Preparation, Characterization, and antioxidative effects of oligomeric proanthocyanidin-I-Cysteine complexes

Article abstract of DOI:10.1021/jf062819n

Controlled acid-catalyzed degradation of proanthocyanidin polymers in grape seeds together with L-cysteine led to oligomeric proanthocyanidin-L-cysteine complexes along with monomeric flavan-3-ol derivatives being isolated, and their structures were confi

Full text of DOI:10.1021/jf062819n

J. Agric. Food Chem. 2007, 55, 1525−1531 1525
Preparation, Characterization, and Antioxidative Effects of
Oligomeric Proanthocyanidin -Cysteine Complexes
−L
HAJIME FUJII,*,† TAKASHI NAKAGAWA,† HIROSHI NISHIOKA,† ERI SATO,†
AYA HIROSE,† YASUHIRO UENO,† BUXIANG SUN,† TAKAKO YOKOZAWA,‡ AND
§
GEN-ICHIRO NONAKA
Amino Up Chemical Company, Ltd., High Tech Hill Shin-ei, 363-32 Kiyota, Sapporo 004-0839,
Japan; Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan;
and Usaien Pharmaceutical Company, Ltd., 1-4-6 Zaimoku, Saga 840-0055, Japan
Controlled acid-catalyzed degradation of proanthocyanidin polymers in grape seeds together with
L-cysteine led to oligomeric proanthocyanidin-L-cysteine complexes along with monomeric flavan-
3-ol derivatives being isolated, and their structures were confirmed on the basis of spectroscopic
data and by chemical means. In addition, comparative studies on the antioxidative and survival effects
of oligomeric proanthocyanidin-L-cysteine complexes and proanthocyanidin polymers were performed.
The oligomeric proanthocyanidin-L-cysteine complexes showed higher bioavailability and antioxidant
capacity and enhanced survival time in the animal test groups. In addition, it is suggested that the
oligomeric complexes may help to prevent oxidative stress and may reduce free radical production.
KEYWORDS: Proanthocyanidin; oligomer; polymer; proanthocyanidin-L-cysteine complex; L-cysteine;
polyphenol; flavan-3-ol; oligomerization; grape seed polyphenol; antioxidative activity; survival study
INTRODUCTION
the flavan-3-ol units and the degree of polymerization to be
confirmed (16). Benzylthiol or 2-hydroxyethylthiol has typically
been used as a thiolytic reagent. It is, however, questionable as
to whether such sulfur-containing products resulting from this
process are suitable to be utilized as foods. As an alternative,
monomeric flavan-3-ol-L-cysteine complexes were previously
obtained by depolymerization of proanthocyanidin in the
presence of L-cysteine by Torres et al. (17). L-Cysteine itself is
safe for food supplements; thus, L-cysteine derivatives herein
may be also safe for food application.
On the basis of this background, the purposes of this study
were to establish the most suitable method to prepare proan-
thocyanidin oligomers from polymers through acid-catalyzed
degradation in the presence of L-cysteine and to compare the
antioxidative effects and rate of animal survival when using
the oligomers with those of original polymers.
Among plant polyphenols, proanthocyanidins are increasingly
attracting attention for their antiaging effects and preventive
properties in relation to the development of multiple human
ailments, such as hypertension, allergies, infection, cardiovas-
cular diseases, and a variety of infections (1-8). Naturally
occurring proanthocyanidins are complicated mixtures, consist-
ing primarily of flavan-3-ol polymers. They are, because of their
large molecular sizes, regarded as not being readily absorbed
through the intestines. On the other hand, it has been reported
that radiolabeled monomeric flavan-3-ols and proanthocyanidin
dimers and trimers can be readily transported through a layer
of colonic carcinoma (Caco-2) cells of human origin (9). The
dimer, procyanidin B-2, is also described as being readily
detectable in human plasma after flavan-3-ol-rich cocoa has been
consumed (10). Furthermore, proanthocyanidin oligomers have
been reported to have much stronger bioactivities than flavan-
3-ol monomers and polymers (11-15). Therefore, it would be
beneficial to develop an available source of oligomers that could
be easily taken up by cells.
