)
3.18 (dd, 4.6, 17.4)
2.91 (br d, 17.4)
4a′′, 5′′, 8a′′
4a′′, 5′′, 8a′′
2
5
′′, 3′′, 4a′′
′′, 8a′′
,
2
5
′′, 3′′, 4a′′
′′, 8a′′
,
′′
4a′′
99.30
157.5b
96.5
98.9
5′′
6′′
7′′
8′′
157.7b
96.8
6.06 (d, 2.3)
6.00 (d, 2.3)
4a′′, 5′′, 7′′, 8′′
4a′′, 7′′, 6′′
6.13 (d, 2.3)
6.04 (d, 2.3)
4a′′, 5′′, 7′′, 8′′
4a′′, 7′′, 6′′, 8a′′
157.4b
95.8
157.4b
95.6c
156.2
136.8
54.0
8a′′
157.3b
129.6
112.8
1
2
′′′
′′′
3.17 (br d,
1.1)
2, 5
′
, 1′′′, 3′′′
,
2
′
, 2′′, 3′′
,
4
′′′, 5′′′, 6′′′
3
′′′-OH
3
4
5
6
′′′
′′′
′′′
′′′
152.8
129.9
151.2
112.3
79.7
110.1
97.4
7.10 (s)
8.77 (s)
2
3
2
′
, 2′′, 1′′′, 2′′′
′′′, 4′′′, 5′′′
′, 3′, 4′
,
127.7
6.03 (br d,
1.8)
2
2
′′, 1′′′, 4′′′
6
′
′
, 2′′, 3′′
, 2′′′
3
3
′
-OH
′′′-OH
4.95 (s)
′, 3′′′
2
1
2, 6
121.9, 121.2
110.3, 109.9
121.3, 121.0
110.6, 110.5
7.20, 7.07
(each 2H, s)
gal-1, 2, 3, 4,
5, 6, 7
7.19, 7.02
(each 2H, s)
gal-1, 2, 3, 4,
5, 6, 7
galloyl
4
3, 5
7
139.4, 138.7
146.1, 145.9
166.1, 165.9
139.3, 139.2
145.8, 145.6
167.6, 166.8
{
a Compounds 8 and 9 were measured in acetone-d6 and in acetone-d6
+
benzene-d6 (9:1), respectively. b,c Assignments may be interchanged in each column.
1770, 1683, 1608, 1519, 1465. Anal. calcd for C44H34O23‚7/2H2O: C,
53.18; H, 4.16. Found: C, 53.21; H, 4.00. 1H and 13C NMR data: See
Table 1.
2.93 (1H, brd, J ) 17.9 Hz, H-4′), 2.85 (1H, brd, J ) 17.2 Hz, H-4),
2.81 (1H, brd, J ) 17.2 Hz, H-4′′), 2.56 (1H, dd, J ) 4.8, 17.9 Hz,
H-4′), 2.43 (1H, dd, J ) 4.6, 17.2 Hz, H-4), 2.31 (1H, dd, J ) 3.9,
17.2 Hz, H-4′′). 13C NMR (125 MHz, in acetone-d6): δ 166.53(2C)
(G-7, 7′′), 166.20 (G-7′), 158.95, 158.71, 158.41(2C), 157.47(2C),
157.28(3C) (C-5, 7, 8a, 5′, 7′, 8a′, 5′′, 7′′, 8a′′), 146.36, 146.12, 145.87-
(2C), 145.81(3C), 145.53, 144.96 (B-3, 5, 3′, 5′, 3′′, 5′′, G-3, 5, 3′, 5′),
144.55 (G-5′), 144.29 (G-3′), 138.82(2C) (G-4, G-4′′), 138.20 (G-4′),
139.95 (B-5′), 133.75 (B-4), 133.45 (B-4′′), 129.39 (B-1), 129.15 (B-
1′), 128.81 (B-1′′), 122.85 (G-1′), 122.01, 121.67 (G-1, 1′′), 117.09
(G-2′), 114.62 (B-2′′), 112.40 (B-2′), 111.95 (B-2), 111.68 (G-6′),
110.14(2C), 109.97(2C) (G-2, 6, 2′′, 6′′), 108.42 (B-6′), 108.05 (B-6),
107.72 (B-6′′), 99.12 (C-4a′′), 98.88 (C-4a′), 98.57 (C-4a), 96.51,
96.35(3C), 96.02, 95.75 (C-6, 8, 6′, 8′, 6′′, 8′′), 76.46 (C-2′′), 76.12
25
EGCg Trimer (14). White amorphous powder; [R]D -205.9° (c
0.18, acetone). MALDI TOF-MS m/z: 1393 [M + Na]+. FAB-MS
(positive mode) m/z: 1371 [M + H]+, (negative mode) m/z: 1369 [M
- H]-. HR FAB-MS m/z: 1371.2312 (calcd for C66H51O33, 1371.2310).
