Proanthocyanidin glycosides and related polyphenols from cacao liquor and their antioxidant effects.

Article abstract of DOI:10.1016/S0031-9422(02)00051-1

Purification of polar fractions from cacao liquor extracts gave 17 phenolics including four new compounds. The new compounds were characterized as a C-glycosidic flavan, an O-glycoside of a dimeric and two O-glycosides of trimeric A-linked proanthocyanidins, on the basis of spectroscopic data. Isolated polyphenols showed inhibitory effects on nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation in microsomes and on the autoxidation of linoleic acid. These effects were attributed to the radical-scavenging activity in the peroxidation chain reactions, based on the findings that the cacao polyphenols effectively scavenged the 1,1-diphenyl-2-picrylhydrazyl radical.

Full text of DOI:10.1016/S0031-9422(02)00051-1

Phytochemistry 59 (2002) 749–758
www.elsevier.com/locate/phytochem
Proanthocyanidin glycosides and related polyphenols from cacao
liquor and their antioxidant effects
Tsutomu Hatanoa, Haruka Miyatakea, Midori Natsumeb, Naomi Osakabeb,
Toshio Takizawab, Hideyuki Itoa, Takashi Yoshidaa,*
aFaculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan
bFunctional Foods R&DLaboratory, Meiji Seika Kaisha Ltd., Japan
Received 18 October 2001; received in revised form 10 January 2002
Abstract
Purification of polar fractions from cacao liquor extracts gave 17 phenolics including four newcompounds. The newcompounds
were characterized as a C-glycosidic flavan, an O-glycoside of a dimeric and two O-glycosides of trimeric A-linked proanthocyani-
dins, on the basis of spectroscopic data. Isolated polyphenols showed inhibitory effects on nicotinamide adenine dinucleotide
phosphate-dependent lipid peroxidation in microsomes and on the autoxidation of linoleic acid. These effects were attributed to the
radical-scavenging activity in the peroxidation chain reactions, based on the findings that the cacao polyphenols effectively sca-
venged the 1,1-diphenyl-2-picrylhydrazyl radical. # 2002 Published by Elsevier Science Ltd.
Keywords: Cacao liquor; Theobroma cacao; Sterculiaceae; Tannin; Polyphenol; Proanthocyanidin glycosides; Antioxidant; Lipid peroxidation;
Radical scavenger
1. Introduction
compounds and the antioxidant effects of the poly-
phenols.
Cacao liquor, which is produced by fermentation and
roasting of cacao beans (Theobroma cacao L., Stercu-
liaceae), is rich in polyphenols. Flavan monomers and
oligomers, flavonol glycosides and hydroxycinnamic
acid derivatives have all been isolated from cacao beans
or cacao liquor (Porter et al., 1991; Sanbongi et al.,
1998). Antioxidant effects of the polyphenol fractions
from cacao liquor have also been reported (Osakabe et
al., 2000). We further investigated the constituents of
cacao liquor, and isolated 13 polyphenolic constituents
including four newcompounds. The isolated poly-
phenols showed inhibitory effects on nicotinamide ade-
nine dinucleotide phosphate (NADPH)-dependent lipid
peroxidation in rat liver microsomes, and on the auto-
xidation of linoleic acid. They also showed activity to
scavenge the l,1-diphenyl-2-picrylhydrazyl (DPPH)
radical. This paper deals with the structures of the new
2. Results and discussion
2.1. Isolation of polyphenols from cacao liquor
Cacao liquor prepared from Ghana beans was defat-
ted with n-hexane, and then homogenized in 70% aq.
acetone. The concentrated filtrate from the homogenate
was extracted with EtOAc and n-BuOH, successively,
and the EtOAc and n-BuOH extracts were respectively
subjected to Sephadex LH-20, MCI-gel CHP-20P, Dia-
ion HP-20 and/or Toyopearl HW-40 chromatographies,
to give four newcompounds ( 1)–(4), together with 13
known compounds. The known compounds were iden-
tified as procyanidin B2 (5) (Porter et al., 1991), proto-
catechuic acid (6), (+)-catechin (7), (ꢀ)-epicatechin (8),
3T-O-b-d -galactopyranosyl-ent-epicatechin-(2a!7,
4a!8)-epicatechin1 (9) (Porter et al., 1991), procyani-
* Corresponding author. Tel./fax: +81-86-251-7936.
address:
E-mail
(T. Yoshida).
yoshida@pheasant.pharm.okayama-u.ac.jp
1
‘‘T’’ and ‘‘ent’’ mean the top unit of the trimeric proanthocyani-
din and enantiomer, respectively.
