Oligomerization mechanism of tea catechins during tea roasting

Article abstract of DOI:10.1016/j.foodchem.2019.01.163

Roasting of green tea causes oligomerization of tea catechins, which decreases the astringency. The aim of this study was to elucidate the oligomerization mechanism. The ¹³C NMR spectrum of the oligomer fraction showed signals arising from catechin and sugar residues. Heating of epigallocatechin-3-O-gallate with ¹³C-labeled glucose (150 °C for 2 h) suggested that condensation of sugars with catechin A-rings caused the oligomerization. The dimeric product obtained by heating for a shorter period (30 min) suggested cross-linking occurred between sugars and catechin A-rings. Furthermore, heating of phloroglucinol, a catechin A-ring mimic, with glucose, methylglyoxal, and dihydroxyacetone, confirmed that the basic mechanism included reaction of the catechin A-ring methine carbons with carbonyl carbons of glucose and their pyrolysis products.

Full text of DOI:10.1016/j.foodchem.2019.01.163

Accepted Manuscript
Analytical Methods
Oligomerization mechanism of tea catechins during tea roasting
Hitomi Morikawa, Keita Okuda, Yuji Kunihira, Aoi Inada, Chika Miyagi,
Yosuke Matsuo, Yoshinori Saito, Takashi Tanaka
PII:
S0308-8146(19)30238-9
DOI:
https://doi.org/10.1016/j.foodchem.2019.01.163
FOCH 24258
Reference:
To appear in:
Food Chemistry
Received Date:
Revised Date:
Accepted Date:
19 October 2018
29 December 2018
24 January 2019
Please cite this article as: Morikawa, H., Okuda, K., Kunihira, Y., Inada, A., Miyagi, C., Matsuo, Y., Saito, Y.,
Tanaka, T., Oligomerization mechanism of tea catechins during tea roasting, Food Chemistry (2019), doi: https://
doi.org/10.1016/j.foodchem.2019.01.163
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Food Chemistry (Research paper)
Oligomerization mechanism of tea catechins during tea roasting
a
a
a
a
b
Hitomi Morikawa , Keita Okuda , Yuji Kunihira , Aoi Inada , Chika Miyagi , Yosuke
a
a
a,*
Matsuo , Yoshinori Saito , Takashi Tanaka
a
Department of Natural Product Chemistry, Graduate School of Biomedical Sciences,
Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
b
Department of Natural Product Chemistry, School of Pharmaceutical Sciences,
Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
*
Corresponding author: Department of Natural Product Chemistry, Graduate School of
Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521,
Japan, Tel: +81 95 8192433, E-mail address: t-tanaka@nagasaki-u.ac.jp (T. Tanaka)
1

Abstract: Roasting of green tea causes oligomerization of tea catechins, which
decreases the astringency. The aim of this study was to elucidate the oligomerization
1
3
mechanism. The C NMR spectrum of the oligomer fraction showed signals arising
from catechin and sugar residues. Heating of epigallocatechin-3-O-gallate with
1
3
C-labeled glucose (150°C for 2 h) suggested that condensation of sugars with catechin
A-rings caused the oligomerization. The dimeric product obtained by heating for a
shorter period (30 min) suggested cross-linking occurred between sugars and catechin
A-rings. Furthermore, heating of phloroglucinol, a catechin A-ring mimic, with glucose,
methylglyoxal, and dihydroxyacetone, confirmed that the basic mechanism included
reaction of the catechin A-ring methine carbons with carbonyl carbons of glucose and
their pyrolysis products.
Keywords: roasted tea; catechin; sugar; methylglyoxal; dihydroxyacetone;
phloroglucinol; polyphenol
2

1
. Introduction
Roasting is an important method for processing, cooking, and preserving foods, and is
essential in the production of cocoa and coffee as it add characteristic aromas and
flavors. However, chemical changes occur in phenolic substances during the roasting
process and these changes are complex and not clearly understood (Taeye, Bodart,
Caullet, & Collin, 2017; Moreira, et al., 2017). In Japan, roasted green tea is a popular
processed tea, and it is served in traditional Japanese multi-course dinners for the elderly.
Roasting adds characteristic flavor via caramelization and Maillard reactions and
decreases the caffeine content by sublimation, which is beneficial to reduce the effect of
tea on sleep. In addition, roasted green tea has a milder taste than non-roasted green tea,
and this is thought to be caused by a decrease in the content of astringent tea catechins.
A comparison of HPLC profiles of an original green tea product and a roasted green tea
(
Fig. 1) showed roasting resulted in epimerization at the catechin C-2 position (Suzuki,
et al., 2003; Seto, Nakamura, Nanjo, & Hara, 1997) and generation of oligomeric
products, which were detected as a broad hump on the baseline. Astringency is caused
by interactions with salivary proteins (Haslam, 1996; Baxter, Lilley, Haslam, &
Williamson, 1997); thus, the epimerization products (1a–4a) do not contribute to the
decrease in astringency because the affinities of 2,3-trans epimerization products in
human serum albumin are stronger than in the original 2,3-cis tea catechins (Ishii et al.,
2
010). Therefore, oligomerization of tea catechins is mainly responsible for the decrease
in tea astringency with roasting; however, the chemical mechanisms for this are
unknown. The aim of this study was to elucidate the oligomerization mechanism using
model experiments and spectroscopic methods.
3

2
. Materials and methods
2
.1. Materials
Green tea leaves were supplied by Nagasaki Agriculture and Forestry Technical
Development Center, Higashisonogi Tea Research Station (Nagasaki, Japan).
1
3
13
1
- C-D-Glucose and U- C -D-glucose were purchased from Cambridge Isotope
6
Laboratories, Inc. (MA, USA). Epigallocatechin-3-O-gallate was isolated from
commercial green tea according to a reported method (Nonaka, Kawahara, & Nishioka,
1
983).
2
.2. Analytical procedures
Ultraviolet (UV)–visible spectra were obtained using a JASCO V-560
1
13
spectrophotometer (Jasco Co., Tokyo, Japan). H and C NMR spectra were recorded in
acetone-d , CD OD, and DMSO-d (Kanto Chemical Co. Inc., Tokyo, Japan) at 27°C
with a JEOL JNM-AL400 spectrometer (JEOL Ltd., Tokyo, Japan) operating at 400 and
6
3
6
1
13
1
00 MHz for H and C nuclei, respectively. The coupling constants are expressed in
hertz, and chemical shifts are presented on the delta (ppm) scale. Column
chromatography was performed using Sephadex LH-20 (25100 μm, GE Healthcare
Bio-Science AB, Uppsala), Diaion HP20SS (Mitsubishi Chemical, Japan), MCI-gel
CHP 20P (75–150 m; Mitsubishi Chemical, Tokyo, Japan), Chromatorex ODS
(
100–200 mesh; Fuji Silysia Chemical, Kasugai, Japan), and silica gel 60N (100–250
μm, Kanto Chemical Co., Tokyo, Japan) columns. Thin layer chromatography (TLC)
was performed on precoated Kieselgel 60 F254 plates (0.2-mm thick, Merck KGaA,
Darmstadt, Germany) with toluene:ethyl formate:formic acid (1:7:1, v/v/v) and
CHCl
3
:MeOH:water (14:6:1, v/v/v). Spots were detected under UV illumination after
4