EtOH
1
UV λmax
nm (log ꢀ): 275 (4.48). H NMR (500 MHz, in acetone-
d6): δ 7.14 (1H, s, G-6′), 7.09, 6.97 (each 2H, s, G-2, 6, 2′′, 6′′), 6.95
(1H, d, J ) 0.5 Hz, B-6′), 6.88 (1H, d, J ) 0.5 Hz, B-6′′), 6.79 (1H,
d, J ) 0.5 Hz, B-6), 6.03, 6.00 (each 1H, d, J ) 2.2 Hz), 5.95 (3H, br
d, J ) 2.2 Hz), 5.92 (1H, d, J ) 2.2 Hz), 5.35 (1H, br d, J ) 4.6 Hz,
H-3), 5.14 (1H, br d, J ) 4.8 Hz, H-3′), 5.03 (1H, br d, J ) 3.9 Hz,
H-3′′), 4.78 (1H, br s, H-2), 4.68 (1H, br s, H-2′), 4.60 (1H, br s, H-2′′),

Oxidation of Epigallocatechin Gallate
J. Agric. Food Chem., Vol. 53, No. 19, 2005 7575
with C-2′, C-4′, and a remaining sp2 carbon at δ 142.5 (C-3′).
These HMBC correlations permitted construction of a 3-hy-
droxy-3-cyclohexene-1,2-dione structure for this B ring. The
unsaturation index (21) calculated from the molecular formula
and chemical shifts of C-1′ (δ 88.6) and C-3′′′ (δ 152.8)
suggested occurrence of an ether linkage between C-1′ and
C-3′′′. The planar structure of 8 was consequently deduced from
these spectroscopic data.
The presence of a 1,2-diketone structure was confirmed by
reaction with o-phenylenediamine, which yielded the quinoxa-
line derivative 8a. Interestingly, in the H NMR spectrum of
Figure 4. Structures of compounds 12 and 13.
1
8a, the A ring H-8 resonated at a significantly higher field (δ
4.99, ∆δ about 1.0 ppm) as compared to those of usual flavan-
3-ols (around δ 6.0). This unusual upfield shift could only be
explained by the shielding effect caused by the quinoxaline
residue. As shown in Figure 5, there are two possible
configurations at C-1′ (A and B). The bulky galloyl group was
oriented to the opposite side of the other flavan-3-ol unit owing
to steric hindrance; therefore, in structure B (C-1′ S), H-8 could
not be shielded by the quinoxaline moiety. On the other hand,
in structure A (C-1′ R), where the galloyl group is oriented
outside the molecule, H-8 is located directly underneath the
quinoxaline plane and was strongly shielded. The configuration
of C-1′ was thus concluded to be R.
(C-2′), 76.02 (C-2), 68.70 (C-3′′), 68.59 (C-3), 68.37 (C-3′), 27.36 (C-
4′′), 26.90(2C) (C-4, 4′). Important HMBC correlations (HfC):
G-6′fG-1′, 2′, 5′, 4′ 7′; H-3′fG-7′, C-4a′; B-6′′fC-2′′, B-1′′, 2′′, 3′′,
4′′, 5′′; H-2′′fB-1′′, 2′′, 6′′, C-3′′; H-3fG-7, C-4a; H-2fB-1, 2, 6;
H-2′fB-1′, 2′, 6′.
RESULTS AND DISCUSSION
(-)-Epigallocatechin 3-O-gallate (1) was enzymatically oxi-
dized with Japanese pear fruit homogenate, which was previ-
ously shown to have a strong ability to convert epigallocatechin
and epicatechin into theaflavin (19). HPLC analysis of the
reaction mixture showed peaks attributable to 1, dehydro-
theasinensin A (4), quinone dimer A (5), and a new product 9
(Figure 3B). 2-Mercaptoethanol was added to the mixture to
hydrogenate the unstable quinone products into stable phenols,
and HPLC analysis of the resulting solution showed complete
conversion of 4 into theasinensin A (6) (Figure 3C). Preliminary
experiments using several reducing agents, such as ascorbic acid,
sodium cyanoborohydride, and sodium bisulfite, showed 2-mer-
captoethanol to be the most effective for this purpose. Separation
of the products was performed by chromatography over MCI-
gel CHP20P, Sephadex LH-20, and Chromatorex ODS, resulting
in isolation of six products, 5, 6, 8, 9, 14, and epitheaflagallin
3-O-gallate (12) (20) (total: 48%), along with recovery of 1
(0.5%). It should be noted that the black tea pigment 12, which
possesses a benzotropolone ring related to that of theaflavin
(Figure 4), was produced by oxidation of 1 without participation
of catechol type catechins.