0031-9422/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0031-9422(02)00051-1

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T. Hatano et al. / Phytochemistry 59 (2002) 749–758
din C1 (10) (Porter et al., 1991), procyanidin B5 (11)
(Thompson et al., 1972), cinnamtannin A2 (12) (Mor-
imoto et al., 1986), 3T-O-a-l-arabinopyranosyl-ent-epi-
catechin-(2a!7, 4a!8)-epicatechin (13) (Porter et al.,
1991), proanthocyanidin A1 (14) (Nonaka et al., 1987),
proanthocyanidin A2 (15) (Jacques et al., 1974), bis-
8,80-catechinylmethane (16) (Kiatgrajai et al., 1982), and
quercetin 3-O-a-l-arabinopyranoside (17) (Sanbongi et
al., 1998).
spectral data, 1 was presumed to be a C-glycoside of a
1
monomeric flavanol. The H NMR spectrum of 1 (in
MeOH-d4, 40 ꢁC) showed an aromatic singlet [ꢀ 6.00
(1H; A-ring H)], three aromatic proton signals forming
an ABX system [ꢀ 6.77 (1H, d, J=8 Hz; H-50), 6.81 (1H,
dd, J=2, 8 Hz; H-60) and 7.02 (1H, d, J=2 Hz; H-20)]
and four aliphatic proton signals coupled with each
other [ꢀ 4.86 (1H, br s; H-2), 4.14 (1H, br m; H-3), 2.88
(1H, dd, J=4, 17.5 Hz; H-4) and 2.83 (1H, dd, J=2,
17.5 Hz; H-4)]. The spectrum also showed seven ali-
phatic proton signals of a sugar unit [ꢀ 4.86 (1H, d,
J=9.5 Hz; H-1), 4.15 (1H, t, J=9.5 Hz; H-2), 3.60 (1H,
dd, J=3, 9.5 Hz; H-3), 3.93 (1H, br d, J=3 Hz; H-4),
3.57 (1H, dd, J=5.5, 6.5 Hz; H-5), 3.74 (1H, dd, J=6.5,
11.5 Hz; H-6), 3.68 (1H, dd, J=5.5, 11.5 Hz; H-6)]. The
coupling constants indicated that the sugar is b-galac-
topyranose, upon the assumption that it is of the
d-form. The 13C NMR spectrum of 1 confirmed that
this molecule consists of epicatechin and galactose residues
2.2. Structure of monomeric flavan C-glycoside 1
Compound 1 was obtained as a light-brown amor-
phous powder. Electrospray-ionization (ESI) MS of this
compound showed an [M+H]+ ion peak at m/z 453,
and [M+NH4]+ ion peak at m/z 470. These peaks cor-
respond to the molecular formula C21H24O11, which
was substantiated by high-resolution ESI MS. Based on
this molecular formula and the following 1H NMR

T. Hatano et al. / Phytochemistry 59 (2002) 749–758
751
(Experimental). The galactose residue on the epica-
techin moiety was assigned to C-8, based on the rotating
frame Overhauser effect (ROE) between H-1 of the
galactose residue (ꢀ 4.85) and B-ring H-20 (ꢀ 7.16) of the
epicatechin residue in the ROESY experiment (in
Me2CO-d6+D2O, 24 ꢁC). The configurations at C-2
and C-3 of the epicatechin residue of 1 are both R, since
1 showed a CD spectrum similar to that of (ꢀ)-epica-
techin (8). Based on these data, the structure of (ꢀ)-
epicatechin 8-C-b-d-galactoside was assigned for 1.
resolution ESI MS. The 1H NMR spectrum (in Me2CO-
d6+D2O, 40 ꢁC) showed signals for three aromatic
protons of A/D-rings [ꢀ 5.95, 5.97 (1H each, d, J=2 Hz;
upper unit); ꢀ 6.26 (1H, s; lower unit)], two sets of three
aromatic protons of B/E-rings forming ABX systems [ꢀ
6.99 (1H, br s; H-20), 6.81 (2H, s; H-50, H-60) (lower
unit); ꢀ 7.19 (d, J=2.5 Hz; H-20), 6.85, (1H, d; J=8.5
Hz, H-50), 7.05 (1H, dd, J=2.5, 8.5 Hz; H-60) (upper
unit)] and two series of aliphatic protons of C/F-rings [ꢀ
4.28 (1H, d, J=3 Hz; H-3), 4.60 (1H, d, J=3 Hz; H-4)
(C-ring); 4.72 (1H, d, J=8 Hz, H-2), 4.14 (1H, ddd,
J=5.5, 8, 8.5 Hz; H-3), 2.94 (1H, dd, J=5.5, 16 Hz,
H-4), 2.58 (1H, dd, J ¼ 8:5, 16.5 Hz; H-4) (F-ring)].