MATERIALS AND METHODS
Chemicals. L-Cysteine hydrochloride monohydrate and tannase
(from Aspergillus oryzae) were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). L-Ascorbic acid was obtained from
Junsei Chemical Co., Ltd. (Tokyo, Japan). (+)-Catechin, (-)-epicat-
echin, (-)-epigallocatechin, (-)-epicatechin 3-O-gallate, and procya-
nidins B-1 and B-2 were obtained from Funakoshi Co., Ltd. (Tokyo,
Japan). The Sephadex LH-20, DIAION HP-20, and MCI-gel CHP 20
that were used were produced by Amersham Biosciences K.K. (Tokyo,
Japan) and Mitsubishi Chemical Corp. (Tokyo, Japan), respectively.
Analyses. 1H (400 MHz) and 13C (100 MHz) nuclear magnetic
resonance (NMR) spectra were recorded on a Jeol GX-400 using TMS
or corresponding solvent signals as an internal standard at 25-40 °C.
Thiolysis is a known method in which the depolymerization
of proanthocyanidins through a cleavage of interflavanoid
linkages, followed by a nucleophilic attack by a thiol group on
the C-4 position of a flavan moiety, enables the structures of
* Author to whom correspondence should be addressed (telephone +81-
11-889-2233; fax +81-11-889-2375; e-mail hfujii@aminoup.co.jp).
† Amino Up Chemical Co., Ltd.
‡ University of Toyama.
§ Usaien Pharmaceutical Co., Ltd.
10.1021/jf062819n CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/25/2007

1526 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Fujii et al.
High-resolution fast atom bombardment mass spectra (HR-FAB-MS)
and high-resolution electrospray ionization mass spectra (HR-ESI-MS)
were obtained with a JEOL JMS-700TZ. Optical rotations were
measured with a Jasco DIP-370 digital polarimeter at room temperature.
Thin-layer chromatography (TLC) was performed on a precoated 0.25
mm Kieselgel 60 F254 glass plate (Merck KGaA, Darmstadt, Germany)
with benzene/ethyl formate/formic acid (2:7:1) and n-butanol/acetic
acid/H2O (4:1:2) and was detected by irradiation of UV light (254 nm)
and by spraying 2% ethanolic FeCl3, anisaldehyde-sulfuric acid, or
0.2% n-butanolic ninhydrin reagents.
graphed over Sephadex LH-20 (ca. 20 mL) with 70% EtOH to give
gallic acid (3 mg) and hydrolysate (4, 11 mg).
Animal Studies. Animals and Treatment. The “Guidelines for
Animal Experimentation” approved by Amino Up Chemical Co., Ltd.
were followed in these experiments. Specific pathogen-free (SPF) male
9-week-old Wistar rats were purchased from Japan SLC, Inc. (Shizuoka,
Japan) to evaluate the antioxidant capacity, and SPF male 5-week-old
ddY mice were purchased from Japan Laboratory Animals, Inc. (Tokyo,
Japan) for the survival study. They were housed under environmentally
controlled conditions at a temperature of about 23 °C, a relative
humidity of about 55%, and a 12-h alternating cycle of light and dark.
All rats and mice were allowed access to a solid diet (CE-2, comprising
24.0% protein, 3.5% lipid, and 60.5% carbohydrate) (CLEA Japan Inc.,
Tokyo, Japan) and drinking water ad libitum. Following 1 week of
adaptation, 35 10-week-old rats were randomly divided into seven
groups (5 animals per group), which were control (no treatment), GSP,
and cys-OLG groups for experiment 1 (expt 1) and control (no
treatment), fraction I, fraction II, and fraction III groups for experiment
2 (expt 2). Both experiments were independently carried out. Each
material was administered daily by oral gavage at a dose of 10 mg/kg
of body weight for 1 week. Blood was taken from the jugular vein
before the initial administration and 24 h after the last administration.