Dehydrotheasinensin AQ (8) was obtained as a yellow
amorphous powder and showed UV absorptions at 277 and 447
nm. On the basis of numbers of carbon signals observed in the
13C NMR spectrum, FAB-MS [m/z 913 (M + H)+, 935 (M +
Na)+], and elemental analysis, the molecular formula was shown
to be C44H32O22. The 1H and 13C NMR spectra (Table 1)
indicated the presence of two galloyl esters in the molecule.
This was consistent with dark blue coloration with the FeCl3
reagent. The spectra also showed two sets of flavan-3-ol A and
C rings in the molecule, and the coupling patterns of the signals
were similar to those of 1. Large downfield shifts of C rings
H-3 (δ?5.91) and H-3′′ (δ 5.78) confirmed the location of the
two galloyl esters at these positions. In the HMBC spectrum
(Table 1), the H-2′′ signal was correlated with three aromatic
carbons (C-1′′′, C-2′′′, and C-6′′′), and the aromatic methine
proton H-6′′′ showed cross-peaks with five aromatic carbons
(C-1′′′-C-5′′′) in addition to C-2′′. The chemical shifts of these
carbons indicated that a pyrogallol ring was attached to C-2′′′.
H-6′′′ showed a long-range (4J) correlation with an sp2 carbon
at δ 136.1 (C-2′), which correlated with H-2. Because H-2 also
showed cross-peaks with an oxygenated quaternary carbon (δ
88.6, C-1′) and a methylene carbon (δ 47.7, C-6′), another B
ring is not aromatic. In turn, the H-6′ methylene protons showed
correlations with two carbonyl carbons (C-4′ and C-5′).
Furthermore, an enolic hydroxyl proton at δ 8.77 was correlated
HPLC analysis of the crude theaflavin fraction obtained from
commercial black tea demonstrated the presence of 8 (Figure
6). The structure of this new pigment is closely related to that
of dehydrotheasinensin A (4) and was probably produced by
intramolecular addition of a phenolic hydroxyl group at C-3′′′
to the conjugated double bond of 4. The configuration of C-1′
of 8 was consistent with that of 4 and the atropisomerism of its
reduction product 6.
Product 9 was obtained as a white amorphous powder, and
1
its H and 13C NMR spectra showed signals arising from two
sets of 3-O-galloyl-flavan-3-ol A and C rings (Table 1). The
remaining 12 signals were attributable to two carbonyl (C-3′
and C-5′), two olefinic (C-1′′′ and C-6′′′), three aliphatic methine
(C-2′, C-6′, and C-2′′′), one acetal (C-4′′′), and four oxygenated
quaternary (C-1′, C-4′, C-3′′′, and C-5′′′) carbons. The total
number of carbon signals observed in the spectrum indicated
that this compound was a dimer of 1. Results of FAB-MS (m/z
931 [M + H]+), MALDI TOF-MS (m/z: 953 [M + Na]+) and
elemental analysis suggested the molecular formula C44H34O23.
Mutual HMBC correlations (Table 1) between CH-2, C-1′, CH-
2′, and CH-6′ indicated that the C ring C-2 was attached to
C-1′, and this was supported by NOESY correlations (Table
1
1) and long-range H-1H couplings between H-2, H-2′, and
H-6′. On the other hand, the HMBC and NOESY correlations
between CH-2′′, C-1′′′, CH-2′′′, and CH-6′′′ suggested the
relative locations of these carbons shown in formula 9 (Figure
1
2). This was supported by observation of H-1H long-range
couplings between H-2′′ and H-2′′′, H-2′′ and H-6′′′, and H-2′′′
and H-6′′′. In the HMBC spectrum, H-2′ and H-6′ were
correlated with carbonyl carbons C-3′ and C-5′, respectively,
and H-6′ was also correlated with the oxygenated carbon C-4′.
The remaining two oxygenated quaternary carbons C-3′′′ and
C-5′′′ and the acetal carbon C-4′′′ showed correlation peaks with
H-2′′′, while the acetal carbon C-4′′′ was coupled with H-6′′′.