These signals and the absence of H-2 in the upper flavan
unit indicated that this compound is a flavan dimer of
2.3. Structure of dimeric flavan O-glycoside 2
Compound 2 was obtained as a light-brown powder.
Its molecular formula C35H32O16 was derived by high-

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T. Hatano et al. / Phytochemistry 59 (2002) 749–758
proanthocyanidin A series. A 2,3-trans structure was
identified in the lower unit from the coupling constants
of the lower F-ring protons. The spectrum also showed
six aliphatic protons ascribed to a sugar residue (ꢀ, 4.46
(1H, d, J=4.5 Hz; H-1), 3.31 (1H, dd, J ¼ 4:5, 6.5 Hz;
H-2), 3.45 (1H, dd, J ¼ 3:5, 6.5 Hz; H-3), 3.66 (1H, m;
H-4), 3.19 (1H, dd, J ¼ 3:5, 12 Hz, H-5), 3.11 (1H, dd,
J ¼ 6:5, 12 Hz; H-5)]. The sugar in 2 was assigned as an
[aglycone H-3 (C-ring)–arabinose C-1], in the HMBC
spectrum of 2. The HMBC spectrum also showed the
connectivity of ꢀH 4.72ꢀꢀC 152.4ꢀꢀH 4.60 [H-4 (C-
ring)–C-8a (D-ring)-H-2 (F-ring)], indicating the pre-
sence of the C-4 (C-ring)–C-8 (D-ring) interflavan link-
age.
The CD spectrum of compound 2 showed a negative
Cotton effect in the short wavelength region
([ꢁ]228ꢀ6.9ꢂ103), indicating that the orientation of the
interflavan bond at C-4 of the C-ring is a (Barrett et al.,
1979; Botha et al., 1981; Kolodziej et al., 1991). The
orientation of the phenyl group at C-2 of the C-ring,
which is dependent on the C-4 orientation in the proan-
thocyanidin A-type structure, is therefore b. The b-
orientation of the C-3 hydroxyl group of the C-ring was
assumed tentatively, based on analogy to other natural
1
arabinopyranose on the basis of H and the 13C signal
patterns. This assignment was substantiated by acid
hydrolysis of 2 followed by the analysis with HPLC,
which showed the production of arabinose.
The location of the arabinose at O-3 of the upper fla-
van unit on the proanthocyanidin residue was further
supported by the correlations, ꢀH 4.46ꢀꢀC 71.0 [arabi-
nose H-1–aglycone C-3 (C-ring)] and ꢀH 4.28ꢀꢀC 98.9

T. Hatano et al. / Phytochemistry 59 (2002) 749–758
753
dimeric proanthocyanidins of the A-type with OH at C-
5(A) (Balde et al., 1991). Although the coupling constant
J3,4 of the C-ring in 2 (3 Hz) was practically the same as
the reported values for the 3,4-trans A-type dimers (3–4
Hz), the values are not indicative for the relative config-
uration C-3–C-4 (Cronje et al., 1990, 1993).
of arabinose in 3 was substantiated by its formation on
the acid hydrolysis of 3.
The 13C NMR spectrum of 3 showed carbon signals
of three flavan units and the arabinose residue, and the
chemical shifts of the flavan units were similar to those
reported for cinnamtannin B1 (19) (Nonaka et al.,
1983), except for the C-ring carbons of the top flavan
units [ꢀ 104.2 (19)–99.3 (3) (C-2); 66.5 (19)–71.2 (3) (C-
3); 30.0 (19)–24.9 (3) (C-4)]. These chemical shift chan-
ges are attributable to the glycosidation at O-3 of the
top unit, suggesting the structure of 3-T-O-arabino
sylcinnamtannin B1 for compound 3. Duplication of the
1H signals described above coincided with the 4–8 link-
age in 3 between the middle and lower unit, rather than
the less hindered 4–6 linkage. The positive Cotton effect
at 225 nm ([ꢁ]225+4.5ꢂ104) in the CD spectrum of 3
satisfied a b-orientation of the interflavan linkage at C-4
in each of the top and middle flavan units.
