Coupling reactions of catechins with natural aldehydes and allyi alcohols and radical scavenging activities of the triglyceride-soluble products

Article abstract of DOI:10.1021/jf9010998

Hydrophobie catechin derivatives were produced by heating with natural aldehydes or allyl alcohols. (+)-Catechin or (-)-epigallocatechin-3-0-gallate was heated with frans-2-hexenal, citral, (+)-citronellal, geraniol, or phytol. Although each reaction gene

Full text of DOI:10.1021/jf9010998

J. Agric. Food Chem. 2009, 57, 6417–6424 6417
DOI:10.1021/jf9010998
Coupling Reactions of Catechins with Natural Aldehydes and
Allyl Alcohols and Radical Scavenging Activities of the
Triglyceride-Soluble Products
†
,
R
YUSUKE
F
UDOUJI,† TAKASHI
T
ANAKA,*,† TOSHITUGU
†
T
AGURI,§ YOSUKE
M
ATSUO
AND
I
SAO
K
OUNO
†Department of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki
University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan, and §Nagasaki Prefectural Institute for
Environmental Research and Public Health, Ikeda 2-1306-11, Omura, Nagasaki 856-0026, Japan
Hydrophobic catechin derivatives were produced by heating with natural aldehydes or allyl alcohols.
(+)-Catechin or (-)-epigallocatechin-3-O-gallate was heated with trans-2-hexenal, citral, (+)-citro-
nellal, geraniol, or phytol. Although each reaction generated complex mixtures of products, 11
compounds were isolated and characterized by spectroscopic methods. The unsaturated aldehydes
were found to attach to the flavan A-ring. Besides C-C linkage between aldehyde and the C-8 and/
or C-6 of the catechin A-ring, formation of ether linkages between unsaturated carbons of the
aldehydes and phenolic hydroxyl groups was observed. The allyl alcohols, geraniol and phytols,
reacted at the galloyl group as well as the A-ring. After partitioning between triglyceride and water,
the lipid layer of the reaction products showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging activity. In contrast, epigallocatechin-3-O-gallate was not transferred to the lipid layer.
KEYWORDS: Catechin; essential oil; phytol; aldehyde; allyl alcohol; radical scavenging
INTRODUCTION
using special chemical reagents that are unacceptable as food
ingredients; thus, this paper introducesa facile method toproduce
catechin derivatives with hydrophobic moieties. Some of the
products showed increased lipid solubility and 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging activities in the trigly-
ceride (TG) layer following TG/water partitioning of the pro-
ducts.
Recent medical and biological studies indicate that oxidative
stress causes and exacerbates some chronic diseases, such as
atherosclerosis, diabetes, and cancer (1-3). In addition, epide-
miological studies strongly suggest a correlation between a higher
content of polyphenols in the diet and a lower risk of mortality
from cancer and coronary heart diseases (4-7). Therefore, poly-
phenols as antioxidative food constituents have attracted the
attention of food scientists, manufacturers, and consumers.
Catechins, such as (+)-catechins (1) and (-)-epigallocatechin-
3-O-gallate (2), are known to prevent free radical-mediated
damage (8-11). However, the bioavailability of tea catechins
and related tea polyphenols is not sufficiently high to display their
antioxidative abilities observed in in vitro experiments (12-14).
Tea catechins are water-soluble antioxidants. In addition, tea
catechins associate with proteins and lipid bilayers through
hydrophobic interactions and hydrogen bonding (15-18). To
alter the solubility and physicochemical nature by chemical
methods, some derivatives of tea catechins have been synthe-
sized (19-22). Although direct application of these compounds as
food additives may be difficult because of the technical difficulties
associated with production and the current lack of safety evalua-
tions available, these derivatives are interesting from the view-
point of medicinal and food chemistry. The aim of this study is to
develop a new method to prepare catechin derivatives without
MATERIALS AND METHODS
Materials. (+)-Catechin (1) was isolated from commercial gambir
(extract of the leaves and young twigs of Uncaria gambir) and purified by
recrystallization (23). (-)-Epigallocatechin-3-O-gallate (2) was isolated
from commercial green tea by column chromatography and purified by
crystallization (24). Essential oils and phytol were purchased from Tokyo
Chemical Industry Co., Ltd., Japan, and other chemicals and solvents
were of reagent grade.
Analytical Procedures. UV spectra were obtained using a JASCO
V-560 UV-vis spectrophotometer. Optical rotations were measured using
a JASCO DIP-370 digital polarimeter. The 1H and 13C NMR, 1H-1H
correlation spectroscopy (COSY), NOE spectroscopy (NOESY), hetero-
nuclear single quantum coherence (HSQC), and heteronuclear multiple-
bond connectivity (HMBC) spectra were recorded in acetone-d6 at 27 °C
on a Unity plus 500 spectrometer (Varian, Palo Alto, CA) operating at a
1H frequency of 500 MHz. The one-dimensional 1H and 13C NMR spectra
were recorded with a JEOL JNM-AL400 spectrometer operating at a 1H
frequency of 400 MHz. The matrix-assisted laser desorption time-of-flight
mass spectra (MALDI TOF MS) were recorded on a Voyager-DE Pro
spectrometer (Applied Biosystems, Fullerton, CA), and 2,5-dihydrohy-
droxybenzoic acid (10 mg/mL in 50% acetone containing 0.05% trifluor-
oacetic acid) was used as the matrix. Fast atom bombardment (FAB) MS
was recorded on a JMS 700N spectrometer (JEOL Ltd., Japan), and
*Author to whom correspondence should be addressed [telephone:
81 (0)95 819 2433; fax: 81 (0)95 819 2477; e-mail: t-tanaka@nagasaki-u.
ac.jp].
© 2009 American Chemical Society
Published on Web 06/19/2009
pubs.acs.org/JAFC

6418 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Fudouji et al.
m-nitrobenzyl alcohol or glycerol was used as the matrix. Elemental
analysis was conducted with a Perkin-Elmer 2400R analyzer (Perkin-
Elmer Inc., Waltham, MA).
MALDI TOF MS m/z 683 [M + Na]+, 699 [M + K]+. Anal. Calcd
9
for C36H36O12
5.76%.
/ H2O: C, 61.67%; H, 5.82%. Found: C, 61.73%; H,
4
3
Column chromatography was performed using Sephadex LH-20 (25-
100 μm, GE Healthcare Bio-Science AB, Uppsala, Sweden), Chromatorex
ODS (100-200 mesh; Fuji Silysia Chemical Ltd., Tokyo, Japan), and silica
gel 60N (100-210 μm, Kanto Chemical Co., Inc., Tokyo, Japan) columns.
Thin layer chromatography (TLC) was performed on precoated Kieselgel
60 F254 plates (0.2 mm thick, Merck, Darmstadt, Germany) with toluene/
ethyl formate/formic acid (5:4:1 or 1:7:1 v/v) or CHCl3/MeOH/water
(90:10:1, 80:20:2, or 70:30:5 v/v). Spots were detected using ultraviolet
(UV) illumination and by spraying the plates with 2% ethanolic FeCl3 or
10% sulfuric acid reagents followed by heating. Analytical HPLC was
performed on a Cosmosil 5C18-AR II (Nacalai Tesque Inc., Tokyo, Japan)
column (4.6 mm i.d. ꢀ 250 mm) with a gradient elution from 25 to 90%
(0-40 min) and 90% (40-55 min) of CH3CN in 50 mM H3PO4. A flow
rate of 0.8 mL/min was used. The column temperature was maintained at
35 °C, and detection was achieved using a JASCO photodiode array
Compound 4 was obtained as a brown amorphous powder: [R]2D9 -248.3
(c 0.1 MeOH); UV (MeOH) λmax nm (ε) 276 (10050); IR (dry film) νmax
cm-1 3390, 1694, 1615, 1537, 1448; 1H NMR (500 MHz, acetone-d6) δ
0.85 (3H, s, H-90), 0.95 (3H, s, H-80), 1.29 (1H, m, H-5b0), 1.29 (3H, s,
H-100), 1.52 (1H, br d, J=14.9 Hz, H-5a0), 1.58 (1H, dd, J=4.8, 12.3 Hz, H-
4b0), 1.65 (1H, br d, J=12.6 Hz, H-60), 1.73 (1H, dt, J=3.0, 12.8 Hz,
H-2b0), 1.90 (2H, br d, J=12.8 Hz, H-2a0, H-4a0), 2.95 (1H, dd, J=2.2,
17.3 Hz, H-4b), 3.09 (1H, br d, J=17.3 Hz, H-4a), 3.43 (1H, br s, OH-70),
3.64 (1H, br d, J=2.3 Hz, H-10), 5.15 (1H, br s, H-2), 5.49 (1H, br s, H-3),
6.01 (1H, s, H-6), 6.67 (2H, s, B-ring H-2,6), 7.02 (2H, s, galloyl H-2,6),
13
8.36 (1H, br s, OH-5); C NMR (125 MHz, acetone-d6) δ 22.3 (C-50),
25.7 (C-90), 26.5 (C-4), 27.4 (C-10), 29.0 (C-80,100), 39.4 (C-20), 40.9 (C-
40), 54.7 (C-60), 69.4 (C-3), 72.1 (C-70), 75.0 (C-30), 78.5 (C-2), 96.3 (C-
6), 98.7 (C-4a), 104.2 (C-8), 106.7 (B-ring C-2,6), 110.0 (galloyl C-2,6),
121.7 (galloyl C-1), 129.9 (B-ring C-1), 133.3 (B-ring C-4), 138.7 (galloyl
C-4), 145.8 (galloyl C-3,5), 146.3 (B-ring C-3,5), 152.3 (C-8a), 155.6
(C-5), 156.9 (C-7), 166.0 (galloyl C-7); MALDI TOF MS m/z 633 [M+
detector MD-910. Preparative HPLC was performed on a Cosmosil 5C18
-
AR-II column (20 ꢀ 250 mm) with 90% CH3CN containing 0.1% TFA.