4
In addition, C-5′′′ was correlated through J coupling with the
H-2′′′ located at a homoallylic position. Because C-1′′-C-6′′
and C-1′′′-C-6′′′ originated from pyrogallol B rings of 1, these
HMBC correlations suggested the disposition of these carbons
as shown in formula 9. The 1H NMR spectrum exhibited a D2O
exchangeable singlet due to a hydroxyl group correlated with

7576 J. Agric. Food Chem., Vol. 53, No. 19, 2005
Tanaka et al.
Figure 5. Structures of 8 and 8b. Structures A and B represent 8a and its 1′-S isomer, respectively.
Figure 6. HPLC profile (at 450 nm) of a crude theaflavin fraction of commercial black tea.
C-3′′′ and C-2′ in the HMBC spectrum, suggesting a C-C
linkage between C-2′ and C-3′′′. This was supported by the
appearance of HMBC correlations of H-2′ with C-3′′′ and C-2′′′,
and observation of NOESY correlations of the hydroxyl proton
with H-2′ and H-2′′′. Another C-C linkage between C-3′ and
C-5′′′ was deduced from HMBC correlations of H-5′ with C-4′′′
and C-5′′′. Furthermore, HMBC correlations of H-2′′′ with the
C-5′ carbonyl carbon suggested additional C-C bonding
between C-4′ and C-2′′′. The presence of these C-C linkages
was supported by NOESY correlations between H-6′ and H-6′′′
and H-2′ and H-2′′′, respectively. Taking the unsaturation index28
calculated from the molecular formula and chemical shift of
the acetal carbon C-4′′′ (δ 110.1) into account, formation of an
acetal ring between C-1′ and C-4′′′ was suggested. On the basis
of the above spectroscopic observations, the structure of 9 could
be represented by formula 9 and tentatively named EGCg
quinone dimer B. The structure of this compound was related
to that of EGCg quinone dimer A (5), and a plausible mechanism
for formation of 9 from 1 is illustrated in Figure 7, in which
successive addition of electron-rich carbons to carbonyl groups
forms the caged structure. However, we could not determine
the absolute configuration of this compound.
Compound 14 was isolated as a white amorphous powder,
and MALDI TOF-MS (m/z: 1393 [M + Na]+) and FAB-MS
(positive mode: m/z 1371 [M + H]+; negative mode: m/z 1369
[M - H]-) indicated that it is a trimer of 1. The high resolution
FAB-MS showed a [M + H]+ peak at m/z 1371.2312, indicating
the molecular formula C66H50O33. The 1H and 13C NMR spectra
were related to those of 6 and 7; however, they exhibited signals
arising from three sets of flavan-3-ol A and C rings and two
galloyl groups. In the 1H NMR spectrum, four one-proton
aromatic singlets were observed as well as two two-proton

Oxidation of Epigallocatechin Gallate
J. Agric. Food Chem., Vol. 53, No. 19, 2005 7577
selectively formed by reduction of 4 with 2-mercaptoethanol,
and theasinensin D (7) was not detected in this experiment (15).
In addition, by analogy with formation of theasinensins (6 and
7 in Figure 2), product 14 was presumed to be produced by
reduction of product 14a, which is formed by oxidative coupling
of quinones 4a and 1a, as shown in Figure 8. During the
purification procedure, the presence of an isomer of 14 showing
similar chromatographic behavior was suggested. Although
purification failed, this isomer might be an atropisomer differing
in configuration of the galloyl-B′′ ring biphenyl bond. In the
course of our studies on catechin oxidation, this product
represents the first example showing participation of galloyl
groups in intermolecular oxidative coupling. As for the ben-
zotropolone moiety, some interesting pigments produced by
condensation between galloyl groups and catechol rings have
previously been reported (12, 21).
In this experiment, reduction of unstable quinone metabolites,
including 4, with 2-mercaptoethanol in the initial oxidation
mixture of 1 effectively reduced production of inseparable minor
dismutation products, which interfere with chromatographic
purification of metabolites. As a result, we isolated three new
oxidation products including a new black tea pigment. The new
pigment 8 was structurally related to dehydrotheasinensin E (13)
(Figure 4), which was produced by oxidative coupling of (-)-
epigallocatechin (19). In the structure of 13, one carbonyl carbon
of 1,2-diketone formed an intramolecular hemiacetal ring with
the C ring hydroxyl group. In addition, the absolute configu-
ration of 13 at the benzylic methine was shown to be S, which
is opposite to that of 8.