Serum was immediately separated from the blood samples by centrifu-
gation at 3000 rpm for 10 min. In the experiment of survival study on
normal mice, 36 6-week-old mice were randomly divided into three
groups (12 animals per group), which were control (normal powder
diet, CE-2), GSP, and cys-OLG groups. Each group was daily given
a powder diet containing each material at a dose of 200 mg/kg of body
weight. After 6 months, blood was obtained from five fasted mice of
each group for antioxidant evaluation following anesthesia by diethyl
ether, and serum was prepared by centrifugation at 3000 rpm for 10
min. The remaining mice were continuously treated in the same manner
until seven control animals all died naturally.
Analyses. The polyphenol concentration was measured using a
method based on that of Gulcin (18). One hundred and twenty
microliters of serum was mixed with 40 µL of 60% perchloric acid
and extracted with 600 µL of n-butanol for 10 s. After centrifugation
at 12000 rpm for 10 min at 4 °C, a 200-µL aliquot of the supernatant
fraction was dried under nitrogen gas purge for 15 min at 40 °C. Then,
1.0 mL of 0.1 M FeCl3 was added, and the reaction was initiated by
adding 80 µL of 10 mM K3[Fe(CN)6]. After 20 min of incubation at
room temperature, color intensity was measured at 720 nm. The levels
of Trolox equivalent antioxidant capacity (TEAC) and lipid peroxide
(LPO) were evaluated as parameters of antioxidant capacity. For the
TEAC assay (19), 300 µL of 500 µmol/L 2,2′-azinobis(3-ethylben-
zothiazoline-6-sulfonic acid) (ABTS), 36 µL of metmyoglobin, 487
µL of 5 mM phosphate buffer (pH 7.4), and 10 µL of sample were
mixed, and the reaction was initiated by the addition of 167 µL of 450
µmol/L hydrogen peroxide. The absorbance at 734 nm was measured
after being incubated for 15 min at room temperature. LPO concentra-
tions were evaluated using a LPO-Test Wako assay kit (Wako Pure
Chemical Industries, Ltd., Osaka, Japan).
High-Performance Liquid Chromatography (HPLC). Reverse-
phase HPLC was performed using a Hitachi apparatus equipped with
an L-7420 detector (at 260 nm) and a 150 × 4 mm i.d., 5 µm, ODS
80Ts column (Toso Co., Ltd., Tokyo, Japan). Separation was achieved
with an increasing amount of 0.1% acetic acid in acetonitrile (B) in
0.1% aqueous acetic acid (A): 0-5 min, 5% B, isocratic; 5-20 min,
5-70% B, linear gradient; 20-20.1 min, 70-90% B, linear gradient;
and 20.1-25 min, 90% B, isocratic at a flow rate of 0.8 mL/min.
Normal-phase HPLC was achieved using the same apparatus with a
250 × 4.6 mm i.d., 5 µm, Luna Silica column (Phenomenex, Torrance,
CA) at 40 °C, and was recorded at 280 nm. The mobile phases consisted
of CHCl3/MeOH/H2O/0.5% trifluoroacetic acid (400:90:5:5) (A) and
CHCl3/MeOH/H2O/0.5% trifluoroacetic acid (45:405:45:5) (B). Separa-
tion was achieved at a flow rate of 0.8 mL/min with the following
gradient system: 0-20 min, 0-15% B, linear gradient; 20-45 min,
15-30% B, linear gradient; and 45-70 min, 30% B, isocratic.
Preparation of the Oligomeric Proanthocyanidin-L-Cysteine
Complexes from Grape Seed. Dried grape seeds (8.0 kg) were
powdered and extracted with 80% MeOH (30 L) at room temperature
for 3 days. The insoluble materials were filtered out, and the filtrate
was concentrated and applied to a column of DIAION HP-20 (ca. 25
L). After a washing with H2O (100 L), elution with MeOH (50 L)
gave a mixture of grape seed proanthocyanidins (GSP) as a dark brown
powder (456 g). A mixture of GSP (400 g), L-cysteine hydrochloride
monohydrate (400 g), and l-ascorbic acid (4 g) in H2O (4 L) was kept
with stirring at 60 °C for 48 h. The reaction mixture was subjected to
a column of DIAION HP-20 (ca. 25 L). After a washing with H2O
(100 L), elution with 40% EtOH (50 L) yielded oligomeric proantho-
cyanidin-L-cysteine complexes (cys-OLG) as a reddish brown powder
(408 g).