Na]+, 649 [M+K]+. Anal. Calcd for C32H34O12 2H2O: C, 59.49%; H,
General Reaction Procedures. Aldehyde or allyl alcohol (4.0 g) was
added to a solution of 1 or 2 in acetone (2.0 g/30 mL), and the solvent was
evaporated under reduced pressure using a rotary evaporator. In the case
of the reaction with geraniol and phytol, citric acid (0.4 g) and 0.1 M
ethanolic HCl (0.4 mL), respectively, were added prior to evaporation. The
resulting syrup was left standing at room temperature or heated (50 or
80 °C), and the reaction was monitored by TLC analysis. The mixture was
first separated by Sephadex LH-20 column chromatography with EtOH.
The fractions were monitored by TLC and separated into several fractions
containing starting materials and products. The fractions containing the
products were subjected to Chromatorex ODS column chromatography
with 40-100% MeOH (10% stepwise gradient elution). Generally, the
reactions afforded a complex mixture of the products including diaster-
eomeric isomers and, thus, the yields of the purified compounds were
significantly low. Reaction of 1 (2.1 g) with trans-2-hexanal (4.0 g) at room
temperature for 1 week yielded 3 (113 mg). Reaction of 2 (2.0 g) with citral
(4.1 g) at 100 °C for 20 h yielded 4 (52.2 mg), 5 (70.5 mg), 6 (68.4 mg), and
7 (38.6 mg). Reaction of 2 (2.0 g) with (+)-citronellal (4.0 g) at 80 °C for 5 h
yielded 8 (377 mg) and 9 (56.4 mg). Reaction of 2 (2.0 g) with geraniol
(4.0 g) in the presence of citric acid (0.4 g) at 100 °C for 5 h yielded 11
(39.3 mg) and 12 (16.5 mg). Reaction of 2 (4.0 g) with phytol (2.0 g) in the
presence of 0.1 M HCl (0.4 mL) at 50 °C for 30 h yielded 13 (48.0 mg) and
14 (70.0 mg). Reaction of phloroglucinol (2.0 g) with (+)-citronellal (0.4 g)
at 80 °C for 5 h yielded 10 (210.0 mg). Purification of product 10 was
performed by silica gel column chromatography with CHCl3/MeOH/H2O
(95:5:0 and 90:10:1).
3
5.92%. Found: C, 59.58%; H, 6.12%.
Compound 5 was obtained as a brown amorphous powder: [R]2D9 -135.7
(c 0.1 MeOH); UV (MeOH) λmax nm (ε) 276 (9450); IR (dry film) νmax
cm-1 3405, 1693, 1618, 1537, 1449; 1H NMR (500 MHz, acetone-d6) δ
1.05 (3H, s, H-80), 1.21 (3H, s, H-90), 1.27 (3H, s, H-100), 1.32 (1H, dd,
J=4.2, 12.9 Hz, H-50), 1.53 (2H, m, H-40, H-50), 1.60 (1H, br d, J=12.8 Hz,
H-50), 1.71 (1H, br d, J=12.8 Hz, H-60), 1.86 (1H, dd, J=3.0, 15.1 Hz,
H-20), 1.91 (1H, m, H-40), 2.95 (1H, br d, J=18.0 Hz, H-4), 3.10 (1H, dd,
J=5.3, 18.0 Hz, H-4), 3.51 (1H, br s, H-10), 4.99 (1H, br s, H-2), 5.51 (1H,
d, J=5.3 Hz, H-3), 6.05 (1H, s, H-6), 6.71 (2H, s, B-ring H-2,6), 7.15 (2H,
13
s, galloyl H-2,6); C NMR (125 MHz, acetone-d6) δ 22.5 (C-50), 25.9
(C-80), 27.3 (C-10), 27.7 (C-4), 28.9 (C-100), 29.9 (C-90), 39.3 (C-20),
40.8 (C-40), 54.1 (C-60), 69.2 (C-3), 73.4 (C-70), 75.0 (C-30), 80.4 (C-2),
96.6 (C-6), 99.5 (C-4a), 104.7 (C-8), 107.6 (B-ring C-2,6), 110.2 (galloyl
C-2,6), 121.6 (galloyl C-1), 129.3 (B-ring C-1), 133.8 (B-ring C-4), 138.9
(galloyl C-4), 145.8 (galloyl C-3,5), 146.3 (B-ring C-3,5), 153.0 (C-8a),
155.7 (C-5), 156.9 (C-7), 166.4 (galloyl C-7); MALDI TOF MS m/z 633
[M+Na]+, 649 [M+K]+. Anal. Calcd for C32H34O12 2H2O: C, 58.62%;
3
H, 6.00%. Found: C, 58.63%; H, 6.19%.