The orientation at C-2 of the F-ring was tentaviely
assigned to be ꢂ, since the CD spectral pattern at
around 270–290 nm ([ꢁ]293ꢀ2.3ꢂ103, [ꢁ]271+4.9ꢂ103),
which reflects the orientation of the B/E-rings in the
proanthocyanidins, was similar to that of proanthocya-
nidin 13 with a 2b-phenyl (C-ring)–2a-phenyl (F-ring)
structure. Actually, proanthocyanidins with 2a-phenyl
(C-ring)–2a-phenyl (F-ring) and 2a-phenyl (C-ring)–2b-
phenyl (F-ring) structures showed different CD patterns
in this region {[ꢁ]287ꢀ2.5ꢂ103, [ꢁ]271ꢀ1.8ꢂ104 for pro-
anthocyanidin A-2 (15); [ꢁ]297+1.4ꢂ104, [ꢁ]271ꢀ1.3ꢂ104
for epicatechin-(2b!7, 4b!8)-ent-epicatechin (18)}
(Lou et al., 1999). Based on these data, the structure of
3T-O-arabinopyranosyl-ent-epicatechin-(2a!7, 4a!8)-
catechin, which was the C-3 (F-ring) epimer of com-
pound 13, was assigned for compound 2. The NOESY
spectrum showed a correlation between H-1 and H-3 in
the arabinopyranose residue. Therefore, the a-config-
uration at the anomeric center was assigned upon the
assumption that this sugar is of the l-form.
The presence of the interflavan A-type linkage
between the top and middle flavan units was verified by
the acid-catalyzed degradation of 3 in the presence of
toluene-a-thiol, which gave a benzylthioether (20) of a
1
dimeric flavan, together with (ꢀ)-epicatechin. The H
NMR spectrum of 20 showed arabinose signals as dis-
cussed in experimental and the assignments of the other
signals were based on comparisons with the data repor-
ted for the benzylthioether of proanthocyanidin A-2
(21) (Nonaka et al., 1983).
The ent-epicatechin structure in the upper unit of 2 is
probably derived from the catechin moiety in procyani-
din B-3 upon the oxidative transformation to the A-type
proanthocyanidin (Burger et al., 1990; Kondo et al.,
2000), although procyanidin B-3 was not yet isolated
from cacao beans or cacao liquor.
The arabinose residue in 3 was confirmed to be at O-3
on the top flavan unit, based on the correlations, ꢀH 4.39
(C-ring H-3)–ꢀC 99.5 (arabinose C-1) and ꢀH 4.58 (ara-
binose H-1)–ꢀC 71.2 (C-ring C-3) observed in the HMBC
spectrum of 3. The a-configuration at the anomeric cen-
ter, upon the assumption that the arabinose is of the l-
form, was shown by the NOE correlation between H-1
and H-3 of the arabinopyranose residue in the NOESY
spectrum of 3. The structural formula 3 was thus
assigned for this compound. The aglycone of this com-
pound was reported to be isolated from cinnamon
(Nonaka et al., 1983) and cranberry (Foo et al., 2000).
Compound 4 was obtained as a light-brown amor-
phous powder. The molecular formula C51H47O23 was
2.4. Structure of trimeric flavan O-glycosides 3 and 4
Compound 3 was obtained as a light-brown amor-
phous powder. High-resolution ESI MS indicated its
1
molecular formula to be C50H44O22. Although the H
NMR spectrum measured at 24 ꢁC (in Me2CO-d6
+D2O) showed some duplication of signals, measure-
ment at an elevated temperature rectified this. The
ꢁ
spectrum measured at 40 C showed B/E/H-ring (ꢀ 7.3–
1
6.6), A/D/G-ring [ꢀ 6.22 (1H, s), 5.97 (1H, d, J=2 Hz),
5.95 (1H, br s), 5.90 (1H, d, J=2 Hz)] and C/F/I-ring
protons [ꢀ 4.39 (1H, d, J=3.5 Hz; C-ring H-3), 5.34
(1H, br s; F-ring H-2), 3.95 (1H, br m; F-ring H-3), 4.76
(2H, br s; C/F-ring H-4), 4.96 (1H, br, I-ring H-2), 4.30
(1H, br; I-ring H-3), 2.86 (1H, br d, J=14 Hz, I-ring H-
4), 2.71 (1H, br; I-ring H-4)] of the trimeric flavanoid
skeleton. The absence of one of the H-2 signals indi-
cated the presence of an interflavan linkage at C-2 (to
O-7 or O-5), in addition to ordinary interflavan 4–8 or
4–6 bonds. The spectrum also showed aliphatic proton
signals due to an arabinopyranose residue [ꢀ 4.58 (1H,
d, J=5 Hz; H-1), 3.36 (3H, m; H-2, H-5ꢂ2), 3.50 (1H,
dd, J=3.5, 9 Hz; H-3), 3.72 (1H, m; H-4)]. The presence
derived by high-resolution ESI MS. Although the H
and 13C NMR spectra of 4 were similar respectively to
those of compound 3, the spectra of 4 showed signals
attributable to a b-galactopyranose residue [ꢀH 4.40
(1H, d, J=7.5 Hz; H-1), 3.28 (1H, dd, J=7.5, 9.5 Hz;
H-2), 3.44 (1H, dd, J=3, 9.5 Hz; H-3), 3.77 (1H, br d,
J=3 Hz; H-4), 3.50 (1H, t, J=6 Hz; H-5), 3.57 (2H, d,
J=6 Hz; H-6ꢂ2); ꢀC 101.6 (C-1), 71.6 (C-2), 73.8 (C-3),
69.2 (C-4), 76.0 (C-5), 61.6 (C-6)] instead of the arabi-
nopyranose residue in 3. The presence of the galactose
residue in 4 was verified by the formation of galactose
upon the acid hydrolysis of 4. The HMBC correlations,
ꢀH 4.48 (C-ring H-3)–ꢀC 101.6 (galactose C-1) and ꢀH
4.40 (galactose H-1)–ꢀC 72.1 (C-ring C-3), indicated that

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T. Hatano et al. / Phytochemistry 59 (2002) 749–758
the galactose residue was at O-3 on the top flavan unit.