Separation of Cys-OLG. Cys-OLG (50 g) in H2O (0.15 L) was
partitioned with a mixture of n-butanol, n-propanol, and H2O (2:1:3,
0.6 L) six times. The aqueous layer was concentrated and lyophilized
to give a polar fraction (fraction I) as a brown powder (7.2 g), whereas
the organic layer, after concentration, afforded an oligomeric fraction
as a deep brown powder (42.5 g). A portion (15 g) of this powder was
chromatographed over Sephadex LH-20 with an increasing polarity from
EtOH to EtOH/H2O (4:1) to separate fractions into II (1.37 g) and III
(4.05 g). Repeated chromatography of fraction II (1 g) using Sephadex
LH-20 and MCI gel CHP 20 with an increasing amount of MeOH in
H2O (9:1-1:9) gave compounds 1 (288.1 mg), 2 (14.5 mg), and 3 (48.2
mg), whereas fraction III (3 g) yielded compounds 4 (25.3 mg), 5 (16.2
mg), and 6 (43.4 mg) upon similar treatment. These compounds showed
positive spots with a ninhydrin spray reagent on TLC.
Statistics. Antioxidant capacity data are presented as mean ( SD
and were analyzed by one-way ANOVA. Fisher’s protected least
significant difference (PLSD) was used as post hoc test. Regarding
the survival study, survival rates were assessed using the Kaplan-
Meier method, and differences between the rates were analyzed by the
log-rank test. Significance was considered at p < 0.05.
Compound 1 was obtained as a white amorphous powder: [R]D
-62.7° (c 1.0, 50% acetone); HR-FAB-MS, m/z 410.0925 [M + H]+.
Compound 3 was obtained as a pale brown amorphous powder: [R]D
-64.1° (c 1.0, 50% acetone); HR-FAB-MS, m/z 426.0844 [M + H]+.
Compound 4 was obtained as a pale brown amorphous powder: [R]D
+44.0° (c 1.0, methanol); negative HR-ESI-MS, m/z 696.1360 [M -
H]-.
RESULTS AND DISCUSSION
Compound 5 was obtained as a pale brown amorphous powder: [R]D
+1.0° (c 0.2, 50% acetone); negative HR-ESI-MS, m/z 848.1486 [M
- H]-.
Compound 6 was obtained as a brown amorphous powder: [R]D
+56.2° (c 1.0, 50% acetone); negative HR-ESI-MS, m/z 984.1956 [M
- H]-.
Enzymatic Hydrolysis. A solution of compound 5 (18 mg) in H2O
(4.5 mL) was incubated for 3 h with tannase (3.6 mg) at 37 °C. The
solvent was evaporated under reduced pressure, and the residue was
treated with MeOH. The soluble portion of MeOH was chromato-
Preparation of Cys-OLG. The ratio of cysteine moiety in
cys-OLG was estimated by microanalysis and displayed 2.70%
sulfur content. Considering the molecular weights of sulfur
(32.1), cysteine (121.16; C3H7NO2S), and catechin (290.27), the
ratio of cysteine and catechin units is calculated as 1:3.67.
Characterization of Compounds. Compound 1 showed an
[M + H]+ peak in the HR-FAB-MS, which was in agreement
with the molecular formula of C18H20O8NS. The 1H NMR data
for 1 (Table 1) showed that it was closely analogous to (-)-

Oligomeric Proanthocyanidin−L-Cysteine Complexes
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1527
Table 1. 1H Chemical Shifts for 1
−5
H no.