Compound 6 was obtained as a brown amorphous powder: [R]2D9 -79.2
(c 0.1 MeOH); UV (MeOH) λmax nm (ε) 276 (9880); IR (dry film) νmax
cm-1 3409, 1692, 1618, 1536, 1448; 1H NMR (500 MHz, acetone-d6) δ
0.47 (1H, m, H-50), 1.00 (3H, s, H-80), 1.11 (1H, m, H-40), 1.28 (3H, s,
H-100), 1.39 (2H, m, H-40, H-50), 1.44 (3H, s, H-90), 1.87 (1H, dd, J=1.6,
13.0 Hz, H-20), 2.00 (1H, m, H-60), 2.09 (1H, m, H-20), 2.77 (1H, br s,
H-10), 2.98 (2H, m, H-4), 5.03 (1H, br s, H-2), 5.51 (1H, dd, J=1.4, 3.4 Hz,
H-3), 6.07 (1H, s, H-8), 6.63 (2H, s, B-ring H-3,5), 6.98 (2H, s, galloyl
Compound 3 was obtained as a brown amorphous powder: [R]2D9 -59.4
(c 0.1, MeOH); UV (MeOH) λmax nm (ε) 280 (7280); IR (dry film) νmax
cm-1 3389, 1692, 1616, 1449; 1H NMR (500 MHz, acetone-d6) δ 0.42 (3H,
t, J=7.3, Ha-600), 0.69 (3H, t, J=7.3, Hb-600), 0.98 (1H, m, Ha-500), 1.06
(1H, m, Ha-500), 1.16 (1H, m, Hb-500), 1.18 (1H, m, Ha-400), 1.29 (1H, m,
Hb-500), 1.31 (1H, m, Hb-400), 1.55 (1H, m, Ha-400), 1.72 (1H, m, Hb-400),
1.93 (2H, m, H-200), 2.56 (2H, m, Ha-4, Hb-4), 2.78 (1H, m, Hb-300), 2.80
(1H, m, Ha-300), 2.94 (2H, m, Ha-40, Hb-40), 3.92 (2H, m, Ha-3, Hb-3),
4.00 (2H, m, Ha-30, Hb-30), 4.54, 4.54 (each 1H, d, J=8.2 Hz, Ha, b-2),
4.59, 4.63 (each 1H, d, J=7.8 Hz, Ha, b-20), 5.50 (1H, dd, J=2.5, 11.7 Hz,
H-100), 5.53 (1H, dd, J=2.5, 11.7 Hz, H-100), 6.03 (2H, s, H-6, H-60), 6.76
(4H, m, B-ring H-5,50,6,60), 6.90 (2H, m, B-ring H-2,20), 7.64 (1H, s, OH-
7), 7.72 (1H, s, OH-7), 7.89 (4H, m, B-ring OH-3,304,40), 8.33 (1H, s, OH-
50), 8.34 (1H, s, OH-50), 8.40 (1H, s, OH-5), 8.40 (1H, s, OH-5); 13C
NMR (125 MHz, acetone-d6) δ 14.0 (Ca-600), 14.2 (Cb-600), 20.9 (Ca-500),
21.2 (Cb-500), 28.8 (Ca-4, Cb-4), 28.9 (Ca-40, Cb-40), 30.0 (Cb-300), 30.2
(Ca-300), 31.1 (Ca-200), 31.5 (Cb-200), 37.6 (Ca-400), 38.0 (Cb-400), 67.84,
67.9 (Ca-30, Cb-30), 68.3, 68.6 (Ca-3, Cb-3), 70.6, 71.5 (Ca-100, Cb-100),
82.3, 83.0 (C-2, C-20), 95.9, 96.1, 96.9, 97.0 (Ca-6, Cb-6, Ca-60, Cb-60),
101.0, 102.3, 102.7 (Ca-4, Ca-40, Cb-4, Cb-40), 105.1, 105.5 (C-8, C-80),
107.4, 107.7 (C-8a, C-8a0), 114.9, 115.20, 115.2 (B-ring C-2,20), 115.2,
115.5, 115.6, 115.6 (B-ring C-5,50), 119.6, 119.7, 119.9, 119.9 (B-ring C-
6,60), 131.6, 131.8, 132.0, 132.1 (B-ring C-1,10), 145.7, 145.5, 145.5, 145.6,
145.7 (B-ring C-3,30,4,40), 153.1, 153.2 (Ca-8a0, Cb-8a0), 153.3 (C-8a),
153.9, 154.0 (Ca-50, Cb-50), 155.9, 156.0 (C-7, C-70), 156.4 (C-5);
13
H-2,6); C NMR (125 MHz, acetone-d6) δ 22.6 (C-50), 24.0 (C-90), 26.2
(C-4), 28.9 (C-10), 29.2 (C-100), 29.9 (C-80), 35.7 (C-20), 38.0 (C-40),
47.2 (C-6), 69.1 (C-3), 74.5 (C-30), 78.2 (C-2), 84.1 (C-70), 98.7 (C-8),
101.1 (C-4a), 106.6 (B-ring C-2,6), 109.8 (galloyl C-2,6), 110.00 (C-6),
121.9 (galloyl C-1), 130.7 (B-ring C-1), 133.0 (B-ring C-4), 138.6 (galloyl
C-4), 145.8 (galloyl C-3,5), 146.2 (B-ring C-3,5), 155.1 (C-8a), 155.8
(C-5), 156.0 (C-7), 166.1 (galloyl C-7); MALDI TOF MS m/z 633 [M+
Na]+, 649 [M + K]+. Anal. Calcd for C32H34O12 3/2H2O: C, 60.28%; H,
3
5.85%. Found: C, 60.20%; H, 5.94%.
Compound 7 was obtained as a brown amorphous powder: [R]2D9 52.2
(c 0.1, MeOH); UV (MeOH) λmax nm (ε) 281 (13170); IR (dry film)
ν
max cm-1 3389, 1692, 1614, 1536, 1446; 1H NMR (500 MHz, acetone-d6)
δ 1.30 (3H, s, H-100), 1.57 (3H, d, J=0.7 Hz, H-80), 1.63 (3H, d, J=0.7 Hz,
H-90), 1.67 (2H, t, J=8.2 Hz, H-40), 2.10 (2H, m, H-50), 2.87 (1H, dd,
J=2.2, 17.4 Hz, H-4), 3.02 (1H, dd, J=4.8, 17.4 Hz, H-4), 5.05 (1H, s,
H-2), 5.11 (1H, m, H-60), 5.41 (1H, d, J=10.0 Hz, H-20), 5.54 (1H, m,
H-3), 6.07 (1H, s, H-8), 6.59 (1H, s, B-ring H-2,6), 6.63 (1H, d, J=10.0 Hz,
H-10), 7.01 (2H, s, galloyl H-2,6); 13C NMR (125 MHz, acetone-d6)
δ 17.6 (C-80), 23.4 (C-50), 25.8 (C-90), 26.3 (C-4), 26.5 (C-100), 41.7
(C-40), 69.0 (C-3), 78.2 (C-2), 79.1 (C-30), 96.0 (C-8), 99.8 (C-4a), 103.9
(C-6), 106.6 (B-ring C-2,6), 109.9 (galloyl C-2,6), 118.3 (C-10), 121.7
(galloyl C-5), 124.9 (C-20), 125.1 (C-60), 130.4 (B-ring C-1), 131.8
(C-70), 133.1 (B-ring C-4), 138.7 (galloyl C-4), 145.8 (galloyl C-3,5),

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6419
146.2 (B-ring C-3,5), 152.8 (C-7), 153.2 (C-5), 156.4 (C-8a), 166.0
J=2.5, 17.6 Hz, H-4), 3.03 (1H, dd, J=4.8, 17.6 Hz, H-4), 3.56 (1H, dd,
J=6.0, 14.0 Hz, H-1a0), 3.64 (1H, dd, J=7.6, 14.0 Hz, H-1b0), 5.05 (2H, br
s, H-2 and H-60), 5.12 (1H, br t, J=6.5 Hz, H-20), 5.55 (1H, br s, H-3),
5.98 (1H, d, J=2.3 Hz, H-8), 6.04 (1H, d, J=2.3 Hz, H-6), 6.60 (2H, s, B-
ring H-2,6), 6.93 (1H, s, galloyl H-6); 13C NMR (125 MHz, acetone-d6) δ
16.3 (C-90), 17.7 (C-100), 23.6 (C-10), 25.5 (C-80), 26.5 (C-4), 27.4
(C-50), 40.5 (C-40), 69.0 (C-3), 77.9 (C-2), 95.7 (C-8), 96.3 (C-6), 99.1
(C-4a), 106.7 (B-ring C-2,6), 110.4 (galloyl C-6), 121.1 (galloyl C-1),
124.1 (galloyl C-2), 124.9 (C-20), 125.3 (C-60), 130.6 (B-ring C-1), 131.8
(C-70), 133.1 (B-ring C-4), 134.3 (C-30), 137.4 (galloyl C-4), 142.8
(galloyl C-5), 144.8 (galloyl C-3), 146.1 (B-ring C-3,5), 156.9 (C-8a),
157.4 (C-5), 157.6 (C-7), 167.0 (galloyl C-7); MALDI TOF MS m/z 617
(gallyol C-7); MALDI TOF MS m/z 615 [M + Na]+. Anal. Calcd for
C32H32O11 5/2H2O: C, 60.28%; H, 5.85%. Found: C, 60.25%; H, 5.96%.
3
Compound 8 was obtained as a tan amorphous powder: [R]2D2 -197.7
(c 0.1, MeOH); UV (MeOH) λmax nm (ε) 275 (12393); IR (dry film)
ν
max cm-1 3401, 2921, 2865, 1693, 1608, 1536, 1453; 1H NMR (acetone-d6,
500 MHz) δ 0.64 (m, H-2a0), 1.30, 1.38 (s, H-80), 5.06, 5.08 (br s, H-2),
5.55 (m, H-3), 6.66 (d, J=0.5 Hz, H-B-2,6), 6.96 (2H, s, galloyl H-2,6);
13C NMR (acetone-d6, 125 MHz) δ 19.3 (C-9), 22.9, 23.1 (C-100), 26.9,
27.0 (C-4), 27.9, 28.0 (C-80), 28.7, 28.8 (C-50), 33.6, 33.7 (C-30), 36.4,
36.5 (C-10, C-40), 40.5, 40.8 (C-20), 50.4 (C-60), 69.4 (C-3), 77.1, 77.2,
77.3 (C-2, C-70), 100.3 (C-4), 106.8, 106.9, 107.5 (C-6, C-8), 106.1 (B-
ring C-2,6), 109.9, 110.1 (galloyl C-2,6), 122.0 (galloyl C-1), 130.9 (B-ring
C-1), 132.7, 132.8 (B-ring C-4), 138.6, 138.8 (galloyl C-4), 145.8, 146.3
(galloyl C-3, 5, B-ring C-3, 5), 151.2, 152.8, 153.0 (C-5, C-7, C-8a), 166.2
(galloyl C-7); MALDI TOF MS m/z 753 [M + Na]+. Anal. Calcd for
[M + Na]+. Anal. Calcd for C32H34O11 2H2O: C, 60.95%; H, 6.07%.