spraying with 2% ethanolic FeCl
HPLC was performed using a Cosmosil 5C18-AR II (Nacalai Tesque Inc., Kyoto, Japan)
column (4.6 mm i.d. × 250 mm) with a gradient elution from 4%–30% CH CN (39 min)
and 30%–75% CH CN (15 min) in 50 mM H PO . The mobile phase flow rate was 0.8
mL/min, and detection was achieved using a Jasco MD-910 photodiode array detector.
3
or a 5% sulfuric acid reagent and heating. Analytical
3
3
3
4
2
.3. Heating of (−)-epigallocatechin-3-O-gallate (2)
An aqueous solution (200 mL) of (−)-epigallocatechin-3-O-gallate (2) (1.0 g)
was lyophilized, and the resulting white powder was heated at 150°C for 60 min in an
electric furnace (Yamato Scientific co., ltd., Tokyo, Japan). The reaction mixture was
subjected to Sephadex LH-20 column chromatography (3 cm i.d. × 25 cm) with
0
–100% MeOH containing 1% trifluoroacetic acid (10% stepwise, each 100 mL) to
produce (−)-gallocatechin-3-O-gallate (142.5 mg), gallic acid (18.2 mg), and tricetinidin
12.2 mg) (Kuhnert N., Clifford M. N., & Radenac A-G., 2001; Coggon, Moss, Graham,
Sanderson, 1973) and recover 2 (756 mg).
(
&
2
.4. Roasting of green tea leaves and separation of oligomeric polyphenols
Green tea leaves (20 g) were heated at 180°C in an electric furnace for 30 min.
After cooling, the leaves were extracted twice with acetone:H O (3:2, v/v, 300 mL) at r.t.
2
The extract was concentrated using a rotary evaporator, and the resulting aqueous
solution (150 mL) was partitioned with EtOAc (150 mL) twice to produce an EtOAc
fraction (2.97 g). The aqueous layer was first concentrated to remove residual EtOAc
and then subjected to Diaion HP20SS column chromatography (3 cm i.d. × 20 cm) with
H
2
O:MeOH (0–100%, 20% stepwise, each 100 mL). The eluate was monitored by TLC
5

and separated into two fractions (Fr.). Fraction 1 mainly contained sugars (3.1 g), and Fr.
contained catechins and caffeine (2.1 g). Fraction 2 was subjected to size-exclusion
2
column chromatography using Sephadex LH-20 (2 cm i.d. × 55 cm) with 7 M
urea:acetone (2:3, v/v, containing conc. HCl at 5 mL/L) (Yanagida, Shoji, & Shibusawa,
2
003) to yield oligomers (241 mg) and a mixture of catechins, caffeine, and oligomers
1.6 g). The oligomers were detected at the origin in TLC analysis and as a broad hump
on the baseline in HPLC analysis (Fig. S1).
(
2
.5. Heating of (−)-epigallocatechin-3-O-gallate (2) with sucrose
−)-Epigallocatechin-3-O-gallate (2) (1.0 g) and sucrose (1.0 g) were
dissolved in H O (10 mL) and heated on a hot plate until most of the water evaporated.
(
2
The resulting paste was heated at 150°C for 2 h in an electric furnace, and the products
were separated by Sephadex LH-20 column chromatography (3 cm i.d. × 12 cm) with
0
–100% MeOH (20% stepwise, each 100 mL) and then MeOH:H O:acetone (60:20:20
2
and 0:1:1 v/v/v steps, each 100 mL) to produce two fractions. The first fraction was
purified by Diaion HP20SS column chromatography (3 cm i.d. × 12 cm) with 0–100%
MeOH (10% stepwise, each 100 mL) to produce the pure oligomers (307 mg). The
second fraction (1.16 g) also contained oligomers as the major constituents as well as
gallic acid and minor impurities (Fig. S2).
2
.6. Heating of (−)-epigallocatechin-3-O-gallate (2) with glucose
−)-Epigallocatechin-3-O-gallate (2) (510 mg) and D-glucose (386 mg) were
dissolved in H O:acetone (4:1, v/v, 2.5 mL) in a petri dish and concentrated by heating
(
2
at 75°C. The resulting paste was heated at 150°C for 2 h in the electric furnace, and the
6

products were separated by Sephadex LH-20 column chromatography (2 cm i.d. × 25
cm) with 60%–100% MeOH (20% stepwise, each 100 mL) and then
MeOH–H O–acetone (90:5:5, 80:10:10, 60:20:20, and 0:1:1 v/v/v steps, each 100 mL)
2
to give five fractions: Fr. 1 (172 mg), Fr. 2 (186 mg), Fr. 3 (290.4 mg), Fr. 4 (77 mg),
and Fr. 5 (192 mg). HPLC of Fr. 5 showed that it only contained oligomeric products.
1
3
2
.7. Heating of (−)-epigallocatechin-3-O-gallate (2) with C-labeled glucose
Similarly, 2 (510 mg) was heated with a 9:1 mixture of glucose and
1
3
1
- C-D-glucose (total of 386 mg) to yield the oligomers (182 mg). Experiments using 2
1
3
(
510 mg) and a 9:1 mixture of glucose and U- C6-D-glucose (total of 386 mg) afforded
the oligomers (190 mg).
2
.8. Monomeric and dimeric products of 2 produced by heating with glucose
An aqueous solution (400 mL) of 2 (2.0 g) and D-glucose (1.6 g) was
lyophilized, and the resulting white powder was heated at 150°C for 30 min. The
reaction mixture was subjected to MCI-gel CHP20P column chromatography (3 cm i.d.
×
20 cm) with 0–100% MeOH (10% stepwise, each 100 mL) to give five fractions: Fr. 1
773 mg), Fr. 2 (246 mg), Fr. 3 (654 mg), Fr. 4 (402 mg), and Fr. 5 (139 mg). Separation
of Fr. 1 using the Sephadex LH-20 column (2 cm i.d. × 20 cm) with 0–100 % MeOH in
O (10% stepwise, each 100 mL) gave 5 (32.2 mg), 6 (95.7 mg), and 7 (12.3 mg).
(
H
2
Fraction 3 was subjected to Sephadex LH-20 column chromatography (3 cm i.d. × 20
cm) with 0–100% MeOH (10% stepwise, each 100 mL) to produce 2 (93.8 mg) and 2a
(
71.9 mg). Fractions 4 and 5 mainly contained oligomeric products, and separation of Fr.
4
using the Sephadex LH-20 column (3 cm i.d. × 20 cm) with 20–100% MeOH (10%
7