H-2
H-3
H-4
H-6
H-8
B-H-2
B-H-5
B-H-6
1a
2b
3b
4a
5b
5.09 (br s)
4.86 (d, 9.6 Hz)
4.22 (dd, 9.6, 4.3 Hz)
4.23 (d, 4.3 Hz)
6.04 (d, 2 Hz)
5.81 (d, 2 Hz)
6.94 (br s)
5.13 (s)
4.07 (s)
3.92 (s)
4.96 (br s)
4.22 (br m)
4.54 (br s)
5.92
5.3
5.4
4.6
4.07 (m)
3.86 (d, 2 Hz)
5.99 (d, 2 Hz)
5.87 (d, 2 Hz)
6.99 (d, 2 Hz)
6.76 (d, 8.3 Hz)
6.81 (dd, 8.3, 2 Hz)
6.03 (br s)
5.96 (br s)
6.57 (s)
5.9 or 6.1
5.9 or 6.1
5.92
6.60
6.60
6.60
−
−
−
7.09
7.09
7.09
6.5−
6.5−
6.5−
5.3
4.0
4.0
6.75
6.75
6.75
6.86 (br s)
6.86 (br s)
6.57 (s)
H-2
H-3
H-4
H-6
′
′
′
′
5.20 (br s)
3.90 (s)
3.83 (s)
5.92
5.9 or 6.1
B
B
B
′
′
′
-H-2
-H-5
-H-6
6.60
6.67 (d, 8.3 Hz)
6.60 7.09
−
7.09
6.5−
6.5−
6.5−
6.75
6.75
6.75
−
gal-H-2
gal-H-6
cys-H-2
cys-H-3
6.8 (br m)
6.8 (br m)
4.07 (dd, 9, 3.9 Hz)
3.0 (br m)
3.6 (br m)
4.13 (dd, 9, 4 Hz)
2.95 (dd, 15, 9 Hz)
3.43 (dd, 15, 4 Hz)
4.07 (dd, 9, 4 Hz)
3.02 (dd, 15, 9 Hz)
3.55 (dd, 15, 4 Hz)
4.10 (br m)
3.00 (dd, 15, 9 Hz)
3.45 (dd, 15, 4 Hz)
4.14 (dd, 9, 4 Hz)
2.95 (br m)
3.44 (br m)
a Measured in acetone-d6 D2O. b Measured in CD3OD at 25
− °C.
Figure 1. Structures of compounds 1−6.
epicatechin (2,3-cis orientation) as it displayed a broad singlet
signal at δ 5.09 due to H-2. This was supported by the 13C
NMR chemical shift (δ 74.7) due to the epicatechin C-2 carbon
(20). In the 1H NMR spectra, the appearance of a set of aliphatic
signals at δ 5.09 (1H, s), 4.07 (1H, m), and 3.86 (1H, d, J ) 2
Hz), assignable to flavan H-2, -3, and -4, respectively, indicated
the presence of a 4-substituted flavan ring with 2,3-cis and 3,4-
trans configurations in the C-ring (21). In addition, the
appearance of ABX signals at δ 4.13 (1H, dd, J ) 9, 4 Hz),
2.95 (1H, dd, J ) 15, 9 Hz), and 3.43 (1H, dd, J ) 15, 4 Hz)
suggested the presence of a cysteine moiety. On the basis of
these findings, the structure of 1 was deduced as 4â-(S-L-
cysteinyl)-(-)-epicatechin as shown in Figure 1. These spec-
troscopic data are identical with those reported by Torres et al.
(17).
the structure of 2 was concluded to be 4â-(S-L-cysteinyl)-(+)-
catechin. These spectroscopic data are also identical with those
reported by Torres et al. (17).
Compound 3 gave a blue coloration with the FeCl3 reagent,
suggesting the presence of a pyrogallol ring in the molecule
(22). The HR-FAB-MS of 3 showed an [M + H]+ peak, which
was 16 mass units more than that of 1. The 1H NMR spectrum
of 3 (Table 1) was similar to that of 1, except for the two proton
singlet signals at δ 6.57 due to the 2- and 6-positions on the
B-ring, thus leading to the conclusion that the structure was
4â-(S-L-cysteinyl)-(-)-epigallocatechin (3).