3
Found: C, 60.95%; H, 6.09%.
Compound 13 was obtained as a brown amorphous powder: [R]2D0 -79.2
(c 0.1, MeOH); UV (MeOH) λmax nm (ε) 276 (12358); IR (dry film)
C42H50O11 3/2H2O: C, 66.56%; H, 7.05%. Found: C, 66.59%; H, 7.03%.
ν
max cm-1 3425, 1694, 1626, 1455; 1H NMR (500 MHz, acetone-d6) δ 0.83,
3
0.84 (each 3H, d, J=6.6 Hz, H-180, H-190), 0.86 (6H, d, J=6.6 Hz, H-160,
H-200), 1.02-1.41 (m, H-50-H-140), 1.52 (1H, septet, J=6.6 Hz, H-150),
1.74 (3H, d, J=0.9, H-170), 1.91 (2H, m, H-40), 2.91 (1H, dd, J=2.1, 17.2
Hz, C-4a), 3.08 (1H, dd, J=4.8, 17.2 Hz, H-4b), 3.33 (2H, m, H-10), 5.01
(1H, s, H-2), 5.24 (1H, br t, J=7.1 Hz, H-20), 5.54 (1H, m, H-3), 6.13
(1H, s, H-8), 6.60 (2H, d, J=0.5 Hz, B-ring H-2,6), 7.00 (1H, s, galloyl
H-2,6), 7.11 (1H, s, 5-OH), 8.16 (1H, s, 7-OH); 13C NMR (125 MHz,
acetone-d6) δ 16.1 (C-170), 20.0 (C-160,200), 22.8, 22.9 (C-180,190), 22.6
(C-10), 27.0 (C-4), 25.1, 25.4, 26.0, 28.6, 33.3, 33.4, 37.4, 37.9, 38.0, 38.1,
40.0 (C-50-C-150), 40.7 (C-40), 69.2 (C-3),77.7 (C-2), 96.2 (C-8), 99.4
(C-4a), 106.6 (B-ring C-2.6), 108.9 (C-6), 109.9 (galloyl C-2,6), 121.7
(galloyl C-1), 124.2 (C-20), 130.6 (B-ring C-1), 133.1 (B-ring C-4), 135.1
(C-30), 138.7 (galloyl C-4), 145.8 (galloyl C-3,5), 146.2 (B-ring C-3,5),
154.2 (C-8a), 154.7 (C-5), 155.0 (C-7), 167.0 (galloyl C-7); MALDI
Compound 9 was obtained as a brown amorphous powder: [R]2D0 -200.6
(c 0.1, MeOH); UV (MeOH) λmax nm (ε) 276 (11597); IR (dry film)
ν
max cm-1 3373, 1682, 1615, 1455; 1H NMR (acetone-d6, 500 MHz) δ 0.62,
0.64 (q, J = 11.5 Hz, H-2a0), 0.81, 0.86 (d, J=9.8 Hz, H-100), 1.04, 1.07 (s,
H-90), 1.06 (m, H-4a0), 1.13 (m, H-5a0), 1.30, 1.31 (s, H-80), 1.37, 1.39 (m,
H-60), 1.58 (m, H-30), 1.82 (m, H-4b0, H-5b0), 2.48, 2.55 (dt, J=2.5, 11.0 Hz,
H-10), 2.93 (dd, J=2.4, 14.0 Hz, H-4a), 3.02, 3.19 (br d, J=13.1 Hz,
H-2b0), 3.08 (dd, J=4.5, 14.0 Hz, H-4b), 5.03, 5.15 (br s, H-2), 5.54, 5.56
(m, H-3), 5.94, 5.95 (s, H-6), 6.61, 6.66 (d, J=0.5 Hz, H-B-2,6), 6.99, 7.02
(2H, s, galloyl-2,6); 13C NMR (acetone-d6, 125 MHz) δ 19.1, 19.3 (C-9),
22.8, 23.0 (C-100), 26.6, 26.9 (C-4), 28.7, 28.8 (C-80), 28.7, 28.8 (C-50),
33.3, 33.6 (C-30), 36.1, 36.3 (C-10), 36.2, 36.3 (C-40), 39.9, 40.3 (C-20),
50.4 (C-60), 69.0, 69.3 (C-3), 77.1, 77.7 (C-2), 77.1, 77.2 (C-70), 97.4, 97.7
(C-6), 100.0, 100.4 (C-4), 105.9, 107.6 (C-8), 106.1, 106.6 (B-ring C-2,6),
109.8, 109.9 (galloyl C-2,6), 121.6, 121.8 (galloyl C-1), 130.5, 130.6 (B-
ring C-1), 132.8, 133.0 (B-ring C-4), 138.6, 138.7 (galloyl C-4), 145.8
(galloyl C-3,5), 146.1, 146.2 (B-ring C-3,5), 154.1, 154.2 (C-7), 154.7,
154.9 (C-8a), 155.2 (C-5), 166.0, 166.1 (galloyl C-7); MALDI TOF MS
TOF MS m/z 759 [M + Na]+. Anal. Calcd for C42H56O11 1/4H2O: C,
3
68.04%; H, 7.68%. Found: C, 68.05%; H, 8.06%.
Compound 14 was obtained as a brown amorphous powder: [R]1D9 -84.9
(c 0.1, MeOH); UV (MeOH) λmax nm (ε) 272 (10106); IR (dry film)
m/z 617 [M + Na]+, 633 [M + K]+. Anal. Calcd for C32H34O11 3/2H2O:
ν
max cm-1 3379, 1692, 1608, 1462; 1H NMR (500 MHz, acetone-d6) δ 0.82,
3
C, 61.83%; H, 6.00%. Found: C, 61.82%; H, 6.41%.
0.86 (each 3H, d, J=6.6 Hz, H-180, H-190), 0.85 (6H, d, J=6.6 Hz, H-190,
H-200), 1.02-1.41 (m, H-50-H-170), 1.52 (1H, septet, J=6.6 Hz, H-150),
1.67 (3H, d, J=0.9 Hz, H-170), 1.85 (2H, m, H-40), 2.89 (1H, dd, J=2.3,
17.4 Hz, C-4a), 3.02 (1H, dd, J=4.5, 17.4 Hz, H-4b), 3.56 (1H, dd, J=6.6,
14.0 Hz, H-1a0), 3.64 (1H, dd, J=6.6, 14.0 Hz, H-1b0), 5.05 (1H, s, H-2),
5.12 (1H, br t, J=6.9 Hz, H-20), 5.55 (1H, m, H-3), 5.98 (1H, d, J=2.3 Hz,
H-8), 6.04 (1H, d, J=2.3 Hz, H-6), 6.60 (2H, d, J=0.7 Hz, B-ring H-6),
6.92 (1H, s, galloyl H-6), 8.13 (1H, br s, 7-OH), 8.32 (1H, br s, 5-OH);
13C NMR (125 MHz, acetone-d6) δ 16.2 (C-170), 20.0 (C-160,200), 22.8,
22.9 (C-180,190), 25.4 (C-10), 26.5 (C-4), 25.1, 25.5, 26.1, 28.6, 33.3, 33.4,
37.4, 37.9, 38.0, 38.1, 40.0 (C-50-C-150), 40.8 (C-40), 68.9 (C-3), 77.9
(C-2), 95.7 (C-8), 96.3 (C-6), 99.1 (C-4a), 106.7 (B-ring C-2.6), 110.4
(galloyl C-6), 121.1 (galloyl C-1), 124.1 (galloyl C-2), 124.7 (C-20), 130.6
(B-ring C-1), 133.0 (B-ring C-4), 134.0 (C-30), 137.3 (galloyl C-4), 142.8
(galloyl C-5), 144.7 (galloyl C-3), 146.1 (B-ring C-3,5), 156.9 (C-8a),
157.3 (C-5), 157.6 (C-7), 167.0 (galloyl C-17); MALDI TOF MS m/z
759 [M + Na]+. Anal. Calcd for C42H56O11: C, 68.46%; H, 7.66%.