stepwise, each 100 mL) and subsequent purification using Chromatorex ODS column
chromatography (3 cm i.d. × 25 cm) with 0–50% MeOH (5% stepwise, each 100 mL)
furnished 8 (10.4 mg).
2
.8.1. 8-C-Glucosyl-(−)-epigallocatechin-3-O-gallate (5)
Brown amorphous powder, [α]
D
−195.4 (c 0.10, MeOH). FAB-MS m/z: 621
16: 621.1456),
+
+
[
M+H] , 643 [M+Na] . HR-FABMS m/z: 621.1465 (Calcd for C28
29
H O
6
43.1293 (Calcd for C28
H28NaO16: 643.1275). UV λmax nm (log ε): 211 (4.88), 276
−
1
1
(
4.02). IR νmax cm : 3388, 1691, 1613, 1537, 1453, 1448. H NMR (acetone-d
6
+ D O)
2
: 2.96 (2H, m, H-4), 3.42–3.88 (m, glc-2-6), 4.98 (1H, d, 9.8 Hz, glc-1), 5.06 (1H, br s,
H-2), 5.34 (1H, m, H-3), 5.98 (1H, s, H-6), 6.75 (2H, s, B-2,6), 6.99 (2H, s, galloyl-H).
1
3
C NMR (acetone-d
6
+ D O) : 26.3 (C-4), 61.6 (glc-6), 69.4 (C-3), 70.8 (glc-4), 73.5
2
(
glc-2), 76.2 (glc-1), 77.9 (C-2), 78.7 (glc-3), 81.6 (glc-5), 96.8 (C-6), 98.4 (C-4a),
1
1
1
2
03.5 (C-8), 106.3 (B-ring-2,6), 109.8 (galloyl-2,6), 121.1 (galloyl-1), 130.6 (B-ring-1),
32.7 (B-ring-4), 138.7 (galloyl-4), 145.6 (galloyl-3,5), 146.0 (B-ring-3,5), 154.0 (C-8a),
56.3 (C-7), 157.0 (C-5), 166.4 (galloyl-7).
.8.2. 6-C-Glucosyl-(−)-epigallocatechin-3-O-gallate (6)
Brown amorphous powder, [α]
D
−66.6 (c 0.10, MeOH). FAB-MS m/z: 621
16: 621.1456),
+
+
[
M+H] , 643 [M+Na] . HR-FABMS m/z: 621.1451 (Calcd for C28
29
H O
6
43.1279 (Calcd for C28
H28NaO16: 643.1275). UV λmax nm (log ε): 211 (4.83), 276
−
1
1
(
3.99). IR νmax cm : 3393, 1681, 1613, 1536, 1453, 1337. H NMR (acetone-d
6
+ D O)
2

4
6
6
: 2.86 (1H, dd, 3, 18 Hz, H-4), 3.00 (1H, dd, 3, 18 Hz, H-4), 3.48–3.82 (m, glc-2-6),
.83 (1H, d, 9.6 Hz, glc-1), 5.01 (1H, br s, H-2), 5.43 (1H, m, H-3), 6.06 (1H, s, H-8),
1
3
.61 (2H, s, B-2,6), 6.99 (2H, s, galloyl-H). C NMR (acetone-d
6
+ D O) : 26.5 (C-4),
2
1.3 (glc-6), 69.5 (C-3), 70.1 (glc-4), 74.6 (glc-2), 76.8 (glc-1), 78.0 (C-2), 79.0 (glc-3),
8

8
1.8 (glc-5), 96.6 (C-8), 100.3 (C-4a), 105.4 (C-6), 106.5 (B-ring-2,6), 109.8
galloyl-2,6), 121.4 (galloyl-1), 130.3 (B-ring-1), 133.0 (B-ring-4), 138.8 (galloyl-4),
45.8 (galloyl-3,5), 146.0 (B-ring-3,5), 155.3 (C-7), 155.6 (C-5), 156.0 (C-8a), 166.5
galloyl-7).
.8.3 6-C-Substituted product of (−)-epigallocatechin-3-O-gallate (7)
(
1
(
2
Brown amorphous powder, [α]
D
−136.8 (c 0.12, MeOH), UV (MeOH) λmax (log
−
1
+
ε): 268 (4.04), IR νmax cm : 3404, 1693, 1619, 1537, 1455, 1339. HR-ESI-MS [M+H]
+
1
m/z: 765.1873 [M+H] (Calcd for C34
H
37
O
20, 765.1873). H NMR (acetone-d
: 2.98 (2H, m, H-4), 5.07 (1H, br s, H-2), 5.33 (1H, br, s, H-3), 6.75 (2H, s, B-ring-2,6),
.98 (2H, s, galloyl-2,6), C-6-sugar unit: 3.01 (1H, d, J = 16.5 Hz,, H-1), 3.20 (1H, d, J
16.5 Hz, H-1), 3.63 (2H, m, H-6), 3.89 (1H, m, H-5), 4.05 (1H, d, J = 8.3 Hz, H-3),
.16 (1H, t, J = 8.3 Hz, H-4), C-8-glucosyl: 3.45 (1H, m, H-5), 3.66 (m, H-4), 3.67 (m,
6
+ D
2
O)