The CD spectrum showed a positive Cotton effect at
234 nm ([ꢁ]234+5.5ꢂ104), indicating the b-orientation of
the two interflavan linkages at C-4 of the flavan units.
Based on these data, the structural formula 4 was
assigned for this compound.
ent from that observed for the NADPH-dependent lipid
peroxidation, and the effects of almost all of the poly-
phenols on the oxidation of linoleic acid were stronger
than the effect of dl-a-tocopherol. The difference may be
attributed to the presence of other biomolecules in the
microsomal solution, which interacted with the poly-
phenol.
2.5. Antioxidant activity of cacao polyphenols
Table 3 shows the scavenging activity of the test
compounds for the DPPH radical. The effects were
similar to or stronger than the effect of a-tocopherol.
The ranking of the polyphenols in terms of scavenging
activity was similar to that for the inhibitory effect on
the autoxidation of linoleic acid.
Inhibitory effects of various tannins and related poly-
phenols on the lipid peroxidation in rat liver mitochon-
dria induced by adenosine 50-diphosphate (ADP) and
ascorbic acid, and on that in liver microsomes induced
by ADP and NADPH, have been reported (Okuda et
al., 1983). The antioxidant effects of the polyphenols are
attributable to their radical-scavenging properties, as
shown by the effects on the diphenylpicryl hydrazyl
(DPPH) radical as a model compound for lipid radicals
or lipid peroxide radicals (Fujita et al., 1988a, b; Yosh-
ida et al., 1989; Hatano et al., 1989). On the other hand,
the polyphenolic constituents of cacao liquor have been
reported to have antioxidant effects (Osakabe et al.,
2000). Effects of the polyphenois isolated from cacao on
the NADPH-dependent lipid peroxidation in rat liver
microsomes, and on the autoxidation of linoleic acid
were therefore examined. We also tested their effects on
the DPPH radical.
The NADPH-dependent lipid peroxidation in rat liver
microsomes was inhibited by almost all of the poly-
phenols tested as shown in Table 1. The polyphenols,
except for (ꢀ)-epicatechin 8-C-galactoside (1), had
inhibitory effects on the lipid peroxidation with IC50
values of 12–68 mM. Although these values are no better
than that of dl-a-tocopherol, the results indicated that
these compounds contribute to the antioxidant activity
of the polyphenolic fraction of cacao.