Found: C, 68.40%; H, 7.94%.
Compound 10 was obtained as a white amorphous powder: 1H NMR
(500 MHz, acetone-d6) δ 0.62 (1H, q, J = 11.5 Hz, H-20), 0.90 (3H, d, J=
6.6 Hz, H-100), 1.01 (3H, s, H-90), 1.08 (1H, m, H-40), 1.11 (1H, m, H-50),
1.28 (3H, s, H-80), 1.34 (1H, m, H-60), 1.57 (1H, m, H-30), 1.83 (2H, m,
H-40, H-50), 2.37 (1H, dt, J=2.8, 11.5 Hz, H-10), 3.19 (1H, br d, J=11.5 Hz,
H-20), 5.76 (1H, d, J=2.5 Hz, H-5), 5.92 (1H, d, J=2.5 Hz, H-3), 7.94 (1H,
s, C-4-OH), 8.17 (1H, s, C-2-OH); 13C NMR (500 MHz, acetone-d6) δ 19.2
(C-90), 23.0 (C-100), 28.0 (C-80), 28.7 (C-50), 33.4 (C-30), 36.2 (C-10), 36.3
(C-40), 40.0 (C-20), 50.3 (C-60), 77.0 (C-70), 96.4 (C-5), 96.6 (C-3), 105.2
(C-1), 156.4 (C-6), 157.4 (C-4), 158.1(C-2); MALDI TOF MS m/z 263
[M+H]+, 285 [M+Na]+
Compound 11 was obtained as a brown amorphous powder: [R]D20
.
-
123.6 (c 0.1, MeOH); UV (MeOH) λmaxnm (ε) 276 (11286); IR (dry film)
νmax cm-1 3389, 1691, 1619, 1454; 1H NMR (500 MHz, acetone-d6) δ
1.53 (3H, d, J=0.9 Hz, H-100), 1.59 (3H, d, J=1.1 Hz, H-80), 1.70 (3H, d,
J=1.1 Hz, H-90), 1.94 (2H, m, H-40), 2.00 (2H, m, H-50), 2.90 (1H, dd,
J=2.1, 15.0 Hz, H-4a), 3.02 (1H, dd, J=4.8, 15.0 Hz, H-4b), 3.32 (1H, dd,
J=6.5, 14.0 Hz, H-1a0), 3.42 (1H, dd, J=7.6, 14.0 Hz, H-1b0), 5.07 (2H, m,
H-2 and H-60), 5.34 (1H, br t, J=7 Hz, H-20), 5.54 (1H, br s, H-3), 6.10
(1H, s, H-6), 6.66 (2H, s, B-ring H-2,6), 7.01 (2H, s, galloyl H-2,6); 13C
NMR (125 MHz, acetone-d6) δ 16.3 (C-90), 17.7 (C-100), 22.5 (C-10),
25.8 (C-80), 26.7 (C-4), 27.5 (C-50), 40.5 (C-40), 69.2 (C-3), 77.9 (C-2),
96.2 (C-6), 98.9 (C-4a), 106.5 (B-ring C-2,6), 107.8 (C-8), 110.0 (galloyl
C-6), 121.8 (galloyl C-1), 124.9 (C-20), 125.3 (C-60), 130.9 (B-ring C-1),
131.3 (C-70), 132.9 (B-ring C-4), 134.1 (C-30), 138.7 (galloyl C-4), 145.8
(galloyl C-3,5), 146.2 (B-ring C-3,5), 154.4 (C-8a), 154.5 (C-5), 154.8 (C-7),
166.1 (galloyl C-7); MALDI TOF MS m/z 617 [M + Na]+. Anal. Calcd
DPPH Radical Scavenging Activity of the Products. Each
compound (0.1 μM) was partitioned between 20 mM phosphate buffer (pH
6.5, 750 μL) and n-octanol (750 μL) or triglyceride (750 μL, Camellia seed
oil, 85% triolein) at 20 °C. After centrifugation, the organic layer (40 or
100 μL) was diluted to 1 mL by adding n-octanol. A portion (100 μL) of the
diluted solutionwas added to a 0.2 mM ethanolicDPPH solution(50μL)in
a 96-well microplate and shaken for 30 min at 25 °C. The concentration of
the DPPH radical was compared by measuring the absorbance at 490 nm.
The scavenging activity (as a percentage) was expressed by [1 - (absor-
bance of the test sample solution/absorbance of the blank solution)] ꢀ 100.
The partition coefficient between n-octanol and the phosphate buffer was
measured by comparing UV absorptions of n-octanol and the aqueous
layer at 280 nm after dilution (from 150 μL to 1.0 mL).
for C32H34O11 2H2O: C, 60.95%; H, 6.07%. Found: C, 61.03%; H,
3
6.10%.
Compound 12 was obtained as a brown amorphous powder: [R]2D0 -92.6
(c 0.1, MeOH); UV (MeOH) λmaxnm (ε) 273 (9801); IR (dry film) νmax
cm-1 3399, 1686, 1607, 1465; 1H NMR (500 MHz, acetone-d6) δ 1.52 (3H,
d, J=0.9 Hz, H-100), 1.58 (3H, d, J=1.2 Hz, H-80), 1.68 (3H, d, J=1.2 Hz,
H-90), 1.86 (2H, t, J=7.2 Hz, H-40), 1.99 (2H, m, H-50), 2.89 (1H, dd,
DPPH Radical Scavenging Activity of the Reaction Mix-
ture. A solution of 2 (2 mg) in acetone (100 μL) was mixed with the test
compound (15 μL) and dried under reduced pressure. The mixture was
heated at 50 °C for 12 h and partitioned between Camellia seed oil

6420 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Fudouji et al.
(triglyceride with a fatty acid composition of 85% oleic acid) and 20 mM
sodium phosphate buffer (pH 6.5) (each 750 μL). After centrifugation, a
portion of the organic layer (50 μL) was diluted to 1 mL by adding
n-octanol. A portion (100 μL) of the diluted solution was used for
comparing the radical scavenging activity. A control in the absence of 2
showed essentially no radical scavenging activity.
RESULTS AND DISCUSSION
The reactions of catechins with various aldehydes in foods or in
vitro model experiments have been performed (25-31) and
indicate that the initial step of the reaction is a simple electrophilic
substitution at the C-8 and/or C-6 position(s) of the catechin
A-ring. To develop a practical method to prepare catechin
derivatives having hydrophobic alkyl moieties, we examined the
condensation of catechins with three aldehydes [green leaf alde-
hyde (trans-2-hexenal), citral (3,7-dimethyl-2,6-octadien-1-al),
and (+)-citronellal (3,7-dimethyloct-6-en-1-al)] and two allyl
alcohols [geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) and
phytol (trans-3,7,11,15-tetramethylhexadec-2-en-1-ol)] (Figure 1).
In the present study, we propose a very simple reaction proce-
dure (29) involving the mixture of catechin and aldehyde or
alcohol heated or left at room temperature in the absence of
solvent. Although the reactions gave mixtures of the products
generated by nonselective condensation, the structures of the
major products were elucidated.