Figure 7. Proposed mechanism for production of quinone dimer 9.
singlets due to galloyl groups. These results suggested that the
B ring C-2 of an additional epigallocatechin-3-O-gallate was
attached to the C-2 of a galloyl group of theasinensin through
a C-C bond. This was supported by HMBC spectroscopic
analysis (Figure 8), which showed a correlation of H-3′ with
the carboxyl group (C-G7′) of the galloyl group attached to
the additional epigallocatechin-3-O-gallate moiety. The aromatic
methine proton of the galloyl group (H-G6′, δ 7.14) showed
HMBC correlations with C-G1′ (δ 122.85), C-G2′ (δ 117.09),
C-G5′ (δ 144.55), C-G4′ (δ 138.20), and C-G7′ (δ 166.2),
and chemical shifts of these carbons were slightly different from
those of usual galloyl groups. Consequently, the structure of
this trimer was determined to be as shown by formula 14. The
absolute configuration of the B-B′ ring biphenyl bond of the
theasinensin moiety in 14 was suggested to be R, because
theasinensin A (6), which has a R biphenyl bond, was stereo-
Production of the quinone dimers 5 and 9 indicated the high
reactivity of quinone 1a. These quinone dimers were unstable
and decomposed when their aqueous solutions were heated;
therefore, it is possible that they do not exist in commercial
black tea. Trimer 14 was probably generated by reduction of
14a, which has a structure analogous to 4. If 14a is produced
at the fermentation stage of black tea production, it will undergo
oxidation-reduction dismutation in a manner similar to that of
4, resulting in generation of a complex mixture of reduction
and oxidation products. Therefore, production of 14 suggested
Figure 8. Proposed mechanism for production of trimer 14 and selected HMBC correlations.

7578 J. Agric. Food Chem., Vol. 53, No. 19, 2005
Tanaka et al.
(12) Sang, S.; Tian, S.; Meng, X.; Stark, R. E.; Rosen, R. T.; Yang,
C. S.; Ho, C.-T. Theadibenzotropolone A, a new type pigment
from enzymatic oxidation of (-)-epicatechin and (-)-epigallo-
catechin gallate and characterized from black tea using LC/MS/
MS. Tetrahedron Lett. 2002, 43, 7129-7133.
that generation and subsequent intermolecular coupling of
galloyl quinones contribute to formation of minor polyphenols
in black tea. Studies to show the evidence of 14a being
generated during tea fermentation are now in progress.
(13) Hashimoto, F.; Nonaka, G.; Nishioka, I. Tannins and related
compounds. LXIX. Isolation and structure elucidation of B,B′-
linked bisflavanoids, theasinensins D-G and oolongtheanin from
oolong tea. (2). Chem. Pharm. Bull. 1988, 36, 1676-1684.
(14) Hashimoto, F.; Nonaka, G.; Nishioka, I. Tannins and related
compounds. CXIV. Structures of novel fermentation products,
theogallinin, theaflavonin and desgalloyl theaflavonin from black
tea, and changes of tea leaf polyphenols during fermentation.
Chem. Pharm. Bull. 1992, 40, 1383-1389.
ABBREVIATIONS USED
COSY, correlation spectroscopy; FABMS, fast atom bom-
bardment mass spectroscopy; HMBC, heteronuclear multiple
bond connectivity; HPLC, high-performance liquid chromatog-
raphy; HSQC, heteronuclear single quantum coherence; NOE-
1
SY, H NMR nuclear Overhauser and exchange spectroscopy.
ACKNOWLEDGMENT
(15) Tanaka, T.; Watarumi, S.; Matsuo, Y.; Kamei, M.; Kouno, I.
Production of theasinensins A and D, epigallocatechin gallate
dimers of black tea, by oxidation-reduction dismutation of
dehydrotheasinensin A. Tetrahedron 2003, 59, 7939-7947.
(16) Tanaka, T.; Mine, C.; Watarumi, S.; Fujioka, T.; Mihashi, K.;
Zhang, Y.-J.; Kouno, I. Accumulation of epigallocatechin
quinone dimers during tea fermentation and formation of
theasinensins. J. Nat. Prod. 2002, 65, 1582-1587.
(17) Tanaka, T. i.; Kouno, I. Oxidation of tea catechins: Chemical
structures and reaction mechanism. Food Sci. Technol. Res. 2003,
9, 128-133.
(18) Valcic, A.; Muders, A.; Jacobsen, N. E.; Liebler, D. C.;
Timmermann, B. N. Antioxidant chemistry of green tea cat-
echins. Identification of products of the reaction of (-)-
epigallocatechin gallate with peroxyl radicals. Chem. Res.
Toxicol. 1999, 12, 382-386.
We are grateful to K. Inada and N. Yamaguchi (Nagasaki
University) for NMR and MS measurements.
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