3. Experimental
3.1. General
1H and 13C NMR spectra were measured on a Varian
1
VXR-500 (500 MHz for H and 126 MHz for 13C) in
ꢁ
Me2CO-d6–D2O at 40 C unless mentioned otherwise,
Table 2
Inhibitory effects of cacao polyphenols on the autoxidation of linoleic
acid initiated by the addition of V-70
Compound
IC50 (mg/ml)
(ꢀ)-Epicatechin (8)
0.62
1.9
2.0
2.2
3T-O-a-l-Arabinopyranosylcinnamtannin B1 (3)
3T-O-b-d-Galactopyranosylcinnamtannin B1 (4)
3T-O-a-l-Arabinopyranosyl-ent-epicatechin-
(2a!7, 4a!8)-epicatechin (13)
Procyanidin B5 (11)
2.3
2.5
3T-O-ꢃ-d-Galactopyranosyl-ent-epicatechin-
(2a!7, 4a!8)-epicatechin (9)
Cinnamtannin A2 (12)
2.7
5.3
9.5
7.7
Procyanidin C1 (10)
(ꢀ)-Epicatechin 8-C-galactopyranoside (1)
dl-a-Tocopherol
The ranking of the polyphenols based on the strength
of their inhibitory effects on the autoxidation of linoleic
acid, which are shown in Table 2, was somewhat differ-
Table 1
Inhibitory effects of cacao polyphenols on lipid peroxidation in rat
liver microsomes
Table 3
Radical-scavenging effects of cacao polyphenols on the DPPH radical
Compound
IC50 (mg/ml)
Compound
EC50 (mM)
Procyanidin B2 (5)
Procyanidin B5 (11)
12
12
18
Procyanidin B2 (5)
Cinnamtannin A2 (12)
1.4
2.1
2.1
2.3
2.3
2.4
6.2
3.9
3.9
3T-O-b-d-Galactopyranosyl-ent-epicatechin-
(2a!7, 4a!8)-epicatechin (9)
3T-O-a-l-Arabinopyranosyl-ent-epicatechin-
(2a!7, 4a!8)-epicatechin (13)
Cinnamtannin A2 (12)
(ꢀ)-Epicatechin (8)
3T-O-a-l-Arabinopyranosylcinnamtannin B1 (3)
Procyanidin B5 (11)
21
3T-O-b-d-Galactopyranosylcinnamtannin B1 (4)
Procyanidin C1 (10)
25
28
3T-O-a-l-Arabinopyranosylcinnamtannin B1 (3)
(ꢀ)-Epicatechin (8)
(ꢀ)-Epicatechin 8-C-galactopyranoside (1)
3T-O-b-d-Galactopyranosyl-ent-epicatechin-
(2a!, 4a!8)-epicatechin (9)
3T-O-a-l-Arabinopyranosyl-ent-epicatechin-
(2a!7, 4a!8)-epicatechin (13)
dl-a-Tocopherol
29
36
3T-O-b-d-Galactopyranosylcinnamtannin B1 (4)
Procyanidin C1 (10)
68
>100
5.6
3.9
4.6
(ꢀ)-Epicatechin 8-C-galactopyranoside (1)
dl-a-Tocopherol

T. Hatano et al. / Phytochemistry 59 (2002) 749–758
755
and chemical shifts are given in ꢀ values (ppm), based
on those of the solvent signals (ꢀH 2.04 and ꢀC 29.8 for
Me2CO-d6, and dH 3.30 and ꢀC 49.8 for MeOH-d4).
NMR (in Me2CO-d6+D2O, 24 ꢁC) ꢀ: 2.80 [1H, dd,
J=2.5, 17 Hz, epicatechin (EC) H-4], 2.86 (1H, dd,
J=4, 17 Hz, EC H-4), 3.62 [1H, br dd, J=5, 6.5 Hz,
galactose (Gal) H-5], 3.66 (1H, dd, J=3, 9.5 Hz, Gal
H-3), 3.70 (1H, dd, J=5, 11.5 Hz, Gal H-6), 3.73 (1H,
dd, J=6.5 Hz, 11.5 Hz, Gal H-6), 4.03 (1H, br d, J ¼ 3
Hz, Gal H-4), 4.05 (1H, t, J=9.5 Hz, Gal H-2), 4.10
(1H, m, EC H-3), 4.85 (1H, d, J=9.5 Hz, Gal H-1), 4.92
(1H, br s, EC H-2), 5.98 (1H, s, EC H-6), 6.76 (1H, d,
J=8 Hz, EC H-50), 6.82 (1H, dd, J=2, 8 Hz, EC H–60),
7.16 (1H, d, J=2 Hz, EC H-20). 13C NMR (in MeOH-
3.2. Isolation of polyphenols from cacao liquor
The cacao liquor used in this study was prepared from
fermented cacao beans imported from Ghana. A pre-
paration of the liquor (420 g) defatted with n-hexane
was homogenized in 70% aq. acetone (4.5 l), and the
filtrate concentrated from the homogenate was extrac-
ted with CHCl3, EtOAc and n-BuOH, successively. The
EtOAc extract (4.5 g) was subjected to column chro-
matography on Toyopearl HW-40C (eluants, H2O–30%
EtOH–70% EtOH–70% acetone), to give procyanidin
B2 (5) (132 mg) and other fractions rich in polyphenols.