Structures of the Products. Initially the reaction of (+)-catechin
(1) with trans-2-hexenal was examined. The aldehyde is known as
green leaf aldehyde and produced in the green tea leaf (32). The
coupling reaction proceeded slowly at room temperature. After 1
week, TLC analysis indicated that the products were primarily
less polar compared to 1. However, purification of the less polar
products was difficult because of the complex composition, and
only compound 3 was isolated (Figure 2). MALDI TOF MS
(m/z 683 [M+Na]+) indicated that the molecular weight of this
species was 660, which showed that two molecules of 1 were
linked to a trans-2-hexenal unit in 3. The 1H and 13C NMR and
1H-1H COSY spectra supported the molecular composition;
however, the appearance of duplicate signals suggested the
presence of two diastereomeric isomers differing in the config-
uration at the asymmetric centers (C-100 and C-300) of the hexane
unit. HMBC correlations shown in Figure 3 revealed that the C-8
carbons of both catechin units were substituted. The H-100 of the
hexane unit (δ 5.53 and 5.50, each dd, 2.5, 11.7 Hz) correlated to
C-8 (δ 105.1, 105.5), C-8a (δ 153.3), and C-7 (δ 155.9, 156.0),
indicating a C-C linkage between the C-8 and C-100. In turn, the
C-80 correlated with the H-200 methylene protons of the hexane
unit. This correlation indicated that the C-300 methine carbon was
attached to the C-80 of the other catechin unit. The molecular
formula C36H36O12 deduced from the molecular weight and
elemental analysis indicated the degree of unsaturation of 3 was
19. This indicated the presence of an additional ring system
besides the two catechin units. On the basis of the chemical shift
of the C-100 (δ 70.6, 71.5), the ring was determined to be formed by
an ether linkage between the C-70 hydroxyl group and the C-100 of
the hexane unit. The H-100 of both diastereomeric isomers showed
NOE correlations with the H-400 methylene protons. Therefore,
H-100 and C-400 were oriented to the same side of each molecule,
and the relative configuration of the dihydropyran ring, including
C-100 and C-300 of the isomers, was the same. Therefore, product 3
is a mixture of diastereomeric isomers differing in the absolute
configuration of the C-100 and C-300 (Figure 2).
Figure 1. Structures of essential oils and phytol.
position. The reaction proceeded at 100 °C without solvent, and
chromatographic separation of the products yielded four pure
compounds, 4-7.
The molecular formula of product 4 was shown to be
C32H34O12 on the basis of the [M+Na]+ peak at m/z 633 and
the [M + K]+ peak at m/z 649 in the MALDI TOF MS and
elemental analysis. The 1H NMR spectrum showed signals as a
result of the galloyl (δ 7.02, 2H, s), B-ring (δ 6.67, 2H, s), and
C-ring (δ 5.49, br s, H-3; δ 5.15, br s, H-2; δ 3.09, br d; δ 2.95, dd,
H-4) protons, which were similar to those of 2. In addition, three
singlet methyl signals at δ 0.85 (H-90), 0.95 (H-80), and 1.29
(H-100) were observed. Besides signals arising from the unit of 2,
the 13C NMR spectrum showed 10 aliphatic carbon signals
including two oxygen-bearing quaternary carbons [δ 72.1 (C-70),
75.0 (C-30)]. In the HMBC spectrum, a methine proton signal
resonating at δ 3.64 (H-10) correlated to the C-30 and three A-ring
carbons (C-7, C-8, and C-8a) of the epigallocatechin unit. The
two methylene (H-20 and H-40) and one methyl (H-100) proton
also correlated to the C-30. The H-100 methyl proton signal
showed a long-range (4J) coupling to the C-7 of the A-ring. These
HMBC correlations indicated that C-10 of the terpenoid unit was
attached to C-8 of 2 and the C-30 linked to the C-7 through an
ether linkage. The presence of a terminal hydroxyisopropyl group
(C-70-C-90) at C-60 was revealed by the observation of HMBC
correlations between H-60 and C-70 and between H-80(90) and
C-60. This C-60 showed a correlation to the H-10 and indicated the
presence of a C-C bond between C-10 and C-60. This is consistent
with the degree of unsaturation (16) calculated from the mole-
cular formula. The result indicated that a menthane skeleton
formed from the original citral molecule was attached to the C-8
and C-7 hydroxyl group in molecule 4. As for the stereochemistry,
the NOE correlation of the H-90 to the H-2 and H-4β of the flavan
C-ring (Figure 3) indicated that the configurations at the C-10 and
C-60 were S and R, respectively (Figure 2).
Products 5 and 6 were shown to be isomers of 4 on the basis of
1
the MALDI TOF MS, H and 13C NMR, and HMBC spectral
data. The location of the menthane units at C-8 in 5 was
confirmed by the appearance of HMBC correlations of the C-8
(δ 104.7) with H-10 (δ 3.51) and H-2 (δ 4.99). The stereochemical
difference between 4 and 5 was revealed by NOESY spectra,
which showed NOE correlations of the hydroxyisopropyl methyl
proton (H-90) to the B-ring H-2(6) and galloyl H-2(6) protons.
Thus, the configurations at the C-10 and C-60 of 5 were R and S,
respectively. On the other hand, in the structure of 6, the
menthane unit was attached to the C-6. This is because both
the C-ring H-2 (δ 5.03) and the A-ring singlet (δ 6.07, H-8) were
correlated to the C-8a (δ 155.1) in the HMBC spectrum. In
The reaction of epigallocatechin-3-O-gallate (2) and citral was
subsequently examined. In addition to an R,β-unsaturated alde-
hyde structure, which is similar to the above-mentioned 2-
hexenal, this aldehyde has an additional double bond at the C-6

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6421
Figure 2. Structures of catechin (1), epigallocatechin-3-O-gallate (2), and products 3-7 obtained by coupling with trans-2-hexenal and citral.
respectively) were observed. Both of these olefinic protons were
correlated to the C-6 in the HMBC spectrum. Furthermore, the
H-10 (δ 6.63) showed a correlation peak with the C-5, which was
unambiguously assigned by correlation with the C-ring H-4.
These olefinic protons and the C-40 and C-50 methylene protons
showed HMBC correlations with an oxygen-bearing quaternary
carbon (δ 79.1, C-30). The degree of unsaturation (17) indicated
that the C-30 forms a pyran ring with the C-5 or C-7 phenolic
hydroxyl group. The location of the pyran ring was determined to
be C-5 by observation of the 4J HMBC correlation between the
C-5 and C-100 methyl protons. Thus, the arrangement of this
product is represented by structure 7.
Unlike two previous R,β-unsaturated aldehydes, (+)-citronel-
lal does not have a conjugated structure. Two coupling products,
8 and 9, were isolated from the reaction mixture with 2. MALDI
Figure 3. Important COSY, HMBC, and NOESY correlations for com-
TOF MS analysis indicated that two citronellal units were sub-
pounds 3 and 4.
stituted in 8 (m/z 753 [M+Na]+), and one unit was substituted in
addition, the H-100 methyl proton (δ 1.28) correlated to the C-5 (δ
155.8), which also correlated with the C-ring H-4 (δ 2.98). The
configurations of the menthane C-10 and C-60 in 6 were deduced
tobe R and S, respectively, from the NOE correlation between the
H-9 and galloyl H-2(6) protons (Figure 2).
9 (m/z 617 [M+Na]+). Both of the products were shown to be a
mixture of diastereomeric isomers by 1H and 13C NMR spectro-
scopic analysis. That is, 9 was a mixture of two isomers and 8 was
composed of four diastereomers. Comparison of 1H and 13C
NMR chemical shifts with a model compound 10 prepared from
phloroglucinol and citronellal (33) enabled us to deduce the
structures of 8 and 9 (Figure 4). The location of the menthane
moiety in 9 was determined by the observation of HMBC
correlations between H-80 and C-7 (4J correlation) and between
H-10 and C-8 (δ 105.9 and 107.6). The assignment of the A-ring
carbons was confirmed by HMBC correlations of H-2 with C-8a
and H-6 with C-5 and C-7.
The molecular formula of 7 was shown to be C32H32O11 by
MALDI TOF MS and elemental analysis. The HMBC correla-
tions of the C-8a (δ 156.4) with the A-ring aromatic proton (δ
6.07, H-8) and C-ring H-2 (δ 5.05) revealed a substitution at the
C-6 of the epigallocatechin unit. The carbon signals arising from
1
the terpenoid unit were related to those of citral. The H-1H
COSY spectrum indicated the presence of a 4-methylpent-3-enyl
moiety (C-40-C-90). In addition, signals attributable to a cis-
double bond (δ 6.63, 5.41, each d, J = 10.0 Hz, H-10 and H-20,
Geraniol, an essential oil with an allyl alcohol structure,
reacted with 2 under acidic conditions. A mixture of geraniol

6422 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Fudouji et al.
Figure 4. Structures of compounds 8-10 obtained by coupling with citronellal.