6
=
4
1
3
H-3), 3.83 (m, H-2), 3.84 (2H, m, H-6). C NMR (acetone-d
9.2 (C-3), 78.1 (C-2), 94.8 (C-4a), 104.5 (C-6), 104.7 (C-8), 106.9 (B-ring-2,6), 109.8
galloyl-2,6), 121.0 (galloyl-1), 130.4 (B-ring-1), 132.7 (B-ring-4), 138.8 (galloyl-4),
45.5 (B-ring-3,5), 146.0 (galloyl-3,5), 151.6 (C-7), 153.3 (C-5), 157.9 (C-8a), 166.4
galloyl-7); C-6-sugar unit: 34.8 (C-1), 64.3 (C-6), 76.6 (C-4), 80.7 (C-3), 83.9 (C-5),
16.9 (C-2); C-8-glucosyl: 61.4 (C-6), 70.6 (C-4), 73.8 (C-3), 75.4 (C-1), 78.6 (C-2),
6
+ D
2
O) : 26.0 (C-4),
6
(
1
(
1
8
1.6 (C-5).
2
.8.4. Dimeric product (8)
Brown amorphous powder, [α] −168.6 (c 0.10, MeOH), UV (MeOH) λmax (log
D
−
1
ε): 211 (5.02), 269 (4.41), IR νmax cm : 3438, 1697, 1620, 1537, 1445. HR-ESI-MS
+
+
1
[
(
M+Na] m/z: 1065.1955 [M+Na] (Calcd for C50H42NaO25: 1065.1913). H NMR
acetone-d
6
+ D
2
O) : 2.98, 3.09 (each 2H, m, H-4, 4'), 5.06, 5.12 (each 1H, br s, H-2,
9

2
'), 5.51 (2H, br, s, H-3, 3'), 6.13, 6.51 (each 1H, s, A-ring-H), 6.67, 6.68 (each 2H, s,
B-ring-2,6), 7.00, 7.12 (each 2H, s, galloyl-2,6), sugar unit: 3.68 (1H, ddd, J = 2.8, 7.1,
.2, 3.79, H-5), 3.79 (1H, dd, J = 7.1, 11.4 Hz,, H-6), 4.01 (1H, dd, J = 2.8, 11.4 Hz,,
H-6), 4.63 (1H, dd, J = 3.0, 9.2 Hz, H-4), 5.39 (1H, dd, J = 1.6, 3.0 Hz, H-3), 6.52 (1H,
9
1
3
d, J = 1.6 Hz, H-2). C NMR (acetone-d
'), 78.1, 79.1 (C-2, 2'), 91.1, 98.2 (A-ring methine), 99.1, 101.9 (C-4a, 4a'), 102.9,
11.2 (A-ring C-6 or C-8), 106.5 (B-ring-2,6), 109.8, 109.9 (galloyl-2,6), 121.0, 121.3
galloyl-1), 129.9, 130.2 (B-ring-1), 133.0, 133.2 (B-ring-4), 138.8, 139.0 (galloyl-4),
6
+ D O) : 27.1, 27.3 (C-4, 4'), 69.1, 69.5 (C-3,
2
3
1
(
1
1
7
45.7, 145.8 (galloyl-3,5), 146.1, 146.2 (B-ring-3,5), 155.5 (C-8a), 155.3, 157.0 (C-5),
54.1, 156.6 (C-7), 166.1 (galloyl-7); C-6-sugar unit: 38.1 (C-3), 64.3 (C-6), 74.0 (C-4),
4.4 (C-5), 101.7 (C-2), 148.2 (C-1).
2
.9. Reaction of phloroglucinol and glucose
Phloroglucinol (6.3 g) and D-glucose (4.5 g) were dissolved in dimethyl
formamide (5 mL) and heated at 80°C for 24 h. The mixture was poured into H O (100
2
mL), and the resulting phloroglucinol precipitate (2.0 g) was removed by filtration. The
filtrate was applied to a Chromatorex ODS column (3 cm i.d. × 25 cm) with 0–100%
MeOH (10% stepwise, each 100 mL) to yield Fr. 1 and 10 (227 mg). Fraction 1 was
separated using Sephadex LH-20 column chromatography (4 cm i.d. × 30 cm) with
0
–100% MeOH (20% stepwise, each 200 mL) to give glucose, Fr. 1-1, and Fr. 1-2, and
recover phloroglucinol (1.7 g). Fraction 1-1 was purified via silica gel column
chromatography (CHCl :MeOH:H O, 70:30:5, and 60:40:10 v/v/v steps) to yield 9
378.7 mg). Similar silica gel column chromatography of Fr. 1-2 yielded an inseparable
mixture of stereoisomers of 11 (346.7 mg). Treatment of 11 (40 mg) with Ac O (0.8
3
2
(
2
10

mL) in pyridine (0.5 mL) at r.t. for 10 h and subsequent silica gel column
chromatography with hexane:acetone (3:2, v/v) afforded an acetate of one of the
isomers of 11 (47 mg).
2
.9.1. Phloroglucinol-C-β-D-glucoside (9)
White amorphous powder. []
D
+37.9 (c 0.1, MeOH). FAB-MS m/z: 289
+
−1
[
MH] . HR-FABMS m/z: 289.0923 (Calcd for C12
H
17
O : 289.0918). IR max cm :
8
1
3
3
373, 1620, 1452, 1148, 1040. H NMR (400 MHz, CD
3
OD) : 3.37 (1H, m, glc-5),
.46 (2H, m, glc-6), 3.74 (1H, dd, J = 12.1, 4.8 Hz glc-4), 3.83 (1H, dd, J = 9.8, 4.6 Hz,
glc-2), 3.86 (1H, brd, J = 4.6 Hz, glc-3), 4.79 (1H, d, J = 9.8 Hz, glc-1), 5.86 (2H, s,
1
3
H-3, 4). C NMR (100 MHz, CD
3
OD) : 62.4 (glc-6), 71.4 (glc-4), 73.8 (glc-2), 76.8
glc-1), 79.9 (glc-3), 82.4 (glc-5), 96.4 (C-3, 5), 104.3 (C-1), 158.9 (C-2, 6), 159.6
C-4).
.9.2. Dimeric product 10
Yellow amorphous powder. []
(
(
2
D
−24.4 (c 0.1, MeOH). FAB-MS m/z: 397
+
[
MH] . HR-FABMS m/z: 397.1145 (Calcd for C18
H
21
O10: 397.1134). UV max nm (log
−
1
1