Compound 4 showed an [M - H]- peak corresponding to
C33H30O14NS in the negative HR-ESI-MS, suggesting a dimeric
proanthocyanidin structure with a cysteine moiety. The 1H NMR
data for 4 (Table 1) showed a close analogy to those of
procyanidin B-2, displaying broad singlets at δ 4.96 and 5.20
due to H-2 and -2′, respectively, suggesting the presence of two
epicatechin (2,3-cis) units. This was supported by 13C NMR
chemical shifts of the C-2 signals (δ 74.6 and 76.0) (20).
Because the 13C NMR chemical shifts attributable to the flavan
The 1H NMR spectrum of 2 (Table 1) was similar to that of
1, except for the signals due to flavan H-2, -3, and -4 at δ 4.86
(1H, d, J ) 9.6 Hz), 4.22 (1H, dd, J ) 9.6, 4.3 Hz), and 4.23
(1H, d, J ) 4.3 Hz), respectively, the larger coupling constants
suggesting the 2,3-trans and 3,4-trans configurations (20). Thus,

1528 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Fujii et al.
Table 2. 13C Chemical Shifts Assignments for 1, 3, 4, and 6
C rings were in good agreement with those observed in
procyanidin B-2, the position and the configuration of the
interflavanoid linkages were deduced to be C(4â)-C(8) (23).
C no.
C-2
1a
3b
4a
6c
1
In the H NMR spectrum, the signal at δ 3.83 (1H, d, J ≈ 0
74.7
70.6
75.7
71.9
76.0
72.0
76.4
71.7
C-3
Hz), assignable to flavan H-4 of the lower unit, indicated the
presence of 4-substituted flavan rings with 2,3-cis and 3,4-trans
configurations in the C-ring (24). The signals at δ 2.95, 3.44
(each 1H, br m, cys-H-3), and 4.14 (1H, dd, J ) 9, 4 Hz, cys-
H-2) in the 1H NMR spectrum of 4 were assigned to the cysteine
moiety. Accordingly, the structure of 4 was characterized as
4â-(S-L-cysteinyl)-(-)-epicatechin-(4âf8)-(-)-epicatechin.
C-4
C-5
C-6
41.7
156.1
94.5
42.9
157.2
96.2
36.1
36.7d
155.8e
95.7f
156.6d
95.3
C-7
C-8
157.1
96.4
158.2
99.0
156.5
95.3
157.1g
97.0
C-9
157.7
99.0
159.1
99.6
156.5
99.0
155.9
99.7
C-10
B-C-1
B-C-2
B-C-3
B-C-4
B-C-5
B-C-6
130.8
115.8d
144.6e
144.7e
115.9d
119.1
131.1
107.1
146.7
133.6
146.7
107.1
130.7
114.7e
144.6f
144.2f
115.9e
119.2
74.6
131.1h
114.8i
144.9 j
144.7k
115.8m
118.8
75.2
Compound 5 showed an [M - H]- peak corresponding to
C40H34O18NS by the negative HR-ESI-MS. The 1H NMR
spectrum of 5, although all of the signals appeared to be
broadened, showed a signal at δ 6.8 (2H) due to the gallic acid
moiety. Treatment of 5 with tannase yielded gallic acid and a
hydrolysate (4), which was identified on the basis of HR-ESI-
MS (m/z 696.1407 [M - H]-), 1H NMR (CD3OD), and reverse-
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
′
′
′
′
′
′
′
′
69.9
72.8
42.0
36.6d
155.8
96.3f
156.5d
95.3
1
phase HPLC. The downfield shift of the H-3 signal in the H
156.6
99.0
157.0g
99.7
NMR spectrum of 5 (δ 5.4) indicated the presence of the galloyl
group at C-3 of the upper unit in 5. On the basis of these
findings, the structure of 5 was concluded as being 4â-(S-L-
cysteinyl)-(-)-epicatechin-(4âf8)-(-)-epicatechin gallate (5).