Figure 5. Structures of compounds 11-14 obtained by coupling with geraniol and phytol.
and 2 was heated in the presence of citric acid, and two products,
11 and 12, were isolated (see Figure 5). Both compounds showed
an [M+Na]+ peak at m/z 617, indicating the same molecular
constitution. The 1H and 13C NMR spectra were also related to
each other. Comparison of the 13C NMR chemical shifts with
those of geraniol revealed a 3,7-dimethylocta-2,6-dienenyl (ger-
anyl) group in each molecule. On the basis of the appearance of
the HMBC correlation between H-10 and C-8, the location of the
geranyl group in 11 was shown to be at C-8. The assignmentof the
C-8 carbon signal was based on the observation of correlations
between the A-ring carbons and H-2 (to C-8a), H-4 (to C-4a, C-5,
and C-8a), and H-6 (to C-4a, C-5, C-7, and C-8). In contrast, the
geranyl moiety in 12 was determined to be attached at the C-2 of
the galloyl group. Two doublet signals (J = 2.3 Hz) of the A-ring
H-6 and H-8 were observed at δ 6.04 and 5.98, respectively. In
contrast, the signal of the galloyl group was observed as a one-
proton singlet at δ 7.01. The appearance of six aromatic carbon
signals was attributed to the galloyl moiety in the 13C NMR
spectrum, indicating the presence of asymmetrical substitutions. In
addition, one of the galloyl carbons (δ 124.1, C-2) showed an
HMBC correlation with the H-10 methylene protons of the geraniol
moiety. Thus, the conformation was represented by structure 12.
Phytol, which is a component of chlorophylls, has the same
partial structure as geraniol. Reaction with 2 under acidic con-
ditions (at 50 °C for 30 h in the presence of HCl) gave two
products, 13 and 14 (see Figure 5). MALDI TOF MS of these
products (m/z 759 [M+Na]+) indicated that one molecule of
phytol was attached to molecule 2 in these products. The 1H
NMR signals of 13 were related to those of 11, except for the
appearance of complex signals arising from the phytol moiety.
The A-ring singlet δ 6.13 was assigned to the H-8 proton, because
this signal and the C-ring H-2 showed 3J HMBC correlations to
the C-8a (δ 154.2). Furthermore, the C-10 methylene protons of
the phytol moiety were correlated with A-ring C-5 (δ 154.7), C-6
(δ 108.9), and C-7 (δ 155.0). These HMBC correlations revealed
that the phytol C-1 was attached to the C-6 of the flavan-3-ol
1
moiety. On the other hand, the H NMR spectrum of 14 was
related to that of 12: the appearance of mutually coupled A-ring
proton signals (δ 5.98 and 6.04, d, J = 2.3 Hz) and the one-proton
singlet at δ 6.92, which was assigned to the galloyl proton,
indicated the location of the phytol moiety to be at the galloyl
C-2 position. This was consistent with the HMBC correlations
between the galloyl C-2 (δ 124.1) and the phytol H-10 protons [δ
3.56 (dd, J = 6.6, 14.0 Hz), 3.64 (dd, J = 6.6, 14.0 Hz)].
DPPH Radical Scavenging Activity. The products obtained in
this study were expected to show higher lipid solubility when
compared to the lipid solubility of 2. Therefore, DPPH radical
scavenging activities of the organic layer after partitioning with
phosphate buffer (pH 6.5) were compared. In the presence of n-
octanol, the octanol layers of all products showed similar radical
scavenging activities when compared to the activity of 2 (Table 1).
However, when Camellia seed oil (triglyceride with a fatty acid
composition of 85% oleic acid) was used, the activity of the
organic layer of 2 was very small. In contrast, organic layers of the
products 6-9, 11, and 13 showed relatively higher activities. Each
reaction of catechin with the essential oil gave a complex mixture,
and the products isolated in this study may not represent the total
reaction products. Therefore, the lipid-soluble fractions of the
reaction mixtures of 2 with various aldehydes (trans-2-hexenal, citral,
citronellal, perillaldehyde, cinnamaldehyde, and trans-2-nonenal),

Article
J. Agric. Food Chem., Vol. 57, No. 14, 2009 6423
Table 1. DPPH Radical Scavenging Activity of the Products after Solvent
Partitioning
(2) Jaya, D.; Hitomia, H.; Griendling, K. K. Oxidative stress and
diabetic cardiovascular complications. Free Radical Biol. Med.
2006, 40, 183–192.
scavenging activity (%)
(3) Klaunig, J. E.; Kamendulis, L. M. The role of oxidative stress in
carcinogenesis. Annu. Rev. Pharmacol. 2004, 44, 239–267.
(4) Hertog, M. G. L.; Kroumhout, D.; Aravanis, C.; Blackburn, H.;
Buzina, R.; Fidanza, F.; Giampaoli, S.; Jansen, A.; Menotti, A.;
Nedeljkovic, S.; Pekkarinen, M.; Simic, B. S.; Toshima, H.; Feskens,
E. J. M.; Hollman, P. C. H.; Katan, M. B. Flavonoid intake and
long-term risk of coronary heart disease and cancer in the seven
countries study. Arch. Intern. Med. 1995, 155, 381–386.
(5) Hertog, M. G. L.; Feskens, E. J. M.; Hollman, P. C. H.; Katan, M.
B.; Kroumhout, D. Dietary antioxidant flavonoids and risk of
coronary heart disease: The Zutphen Elderly Study. Lancet 1993,
342, 1007–1011.
n-octanol layer
40 μLa
triglyceride layer
product
100 μLa
40 μLa
log Poctanol
2
32.6 ( 5.6
29.9 ( 3.6
32.0 ( 0.7
23.4 ( 2.0
30.0 ( 2.1
28.7 ( 3.1
44.3 ( 3.7
41.8 ( 0.5
1.2 ( 6.6
9.5 ( 5.7
2.9 ( 5.2
3.7 ( 4.8
38.5 ( 2.3
26.4 ( 2.8
42.3 ( 3.6
17.1 ( 2.1
66.7 ( 3.6
68.8 ( 4.7
1.00
1.85
2.66
2.72
>10
4
6
59.3 ( 6.3
51.9 ( 7.9
68.5 ( 4.9
47.9 ( 8.5
80.1 ( 0.9
78.3 ( 0.9
7
8
9
2.03
2.55
>10
11
13
(6) Keli, S. O.; Hertog, M. G. L.; Feskens, E. J. M.; Kroumhout, D.
Dietary flavonoids, antioxidant vitamins, and incidence of stroke.
The Zutphen study. Arch. Intern. Med. 1996, 156, 637–642.
(7) Renaud, S.; de Lorgeril, M. Wine, alcohol, platelets, and French
paradox for coronary heart disease. Lancet 1992, 339, 1523–1526.
(8) Yokozawa, T.; Cho, E.-J.; Hara, Y.; Kitani, K. Antioxidative
activity of green tea treated with radical initiator 2,20-azobis(2-
amidinopropane) dihydrochloride. J. Agric. Food Chem. 2000, 48,
5068–5073.
(9) Nakagawa, T.; Yokozawa, T.; Sano, M.; Takeuchi, S.; Kim, M.;
Minamoto, S. Activity of (-)-epigallocatechin 3-O-gallate against
oxidative stress in rats with adenine-induced renal failure. J. Agric.
Food Chem. 2004, 52, 2103–2107.
(10) Yokozawa, T.; Dong, E.; Nakagawa, T.; Kashiwagi, H.; Nakagawa,
H.; Takeuchi, S.; Chung, H.-Y. In vitro and in vivo studies on the
radical-scavenging activity of tea. J. Agric. Food Chem. 1998, 46,
2143–2150.
(11) Lotito, S. B.; Fraga, C. G. (+)-Catechin prevents human plasma
oxidation. Free Radical Biol. Med. 1998, 24, 435–441.
(12) Ishikawa, T.; Suzukawa, M.; Ito, T.; Yoshida, H.; Ayaori, M.;
Nishiwaki, M.; Yonemura, A.; Hara, Y.; Nakamura, H. Effect of tea
flavonoid supplementation on the susceptibility of low-density
lipoprotein to oxidative modification. Am. J. Clin. Nutr. 1997, 66,
261–266.
(13) Van het Hof, K. H.; de Boer, H. S. M.; Wiseman, S. H.; Lien, N.;
Weststrate, J. A.; Tijburg, L. B. M. Consumption of green or black
tea does not increase resistance of low-density lipoprotein to oxida-
tion in humans. Am. J. Clin. Nutr. 1997, 66, 1125–1132.
(14) Princen, H. M. G.; van Duyvenvoorde, W.; Buytenhek, R.; Blonk,
C.; Tijburg, L. B.; Langius, J. A.; Meinders, A. E.; Pijl, H. No effect
of consumption of green and black tea on plasma lipid and
antioxidant levels and on LDL oxidation in smokers. Arterioscler.