The fractions were further purified by column chroma-
tography on MCI-gel CHP-20P (eluant, H2O–MeOH),
Sephadex LH-20 (EtOH) and/or YMC-gel ODS-AQ
120-S50 (H2O–MeOH), to give protocatechuic acid (6)
(28 mg), (+)-catechin (7) (15 mg), (ꢀ)-epicatechin (8)
(27 mg), procyanidin B2 (5) (12 mg), 3T-O-arabinopyr-
anosyl-ent-epicatechin-(2a–7, 4a–8)-catechin (2) (2 mg),
3T-O-b-d-galactopyranosyl-ent-epicatechin-(2a–7, 4a–
8)-epicatechin (9) (5 mg), procyanidin Cl (10) (39 mg),
procyanidin B5 (11) (48 mg) and cinnamtannin A2 (12)
(4 mg). The n-BuOH extract was subjected to Diaion
HP-20 chromatography (eluant, H2O–MeOH), and
fractions were purified by column chromatography on
Sephadex LH-20 (EtOH), YMC-gel ODS-AQ 120-S50
(H2O–MeOH) and MCI-gel CHP-20P (H20–MeOH), to
give (ꢀ)-epicatechin 8-C-b-d-galactopyranoside (1) (8
mg), 3T-O-b-d-galactopyranosylcinnamtannin B1 (4)
(28 mg), cinnamtannin A2 (12) (42 mg), procyanidin B2
(5) (9 mg), 3T-O-b-d-galactopyranosyl-ent-epicatechin-
(2a!7, 4a!8)-epicatechin (9) (23 mg), 3T-O-a-l-ara-
binopyranosyl-ent-epicatechin-(2a!7, 4a!8)-epicatechin
(13) (6 mg), procyanidin C1 (10) (6 mg), procyanidin B5
(11) (10 mg) and 3T-O-a-l-arabinopyranosylcinnam-
tannin B1 (3) (11 mg). Another portion of the EtOAc
(14 g) extract was purified in an analogous way on col-
umns of Diaion HP-20, Sephadex LH-20, MCI-gel
CHP-20P and YMC-gel ODS-AQ 120-S50, to yield
proanthocyanidin A1 (14) (5 mg), proanthocyanidin A2
(15) (4 mg), bis-8,80-catechinylmethane (16) (5 mg) and
quercetin 3-O-a-l-arabinopyranoside (17) (18 mg) in
addition to the compounds described above.
ꢁ
d4, 40 C) ꢀ: 30.5 (EC C-4), 63.8 (Gal C-6), 68.2 (EC C-
3), 71.4 (Gal C-4), 73.1 (Gal C-2), 77.0 (Gal C-3), 77.7
(Gal C-1), 80.9 (EC C-2), 81.1 (Gal C-5), 98.2 (EC C-6),
100.5 (EC C-4a), 105.8 (EC C-8), 116.0 (EC C-20), 116.9
(EC C-50), 119.8 (EC C-60), 133.2 (EC C-10), 146.5 (EC
C-40), 146.8 (EC C-30), 155.4 (EC C-8a), 157.9 (EC C-7),
158.9 (EC C-5).
3.4. 3T-O-Arabinopyranosyl-ent-epicatechin-(2a!7,
4ꢂ!8)-catechin (2)
A light-brown amorphous powder, [ꢂ]D ꢀ16.4ꢁ (c=1,
MeOH) ESI MS m/z: 709 ([M+H]+). High-resolution
ESI MS m/z 709.1713 ([M+H]+). Calc. for
C35H32O16+H, 709.1769. UV lMmaexOH (log "): 280 (4.17)
CD (MeOH): [ꢁ]283ꢀ2.3ꢂ103, [ꢁ]271
,
+4.9ꢂ103,
[ꢁ]228ꢀ6.9ꢂ103, [ꢁ]211+1.9ꢂ104. 13C NMR ꢀ: 24.8 [C-4
(C-ring)], 29.3 [C-4 (E)], 63.2 [arabinose (Arb) C-5], 66.2
(Arb C-4), 67.6 [C-3 (E)], 70.9 (Arb C-2), 71.0 [C-3 (C)],
72.5 (Arb C-3), 84.3 [C-2 (E)], 96.0 [C-8 (C)], 96.6 [C-6
(E)], 98.0 [C-6 (C)], 98.9 (Arb C-1), 99.2 [C-4a(E)], 103.2
[C-2(C), C-4a (A)], 105.2 [C-8 (D)], 115.4 (2C) [C-20 (B),
C-5 0(B)], 115.9 (2C) [C-20 (E), C-50 (E)], 119.5 [C-60 (B)],
120.5 [C-60 (E)], 130.5 [C-10 (E)], 131.6 [C-10 (B)], 145.3
[C-40 (B)], 145.7 [C-40 (E)], 146.2 [C-30 (E)], 146.5 [C-30
(B)], 152.1 [C-7 (D)], 152.4 [C-8a (D)], 153.7 [C-8a (A)],
155.5, 155.6 [C-5 (A/D)], 158.6 [C-7 (A)].