156.6d
99.0
155.9
99.7
C-10
′
B′
B′
B′
B′
B′
B′
-C-1
-C-2
-C-3
-C-4
-C-5
-C-6
130.7
115.7
144.6
144.2
115.7
119.2
131.6h
114.8
145.0j
144.9k
115.7m
118.8
76.4
Compound 6 showed an [M - H]- peak for C48H42O20NS
by the negative HR-ESI-MS, which suggested that it was a
trimeric proanthocyanidin structure with a cysteine moiety. In
the 13C NMR spectrum of 6 (Table 2), chemical shifts [δ 75.2
and 76.4 (2C)] of C-2 suggested that compound 6 consists
entirely of epicatechin units (20). The other signals attributable
to the flavan C rings showed a close analogy to those of 4,
suggesting the position and the configuration of the interfla-
vanoid linkage of three epicatechin units to be C(4â)-C(8).
The signal (δ 42.3) assignable to flavan C-4 of the terminal
unit was in good agreement with that (δ 41.7) of 1, suggesting
that the cysteine moiety is located at C-4 of the terminal unit.
On the basis of these findings, the structure of 6 was concluded
as being 4â-(S-L-cysteinyl)-(-)-epicatechin-(4âf8)-(-)-epi-
catechin-(4âf8)-(-)-epicatechin (Figure 1).
Assay of Each Component in Cys-OLG by Normal-Phase
HPLC. As shown in Figure 2A, the peaks due to (+)-catechin
and (-)-epicatechin (overlapped in peak A), (-)-epigallocat-
echin, and procyanidin B-2 were detected in GSP, and their
contents were calculated as 3.5, 1.6, and 1.0%, respectively
(Table 3). On the other hand, in cys-OLG, in addition to the
above flavanols, the extra peaks corresponding to procyanidin
B-1 and compounds 1-6 were newly observed, although
compounds 1-3, 5, and 6 were individually observed as
inseparable peaks (Figure 2B). The content of each component
was calculated as shown in Table 3. It is thought that
procyanidin B-1, which was not detected in GSP, was generated
by cleavage of interflavanoid linkages, followed by a nucleo-
philic attack of the C-8 position of (+)-catechin on the C-4
position of procyanidin B-2 or (-)-epicatechin oligomers. The
total contents of monomers and oligomers of cys-OLG (32.2%)
were approximately 5 times greater than those of GSP (6.1%).
As no monomers or dimers were detected in fraction I, it is
suggested that the majority of fraction I is composed of
proanthocyanidin polymers. Similarly, the main components of
fractions II and III are monomers and oligomers, respectively
(Table 3).
C-2′′
C-3′′
C-4′′
C-5′′
C-6′′
C-7′′
C-8′′
C-9′′
C-10′′
70.5
42.3
155.9e
96.3
156.4
99.7
153.7
95.7
B′′-C-1
B′′-C-2
B′′-C-3
B′′-C-4
B′′-C-5
B′′-C-6
132.0h
115.0i
145.2j
144.7k
115.7
118.8
171.8
54.0
cys-1
cys-2
cys-3
172.1
53.7
32.9
172.9
54.7
34.0
172.2
53.7
32.8
33.3
a Measured in acetone-d6
−
D2O at 25
°
C. b Measured in CD3OD at 25
°
C.
d
-
c Measured in dimethyl sulfoxide-d6
are interchangeable.
−D2O at 40 °
C. m Values within the column
that cys-OLG has a significantly higher antioxidant potential
than GSP. In the same manner, the in vitro antioxidant capacities
of the three fractions derived from cys-OLG were compared
in the TEAC assay. Antioxidant indices of fraction I (0.78 (
0.04 mM), fraction II (0.99 ( 0.09 mM), and fraction III (0.97
( 0.07 mM) were obtained, suggesting that fractions II and
III, which were rich in monomers and oligomers, respectively,
exhibited significantly higher antioxidant capacity than fraction
I, which was rich in polymers.
For the in vivo experiment, GSP and cys-OLG were orally
administered to rats at a dose of 10 mg/kg of body weight for
a week (expt 1). The polyphenol concentrations in the GSP and
cys-OLG groups were higher than that in the control group
with a statistically significant difference. The antioxidant
capacities (TEAC and LPO) for the GSP and cys-OLG groups
were superior to those of the control group, but there was no
significant difference between the control and the GSP groups.