): 216 (4.69). IR max cm : 3373, 1620, 1452, 1148, 1040. H NMR (400 MHz,
CD
.51 (1H, dd, J = 2.3, 7.3 Hz, sug-4), 3.59 (1H, ddd, J = 3.4, 5.8, 7.3 Hz, sug-5), 3.74
1H, dd, J = 2.3, 3.2 Hz, sug-3), 3.82 (1H, dd, J = 3.2, 6.5 Hz, sug-2), 4.53 (1H, d, J =.5
Hz, sug-1), 6.07 (1H, d, J = 2.3 Hz, H-5'), 6.08 (1H, d, J = 2.3 Hz, H-3'), 6.11 (1H, d, J
3
OD) : 3.48 (1H, dd, J = 5.8, 13.6 Hz, sug-6), 3.62 (1H, dd, J = 3.4, 13.6 Hz, sug-6),
3
(
13
=
2.3 Hz, H-3'), 6.12 (1H, d, J = 2.3 Hz, H-3'). C NMR (100 MHz, CD
3
OD) : 33.5
(
glc-1), 64.5 (glc-6), 70.1 (glc-3), 73.0 (glc-5), 75.3 (glc-4), 79.4 (glc-2), 99.4 (C-3),
9
1
8.9 (C-5), 96.3 (C-3'), 96.7 (C-7'), 103.9 (2C, C-1, C-1'), 156.1 (C-2'), 156.2 (C-6'),
56.9 (C-2), 157.1 (C-6), 158.4 (C-4'), 158.5 (C-4).
11

2
.9.3 Acetate of bisphloroglucinolyl glucose (11)
White amorphous powder; [α] +112.2 (c 0.11, MeOH); UV λmax nm (log ε):
80 (0.15), 206 (1.80); IR νmax cm : 3453, 1748, 1211; FAB-MS (positive, matrix;
D
−
1
2
+
+
m-nitrobenzyl alcohol) m/z: 775 [M+H] ; HR-FAB-MS m/z: 775.2088 [M+H] (Calcd
1
for C36
H
39
O
19: 775.2086); H NMR (acetone-d
6
, 500 MHz) δ: 6.89, 6.85 (each 1H, d, J
=
2.2 Hz, B-3,5), 6.48 (1H, d, J = 2.0 Hz, A-3), 6.30 (1H, d, J = 2.0 Hz, A-5), 5.54 (1H,
dd, J = 3.5, 8.2 Hz, glc-4), 5.27 (1H, dd, J = 1.3, 3.5 Hz, glc-3), 5.08 (1H, ddd, J = 3.4,
4
4
2
.2, 8.2 Hz, glc-5), 5.03 (1H, dd, J = 1.3, 5.9 Hz, glc-2), 4.48 (1H, d, J = 5.9 Hz, glc-1),
.23 (1H, dd, J = 3.4, 12.4 Hz, glc-6), 4.20 (1H, dd, J = 4.2, 12.4 Hz, glc-6), 2.39, 2.26,
1
3
.25, 2.13, 2.07, 2.02, 1.99, 1.98, 1.97 (each 3H, s, CH
3
). C NMR (acetone-d , 125
6
MHz) δ: 170.8, 170.6, 169.9, 169.8, 169.5, 168.8, 168.3, 167.9, 167.7 (COO), 161.3
A-2), 151.6 (A-4), 150.2, 149.9, 148.9 (B-2,4,6), 146.6 (A-6), 122.0 (B-1), 117.9 (A-1),
15.5, 113.3 (B-3,5), 107.8 (A-5), 101.1 (A-3), 88.8 (glc-2), 71.2 (glc-3), 69.8 (glc-4),
8.2 (glc-5), 61.3 (glc-6), 40.2 (glc-1), 21.1 (2C), 20.9, 20.8, 20.7, 20.6, 20.4, 20.3, 20.0
CH ).
(
1
6
(
3
2
.10. Reaction of phloroglucinol and dihydroxyacetone dimer
Phloroglucinol (1.26 g) and dihydroxyacetone dimer (0.45 g) were dissolved in
acetone (5 mL), and then the acetone was removed using a rotary evaporator. The
resulting syrup was heated at 130°C for 1 h, and then separated by silica gel column
chromatography with CHCl
3
:MeOH:H O (90:10:1, 85:15:1, 80:20:2, 75:25:3, 70:30:5,
2
and 50:50:0 v/v/v steps) to produce 12 (356 mg), 13 (42 mg), phloroglucinol (348 mg),
and an oligomer fraction (768 mg). A similar experiment using phloroglucinol (1.26 g)
and methylglyoxal (0.36 g) yielded 12 (686 mg) and recovered phloroglucinol (445
12

mg).
2
.10.1 Phloroglucinol dimer (12)
Pale brown amorphous powder; UV λmax nm (log ε): 208 (4.47), 215 (4.61),
−
1
2
23 (4.05), 272 (0.62); IR νmax cm : 3242, 1624, 1487; FAB-MS (positive, matrix:
+
+
glycerol) m/z: 289 [M+H] ; HR-FAB-MS m/z: 289.0710 [M+H] (Calcd for C15
H
13
6
O :
1
2
89.0712); H NMR (acetone-d
6
, 500 MHz) δ: 5.99, 5.98 (each 2H, d, J = 2.0 Hz,
1
3
H-3,5,3',5'), 4.59 (1H, s, H-7), 1.77 (3H, s, H-9). C NMR (acetone-d
6
, 125 MHz) δ:
1
9
2
60.5, 160.2, 153.5 (each 2C, C-2,4,6,2',4',6'), 124.7 (C-8), 106.3 (2C, C-1, 1'), 96.8,
1.2 (each 2C, C-2, 4, 2', 4'), 30.6 (C-7), 24.6 (C-9).
.10.2. Phloroglucinol dimer (13)
−
1
Pale brown powder; UV λmax nm (log ε): 272 (2.20); IR νmax cm : 3360, 1631,
1
514, 1466, 1257, 1133, 928, 822, 452; ESI-MS (positive, matrix: glycerol) m/z: 289
+
+
1
[
M+H] ; HR-FAB-MS m/z: 289.0713 [M+H] (Calcd for C15
H
13
O
6
: 289.0712); H
NMR (acetone-d
6
, 500 MHz) δ: 6.34 (1H, d, J = 7.9 Hz, H-5a), 6.01 (1H, d, J = 2.0 Hz,
H-9), 5.98 (1H, d, J = 2.0 Hz, H-7), 5.85 (1H, d, J = 2.0 Hz, H-2), 5.71 (1H, d, J = 2.0
Hz, H-4), 4.08 (1H, m, H-10b), 3.59 (1H, dd, J = 2.0, 15.1 Hz, H-11), 2.53 (1H, dd, J =
1
3
6
1
1
.3, 15.1 Hz, H-11); C NMR (acetone-d , 125 MHz) δ: 162.0 (C-6a), 159.5 (C-3),
6
57.3 (C-8), 156.3 (C-10), 155.6 (C-1), 155.2 (C-4a), 106.2 (C-5a), 105.4 (C-11a),
05.0 (C-10a), 98.2 (C-7), 96.6 (C-2), 89.7 (C-4), 40.8 (C-10b), 18.6 (C-11).
3
3
. Results and discussion
.1. Pyrolysis of (−)-epigallocatechin-3-O-gallate
Despite the high popularity of roasted green tea in Japan, only a few chemical
studies have been published, and these studies have shown isomerization and
13