Thromb. Vasc. Biol. 1998, 18, 833–841.
(15) Luck, G.; Liao, H.; Murray, N. J.; Grimmer, H. R.; Warminski, E.
E.; Williamson, M. P.; Lilley, T. H.; Haslam, E. Polyphenols,
astringency and proline-rich proteins. Phytochemistry 1994, 37,
357–371.
(16) Zhang, Y.-J.; Tanaka, T.; Betsumiya, Y.; Kusano, R.; Matsuo, A.;
Ueda, T.; Kouno, I. Association of tannins and related polyphenols
with the cyclic peptide gramicidin S. Chem. Pharm. Bull. 2002, 50,
258–262.
a A potion (40 or 100 μL) of organic layer (750 μL) after partition was diluted to
1.0 mL, and 100 μL of the diluted solution was added to 0.2 mM ethanolic DPPH
solution (50 μL).
Table 2. DPPH Radical Scavenging Activity of Triglyceride Layer after
Partitioning of the Reaction Mixture with Triglyceride/Water
scavenging activity (%)
2
7.7 ( 3.9
0.9 ( 4.1
48.1 ( 3.9
88.4 ( 0.6
85.6 ( 0.6
89.5 ( 0.0
38.5 ( 2.6
68.7 ( 1.3
36.3 ( 4.0
87.1 ( 0.4
71.1 ( 1.5
2 + trans -2-hexenal
2 + citral
2 + citronellal
2 + geraniol
2 + phytol
2 + perillaldehyde
2 + cinnnamaldehyde
2 + 3-nonen-2-one
2 + trans-2-nonenal
2 + lemon grass essential oil
alcohols (geraniol and phytol), a ketone (3-nonen-2-one), and com-
mercial lemongrass essential oil (major component is citral) were
compared (Table 2). Except for trans-2-hexenal, the triglyceride-
soluble fractions of the reaction mixtures showed strong radical
scavenging activities compared to the activity of 2.
In this study, we introduced a simple method for the prepara-
tion of coupling products of catechin and aldehydes and allyl
alcohols. The reactions resulted in the production of complex
mixtures resulting from the generation of positional and diaster-
eomeric isomers. Therefore, the practical application of the
condensation products as functional materials may be limited
in the form of a mixture of the isomers. Similar reactions may
occur during food processing, such as the heating of tea leaves,
because aldehydes are generated from unsaturated fatty acids by
oxidative degradation. The chemical modification of polyphenol
molecules is an interesting subject in food and medicinal sciences
because of the attractive biological activities of catechins. In
addition to the increase of DPPH radical scavenging activity in
hydrophobic conditions, a preliminary study on the antibacterial
activity of the products by the disk diffusion method (34) suggests
that compounds 11 and 12 show stronger activities than com-
pound 2 against Staphylococcus aureus. Further studies on
biological activities are currently in progress.
(17) Sirk, T. W.; Brown, E. F.; Sum, A. K.; Friedman, M. Molecular
dynamics study on the biophysical interactions of seven green tea
catechins with lipid bilayers of cell membranes. J. Agric. Food Chem.
2008, 56, 7750–7758.
(18) Kamihira, M.; Nakazawa, H.; Kira, A.; Mizutani, Y.; Nakamura,
M.; Nakayama, T. Interaction of tea catechins with lipid bilayers
investigated by a quartz-crystal microbalance analysis. Biosci.,
Biotechnol., Biochem. 2008, 72, 1372–1375.
ACKNOWLEDGMENT
We are grateful to K. Inada, N. Yamaguchi, and N. Tsuda,
Nagasaki University Joint Research Center, for NMR and MS
measurements and elemental analysis.
(19) Tanaka, T.; Kusano, R.; Kouno, I. Synthesis and antioxidant
activity of novel amphipathic derivatives of tea polyphenol. Bioorg.
Med. Chem. Lett. 1998, 8, 1801–1806.
(20) Park, K. D.; Park, Y. S.; Cho, S. J.; Sun, W. S.; Kim, S. H.; Jung,
D. H.; Kim, J. H. Antimicrobial activity of 3-O-acyl-(-)-epicatechin
and 3-O-acyl-(+)-catechin derivatives. Planta Med. 2004, 70, 272–
276.
LITERATURE CITED
(1) Vogel, R, A. Coronary risk factors, endothelial function, and
atherosclerosis: a review. Clin. Cardiol. 2009, 20, 426–432.

6424 J. Agric. Food Chem., Vol. 57, No. 14, 2009
Fudouji et al.
(21) Matsubara, K.; Saito, A.; Tanaka, A.; Nakajima, N.; Akagi, R.;
Mori, M.; Mizushina, Y. Epicatechin conjugated with fatty acid is a
potent inhibitor of DNA polymerase and angiogenesis. Life Sci.
2007, 80, 1578–1585.
(22) Kajiya, K.; Kumazawa, S.; Nakayama, T. Interaction of tea catechin
derivatives with lipid bilayers and their antibacterial activity. Foods
Food Ingredients J. Jpn. 2004, 209, 834–838.
(23) Nonaka, G.; Nishioka, I. Novel biflavonoids, chalcan-flavan dimers
from gambir. Chem. Pharm. Bull. 1980, 28, 3145–3149.
(24) Nonaka, G.; Kawahara, O.; Nishioka, I. Tannins and related
compounds. XV. A new class of dimeric flavan-3-ol gallates,
theasinensins A and B, and proanthocyanidin gallates from green
tea leaf. (1). Chem. Pharm. Bull. 1983, 31, 3906–3914.
(25) Tanaka, T.; Takahashi, R.; Kouno, I.; Nonaka, G. Chemical
evidence for the de-astringency (insolubilization of tannins)
of persimmon fruit. J. Chem. Soc., Perkin Trans. 1 1994, 3013-
3022.
(26) Es-Safi, N.-E.; Cheynier, V.; Moutounet, M. Study of the reactions
between (+)-catechin and furfural derivatives in the presence or
absence of anthocyanins and their implication in food color change.
J. Agric. Food Chem. 2000, 48, 5946–5954.
(27) Beretta, G.; Furlanetto, S.; Regazzoni, L.; Zarrella, M.; Facino, R.
M. Quenching of R,β-unsaturated aldehydes by green tea polyphe-
nols: HPLC-ESI-MS/MS studies. J. Pharm. Biomed. Anal. 2008, 48,
606–611.
catechin and wood aldehydes. J. Agric. Food Chem. 2005, 53,
9249–9256.
(29) Tanaka, T.; Matsuo, Y.; Yamada, Y.; Kouno, I. Structure of
polymeric polyphenols of cinnamon bark deduced from condensa-
tion products of cinnamaldehyde with catechin and procyanidin. J.
Agric. Food Chem. 2008, 56, 5864–5870.
(30) Totlani, V. M.; Peterson, D. G. Epicatechin carbonyl-trapping
reactions in aqueous maillard systems: identification and structural
elucidation. J. Agric. Food Chem. 2006, 54, 7311–7318.
(31) Yuko Noda, Y.; Peterson, D. G Structure-reactivity relationships
of flavan-3-ols on product generation in aqueous glucose/glycine
model systems. J. Agric. Food Chem. 2007, 55, 3686–3691.
(32) Sekiya, J.; Kajiwara, T.; Hatanaka, A. Seasonal changes in activities
of enzymes responsible for the formation of C6-aldehydes and C6-
alcohols in tea leaves, and the effects of environmental temperatures
on the enzyme activities. Plant Cell Physiol. 1984, 25, 269–280.
(33) Murphy, W. S.; Culhane, A.; Duffy, B.; Tuladhar, S. M. Base-
catalyzed reaction of citronellal with phenols. J. Chem. Soc., Perkin
Trans. 1 1992, 3397-3400.
(34) Taguri, T.; Tanaka, T.; Kouno, I. Antimicrobial activity of 10
different plant polyphenols against bacteria causing food-borne
disease. Biol. Pharm. Bull. 2004, 27, 1965–1969.
Received April 2, 2009. Revised manuscript received May 18, 2009.
Accepted May 20, 2009. This work was supported by Grant-in-aid for
Scientific Research 18510189 from the Japan Society for the Promotion
of Science.
(28) Sousa, C.; Mateus, N.; Perez-Alonso, J.; Santos-Buelga, C.; de
Freitas, V. Preliminary study of oaklins, a new class of brick-red
catechinpyrylium pigments resulting from the reaction between