When the GSP and cys-OLG groups were compared, there was
In Vitro and in Vivo Experiments. The in vitro antioxidant
capacity of cys-OLG was compared with that of GSP in the
TEAC assay. Antioxidant indices of 0.74 ( 0.05 mM for GSP
and 0.90 ( 0.08 mM for cys-OLG were obtained, suggesting

Oligomeric Proanthocyanidin−L-Cysteine Complexes
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1529
Figure 2. Normal-phase HPLC of GSP (A) and cys−OLG (B). Peak identification: A, (
+)-catechin and (−)-epicatechin; B, (−)-epigallocatechin; C,
procyanidin B-2; D, procyanidin B-1; E, 1 3; F, 4; G, 5 +
+
2
+
6.
Table 3. Contents (Percent) of Compounds in Each Fraction
Table 4. Comparison of Polyphenol Concentration and Antioxidant
Capacities (TEAC and LPO) in GSP and Cys OLG Groups
(Experiment 1) and Three Fractions from Cys
OLG (Experiment 2)a
−
−
cys−
fraction fraction fraction
GSP OLG
I
II
III
experi-
ment
polyphenol
g/mL)
TEAC
(mM)
LPO
(nmol/mL)
monomers
NDa
ND
ND
7.6
8.3
65.8
ND
ND
ND
group
(µ
(
(
+
−
)-catechin
)-epigallocatechin
+
(
−)-epicatechin
3.5
1.6
3.8
5.0
8.8
1
control
GSP
68.9
99.1
112.8
±
±
±
10.1
18.2*
8.5*
1.00
1.04
1.21
±
±
±
0.15
8.80
8.64
7.91
±
±
±
0.52
1.10
0.83*
1
+
2
+
3
0.12
oligomers
cys
−OLG
0.05*,#
procyanidin B-1
procyanidin B-2
4
1.8
1.1
7.9
3.8
ND
ND
ND
ND
1.2
0.5
ND
ND
6.7
3.3
18.0
22.0
2
control
79.3
100.6
142.8
155.0
±
±
±
±
11.6
22.7
15.1†
21.8†
1.06
1.11
1.18
1.20
±
±
±
±
0.06
0.03
0.07†
0.02†
9.27
8.61
7.80
7.23
±
±
±
±
0.83
0.81
0.41†
0.27†
1.0
6.1
fraction I
fraction II
fraction III
5
+ 6
total
32.2
0
83.4
50.0
a *, p < 0.05 versus control (experiment 1); #, p < 0.05 versus GSP (experiment
1); †, p < 0.05 versus control, fraction I (experiment 2).
a Not detected.
no significant difference in the polyphenol concentration and
LPO, but for the TEAC assay, the cys-OLG group was superior
to the GSP group, and the difference was statistically significant
(Table 4). In the in vivo assessment of the polyphenol
concentration and antioxidant capacities of the three fractions
from cys-OLG (experiment 2), the monomer-rich fraction (II)
and the oligomer-rich fraction (III) were more potent than the
control and polymer-rich fraction (I) with statistically significant
differences. The polymer-rich fraction (I) showed higher anti-
oxidant capacity than the control, but not significant. Although
there was no significant difference between the monomer-rich
fraction (II) and the oligomer-rich fraction (III) in the in vivo
assessment of polyphenol contents, TEAC, and LPO, the
tendency was for the oligomer-rich fraction (III) to be more
potent than the monomer-rich fraction (II).
These results suggested that monomer and oligomer showed
higher antioxidant capacity compared to the polymer in both in

1530 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Fujii et al.
terms of these effects could be a possible future candidate in
food systems, pharmaceuticals, and cosmetic applications.
ACKNOWLEDGMENT
We express our deep gratitude to Dr. Ken-ichi Komatsu of the
Hokkaido Pharmaceutical University School of Pharmacy and
1
his staff for the measurements of H NMR, 13C NMR, and
optical rotation.
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Aging is an inevitable biological process for most organisms.
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JF062819N