oligomerization of tea catechins occur on roasting (Nakagawa, 1967; Hara & Kubota,
969; Anan, Amano, & Nakagawa, 1981). However, the structures of the oligomers
1
have not been studied. In this study, we examined pyrolysis of
epigallocatechin-3-O-gallate (2) as a preliminary experiment. This catechin was selected
because catechins with pyrogallol-type B-rings and galloyl esters account for over 70%
and 60% of total tea catechins, respectively (Anan et al., 1981). Green tea leaves are
typically roasted at 150–180°C (Nakagawa, 1967; Hara et al., 1969); therefore, a
lyophilized powder of 2 was heated at 150°C for 60 min to afford
(
−)-gallocatechin-3-O-gallate, gallic acid, and an anthocyanidin together with recovered
. The anthocyanidin was identified as tricetinidin, which was produced by elimination
2
of gallic acid and subsequent oxidation. In this experiment, little or no oligomeric
products were produced.
3
.2. Catechin oligomer from roasted green tea leaves
Next, the commercial green tea leaves were heated at 180°C for 30 min, and
oligomeric polyphenols were obtained via solvent partitioning, adsorption column
chromatography, and size-exclusion chromatography (Yanagida, Shoji, & Shibusawa,
2
003). Using HPLC analysis, the oligomeric products were detected as a broad hump on
1
3
the baseline, and the UV absorption was similar to that of 2 (Fig. S1). The C NMR
spectrum showed broad signals, which were closely related to those of 2 (Fig. 2A).
However, the A-ring methine signals in the δ 95–100 range were much smaller than
those of 2 (Fig. 2C). These signals shifted to lower field probably in the δ 105–115
range, which suggests that oligomerization reactions occur at the A-ring methine
carbons. In addition, broad signals in the δ 60–80 range suggest sugars participate in the
14

catechin oligomerization upon roasting.
3
.3. Catechin oligomer production from 2 and sucrose
Because the most abundant sugar in tea leaves is sucrose, 2 was heated with
1
3
sucrose at 150°C for 2 h, and the resulting oligomers were separated (Fig. S2). The C
NMR spectrum (Fig. 2B) was similar to that of the oligomer fraction obtained from
roasted green tea leaves except for the absence of the catechol-type B-ring originating
from 3 and 4. These results confirmed the cross-linking of 2 with the sugars.
13
3
.4. Catechin oligomer produced from 2 and C-labeled glucose
Next, the oligomers generated upon heating (150°C for 2 h) of 2 with glucose
were examined because pyrolysis of sucrose upon roasting produces glucose and
1
3
fructose as major products (Sˇimkovic, Sˇurina, & Vricˇan, 2003). The C NMR
spectrum of the oligomeric polyphenols obtained upon heating of 2 with glucose (Fig. 3
A) was similar to those of oligomers of roasted green tea leaves and oligomers prepared
from 2 and sucrose (Fig. 2). The co-oligomerization was clearly confirmed by
1
3
13
experiments using 1- C-labeled glucose (Fig. 3B) and U- C6-labeled glucose (Fig.
1
3
3
C). The spectrum of the oligomer obtained upon heating with 1- C-labeled glucose
exhibited large signals at δ 100 and δ 33 (Fig. 3B, black arrows), which were attributed
to the carbons of the glucose anomeric carbon. The signal at δ 100 may be explained by
formation of the O-glycosidation linkage at the anomeric position. In the spectrum of
1
3
the oligomer obtained upon heating of the U- C6-labeled glucose, signals at δ 115 and
from δ 60 to 84 (Fig. 3C, black arrows) were enhanced in addition to the δ 100 and δ 33
signals. The large signals between δ 60 and 84 were attributed to glucose C-2 to C-6
15

1
3
carbons. Enhancement of the signals at δ 115 and 33 upon incorporation of the C was
likely related to the oligomerization mechanism.
3
.5. Monomeric and dimeric products of 2 produced upon heating with glucose
To understand the oligomerization mechanisms, lyophilized powder of a
mixture of 2 and D-glucose was heated at 150°C for a shorter period (30 min), and the
products were separated by column chromatography to yield four products. Products 5
and 6 were a pair of isomers of glucose C-glucosides according to FAB-MS, which had
+
1
13
a [M+H] peak at m/z 621. The H and C NMR spectra showed signals arising from 2
and glucopyranose moieties; however, the A-ring proton signals were observed as one
proton singlet signal in each spectrum [5: 5.98 (s, H-6), 6: 6.06 (s, H-8)], and the
glucose C-1 of 5 and 6 resonated at δ 76.2 and δ 76.8, respectively (Fig. S3). These data
confirmed that glucose was attached to the A-ring C-8 or C-6 of 2 via C-glycosidic
linkages. The HMBC spectrum of 6 showed the correlation of the A-ring C-8a with the
A-ring H-8 and C-ring H-2, indicating that the glucose of 6 was located at the C-6
position (Fig. S3). Thus, we concluded the glucose in 5 was located at C-8. From these
spectroscopic data, 5 and 6 were determined to be epigallocatechin-3-O-gallate
8
-C-glucoside and 6-C-glucoside, respectively (Fig. 4).
1
13
The H and C NMR spectra of the other monomeric product, 7, showed a set
of signals of 2 and two sets of signals for the sugar moiety. The absence of the A-ring
1
methine proton signal in the H NMR spectrum and the molecular formula of C34
H
37
20
O ,
+
which was confirmed by HR-ESI-MS ([M+H] m/z 765.1873, Calcd for C34
H
37
O
20
:
1
13
7
65.1873), both indicated that 7 was a disubstituted analog of 5 and 6. H and
C
signals arising from one of the two sugar units were similar to the signals of
16

C-glycosidic glucopyranose moieties of 5 and 6. Another sugar unit showed signals that
were assignable to two methylenes (δ 34.8, C-1'''; and 61.4, C-6'''), three oxygenated
methines (δ 76.6, C-4'''; 80.7, C-3'''; and 83.9, C-5'''), and an acetal quaternary carbon (δ
116.9, C-2'''). The HMBC correlations (Fig. 5) and unsaturation index of this molecule
(
17) suggested the presence of a spiroketal structure involving an ether linkage with an
A-ring phenolic hydroxy group. The location of the hydroxy group participating in the
acetal ring formation was determined by observation of hydrogen–deuterium exchange
1
3
shifts of the A-ring carbons (Pfeffer, Valentine, & Parrish, 1979). The C NMR spectra
of 7 measured in acetone-d + H O and acetone-d + D O were carefully compared, and
6
2
6
2
a distinct chemical shift difference was observed for C-7 (Δδ +0.123). By contrast, the
shifts for C-5 (Δδ −0.008) and C-8a (Δδ +0.025) were minimal (Fig. S4). This
observation indicated the presence of a free hydroxy group at the A-ring C-7. Because
the sugar units originated from D-glucose, the configuration of the sugar carbons was
self-evident except for the spiroketal carbon. We deduced the spiroketal was in the
S-configuration from the NOE between the C-1 and C-3 protons. Based on these results,
the structure of 7 was determined (Fig. 4). The chemical shifts of the spiroketal carbon
(
C-2''', δ 116.9) and benzylic methylene carbon (C1''', δ 34.8) coincided with the values
of enhanced carbon signals (δ 115 and 33) in the spectra of the oligomers obtained upon
1
3
condensation with the C-labeled glucoses (Fig. 3). Therefore, a similar spiroketal
structure may be present in the oligomeric products.
+
According to the [M+Na] peak at m/z 1065.1955 in the HR-FAB-MS (Calcd
1
13
for C50H42NaO25: 1065.1913), product 8 is a dimeric product. The H and C NMR
spectra showed that a sugar residue connected two epigallocatechin gallate moieties.
The two A-ring proton singlet signals at δ 6.13 and 6.51 indicated that the two A-ring
17

methine carbons (C-6 or C-8) were attached to the sugar residue. The sugar residue was
composed of six carbons: a trisubstituted double bond (δ 148.2, C-1; δ 101.7, C-2), a
non-oxygenated methine carbon (δ 38.1, C-3), two oxygenated methines (δ 74.0, C-4; δ
7
4.4, C-5), and an oxygenated methylene carbon (δ 64.3, C-6). Formation of a pyran
1
1
was deduced from the unsaturation index (30), and H- H COSY and HMBC
correlations of the sugar protons with the A-rings of epigallocatechin units showed a
dimeric structure (Fig. 4). It was acceptable that the 4-, 5-, and 6-positions of the sugar
moiety retained the configuration of the D-glucose, and the 3,4-cis configuration was
suggested by the small J2,3 value (3.0 Hz). However, we could not determine where the
sugar residue attached to catechin C-6 or C-8 because of the lack of the HMBC
correlations from the catechin C-ring H-2 to the A-ring C-8a; therefore, the structure in
Fig. 4 is a tentative one. A plausible production mechanism of 8 is proposed in Scheme
S1, which explains the catechin cross-linking with the sugar. Similar reactions may
contribute to oligomerization of 2 in roasted tea, although it was difficult to identify the
1
3
signals assignable to the sugar moiety of 8 in the C NMR spectra of the oligomers (Fig.
3
).
3
.6. Reaction of phloroglucinol with glucose and its pyrolysis products
In addition to the low-field shifts of the A-ring methine carbon signals in the
C NMR spectra of the oligomeric products (Figs. 2 and 3), the results of the
1
3
abovementioned chemical examinations indicated that the A-rings were responsible for
the catechin oligomerization. To investigate the reaction mechanism in more detail, we
selected phloroglucinol as a simple A-ring mimic and examined the reactions with
glucose. Heating of phloroglucinol with glucose afforded C-glycoside 9 (Onodera,
18

Yamamoto, Abe, & Ueno, 1994) and two new dimeric products, 10 and 11 (Fig. 6). The
1
1
structure of product 10 was determined by H- H COSY, HSQC, and HMBC
experiments (Fig. S5). Dimer 11 was obtained as an inseparable mixture of
stereoisomers. The major component of this mixture was purified as an acetate, and its
1
3
structure was confirmed (Fig. S6). In the C NMR spectrum of 10, the C-1 of the
glucose moiety resonated at δ 33.5, and the chemical shift was similar to one of the
enhanced carbon signals of the oligomers obtained in the experiments using the
1
3
C-labeled glucoses (Fig. 3). The structures of 10 and 11 suggested another mechanism
for the cross-linking of 2 with the sugars in addition to that suggested by the production
of 8 (Scheme S2).
Reactions with the pyrolysis products of the sugars should also be considered
because heating of sucrose, the most abundant sugar in tea leaves, is known to afford
glucose and fructose, and further degradation of the monosaccharides generates methyl
glyoxal (MG) and dihydroxyacetone (DHA) (Kabyemela, Adschiri, Malaluan, & Ara,
1
997). Both MG and DHA are also generated in the Maillard reaction (Totlani &
Peterson, 2006). In addition, MG is produced from DHA. In this study, phloroglucinol
was heated separately with MG and the DHA dimer, and 12 was obtained as a major
product in the reaction with MG. Products 12 and 13 were obtained from the reaction
with DHA. The structures were determined by spectroscopic methods and computer
calculations (Figs. S7 and S8). Production of 12 via both reactions supported the
conversion of DHA to MG upon heating. These reactions also afforded oligomeric
1
3
products. The C NMR spectrum of the oligomeric products obtained by heating
1
3
phloroglucinol with 2- C-labeled DHA (Fig. S9) showed broad signals at δ 124
1
3
(
enhanced by incorporation of C), δ 48, and δ 24, which coincided with the C-8 (δ
19

1
24.5), C-7 (δ 49.6), and C-9 (δ 24.7) signals of 12. This indicates that the
oligomerization mechanism of phloroglucinol with DHA is similar to the production
mechanism of 12 (Scheme S3). Furthermore, the broad signal observed in the δ
1
05–115 range is assignable to aromatic carbons where the MG unit connected. In the
1
3
C NMR spectrum of the oligomer obtained from the roasted tea (Fig. 2), signals for
the catechin C6 and C8 carbon that were involved in the oligomerization reaction were
likely present in a similar region.
4
. Conclusions
In this study, we showed that roasting green tea leaves causes epimerization of
the catechin C-ring and co-oligomerization of the catechin with sugar or its pyrolysis
products. The co-oligomerization occurs at the catechin A-ring, which is shown by
low-field shifts of the A-ring methine carbon signals from their original locations at δ
9
5–98 to δ 105–115. Structures of the products obtained via model reactions using 2 and
its A-ring mimic, phloroglucinol, suggest that the oligomerization of catechins does not
proceed uniformly; however, the basic mechanism involves cross-linking at the A-ring
C6 and/or C8 with the carbonyl carbons of sugar or its pyrolysis products. The roasting
process decreases the astringency caused by tea catechins and the astringency of the
oligomer fraction obtained from roasted tea is milder than that of the original green tea
catechins. A similar reaction should occur during roasting of cacao and other catechin-
and proanthocyanidin-containing foods. Evaluation of the biological functions of the
oligomers is now in progress.
Conflict of interest
20

The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the Japan Society for the Promotion of Science KAKENHI (Grant Nos.
17K08338 and 16K07741). The authors are grateful to N. Tsuda, K. Chifuku, and H. Iwata at the
Center for Industry, University and Government Cooperation, Nagasaki University, for recording the
NMR and MS data. We thank Gabrielle David, PhD, from Edanz Group (www.edanzediting.com/ac)
for editing a draft of this manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
https://doi.org/
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23

24

Figure Captions
Fig. 1. HPLC profiles (max abs) of extracts of green tea leaves and roasted green tea
leaves: A, Extract of green tea leaves; B, extract of roasted green tea leaves (0.02 g/mL
in 60% EtOH); and C, vertical axis expansion of B. Compounds: 1,
(
(
(
−)-epigallocatechin; 2, (−)-epigallocatechin-3-O-galalte; 3, (−)-epicatechin; 4,
−)-epicatechin-3-O-galalte; 1a, (−)-gallocatechin; 2a, (−)-gallocatechin-3-O-galalte; 3a,
−)-catechin; 4a, (−)-catechin-3-O-galalte; GA, gallic acid; and caf, caffeine.
1
3
Fig. 2 C-NMR spectra (in DMSO-d ) of oligomeric polyphenols obtained from
6
roasted green tea leaves (180°C) (A), oligomer obtained upon heating of 2 with
sucrose (B), and 2 (C). Labeling: A, A-ring; B, B-ring; C, C-ring; G, galloyl; and
Cat-B, catechol-type B-ring.
1
3
Fig. 3. C-NMR spectra of the oligomer fraction obtained upon heating of 2 with
1
3
13
glucose (A), 1- C-labeled glucose (B), and U- C6-labeled glucose (C). The
1
3
black arrows indicate signals enhanced by incorporation of C.
Fig. 4. Structures of 5–8 produced upon heating of 2 with glucose.
1
1
Fig. 5. Selected H- H COSY and HMBC correlations of 7.
Fig. 6. Products generated from phloroglucinol with glucose (9–11), methyl glyoxal
(
12), and dihydroxyacetone (12 and 13).
25

Houji_cha - CH5
2
Green tea - CH5
Houji_cha - CH5
A
B
C
1
1
1
500000
000000
1500000
1000000
500000
0
50000
5
0000
caf
4
0
0.0
0
10.0
10
20.0
20 Retentio
30.0
40.0
50.0
50
60.0
60
3
n
0
3
2
3
a
2a
caf
1a
oligomeric products
1
5
00000
4
3
1a
GA
4a
GA
0
0
.0
10.0
20.0
30.0
40.0
50.0
60.0
0.0
10.0
20.0
30.0
40.0
50.0
60.0
Retention Time [min]
Retention Time [min]
R1
R1
OH
OH
OH
OH
B
HO
O
2
HO
O
2
A
C
3
OR2
3 OR2
OH
OH
R1
R1
R2
H
G
H
G
R2
H
G
H
OH
OH
OH
1
2
3
4
:
:
:
:
OH
OH
H
1a : OH
2a : OH
3a : H
G =
O
H
4a : H
G
Fig. 1. HPLC profiles (max abs) of extracts of green tea leaves and roasted green tea
leaves.
A: Extract of green tea leaves, B: extract of roasted green tea leaves (0.02 g/mL 60%
EtOH). C: vertical axis expansion of B. 1: (-)-epigallocatechin, 2:
(
(
(
-)-epigallocatechin-3-O-galalte, 3: (-)-epicatechin, 4: (-)-epicatechin-3-O-galalte, 1a:
-)-gallocatechin, 2a: (-)-gallocatechin-3-O-galalte, 3a: (-)-catechin, 4a:
-)-catechin-3-O-galalte, GA: gallic acid, caf: caffeine.
26

cat-B G
G
B
A
A
B
sugars
B
B
G
G
C
G
( A )
C
C
1
80.0
170.0
160.0
150.0
140.0
130.0
120.0
110.0
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
1
80 170 160 150 140 130 120 110 100 90
80
70
60
50
40
30
20
10
G
B
G
G
B
sugars
B
G
G
A
B
B
C
C
C
(
A )
180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0
1
90.0
80.0
80
70.0
70
60.0
60
50.0
50
40.0
40
30.0
30
20.0
20
10.0
10
80 170 160 150 140 130 120 110 100 90
C
GB
A
G B
A
G
B
G
G
C
C
B
C
ppm
1
1
8
8
0.
0
0
17
1
0
7
.0
0
1
1
60
6
.
0
0
1
1
5
5
0.
0
0
1
1
4
4
0.
0
0
1
1
3
3
0
0
.0
1
1
20
2
.
0
0
1
1
10
1
.
0
0
1
1
0
0
0.
0
0
9
9
0
0
.0
8
8
0
0
.0
7
7
0
0
.0
6
6
0
0
.0
5
5
0
0
.0
4
4
0
0
.0
3
3
0
0
.0
2
2
0
0
.0
10.0
1
0
13
Fig. 2 C-NMR spectra (in DMSO-d
roasted green tea leaves (180°C) (A), oligomer obtained upon heating of 2 with sucrose
B), and 2 (C). A: A-ring, B: B-ring, C: C-ring, G: galloyl, Cat-B: catechol-type B-ring.
6
) of oligomeric polyphenols obtained from
(
27

1
3
Fig. 3. C-NMR spectra of oligomer fractions obtained upon heating of 2 with glucose
1
3
13
(
A), 1- C-labeled glucose (B) and U- C6-labeled glucose (C). The black
1
3
arrows indicate signals enhanced by incorporation of C.
28

Fig. 4. Structures of 5–8 produced upon heating 2 with glucose.
29