3.5. 3T-O-ꢂ-l-Arabinopyranosylcinnamtannin B1 (3)
A light-brown amorphous powder, [ꢂ]D +12.6ꢁ (c=1,
MeOH). ESI MS m/z: 997 ([M+H]+). High-resolution
ESI–MS m/z: 1014.2593 ([M+NH4]+). Calc. for
C50H44O22+NH4, 1014.2668. UV lMmaexOH (log "): 280
(4.20). CD (MeOH): [ꢁ]283ꢀ5.2ꢂ103, [ꢁ]272+3.1ꢂ 103,
[ꢁ]257+3.7ꢂ103,
[ꢁ]225+4.5ꢂ104,
[ꢁ]209+1.8ꢂ104,
[ꢁ]202ꢀ9.5ꢂ104. 13C NMR ꢀ: 24.9 [C-4(C)], 36.4 [C-4
(F)], 64.3 (Arb C-5), 66.1 [C-3 (F)], 67.1 (Arb C-4), 71.0
(Arb C-2), 71.2 [C-3(C)], 72.5 (Arb C-3), 72.6 [C-3 (F)],
77.8 [C-2 (F)], 79.1 [C-2 (I)], 95.9 [C-8 (A)], 96.2 [C-
6(D)], 96.9 [C-6 (G)], 97.9 [C-6 (A)], 99.3 [C-2 (C)], 99.5
(Arb C-1), 100.1 [C-4a (G)], 102.6 [C-8 (D)], 104.1 [C-4a
(D)], 105.1 [C-4a (A)], 106.7 [C-8 (G)], 115.0,. 115.4,
115.6 (3C), 115.8 [C-20 (B/E/H), C-50 (B/E/H)], 118.9,
119.2, 119.5 [C-60 (B/E/H)], 131.2, 131.3, 131.4 [C-10 (B/
3.3. Epicatechin 8-C-ꢃ-d-galactopyranoside (1)
A light-brown amorphous powder, [ꢂ]D, ꢀ25.8ꢁ
(c=0.9, MeOH). ESI MS m/z: 453 ([M+H]+), 470
([M+NH4]+). High-resolution ESI MS m/z: 470.1649
((M+NH4]+). Calc. for C21H24O11+NH4, 470.1662.
MeOH
max
UV
l
(log "): 280 (3.32). CD (MeOH):
[ꢁ]207ꢀ4.4ꢂ104, [ꢁ]250+1.8ꢂ103, [ꢁ]279ꢀ3.4ꢂ103. 1H

756
T. Hatano et al. / Phytochemistry 59 (2002) 749–758
E/H)], 144.9–146.3 [C-30 (B/S/H), C-40 (B/E/H)], 152.2–
158.3 [C-5 (A/D/G), C-7 (A/D/G), C-8a (A/D/G)].
pound was identified by HPLC on a Chiralcel OC
column with n-hexane–isopropanol (7:13) containing
0.5% acetic acid at 40 C in an oven.
ꢁ
3.6. 3T-O-ꢃ-d-Galactopyranosylcinnamtannin B1 (4)
3.8.1. 4ꢃ-Benzylthioether (20) of 3T-O-ꢂ-l-arabinosyl-
proanthocyanidin A2
A light-brown amorphous powder, [ꢂ]D +17.1ꢁ (c=1,
MeOH) ESI MS m/z: 1044 ([M+NH4]+), 1027
([M+H]+), 865 ([M-galactose+H]+). High-resolution
ESI MS m/z: 1027.2531 ([M+H]+). Calc. for C51H46
O23+H, 1027.2508. UV MeOH lmax (log "): 281 (5.02).
A light-brown amorphous powder. ESI MS m/z: 848
([M+NH4]+), 831 ([M+1]+), 707 ([M–PhCH2SH+
1
H]+), 575 ((M–PhCH2SH–arabinose+H]+) H NMR
ꢀ: 3.51 (1H, dd, J=4.5, 9 Hz, Arb H-3), 3.79 (1H, m,
Arb H-4), 4.01 (2H, s, –CH2S–), 4.19 [1H, d, J=5 Hz,
H-4 (F)], 4.20 [1H, d, J=5 Hz, H-3 (F)], 4.35 [1H, d,
J=4.5 Hz, H-3(C)], 4.59 (1H, d, J=5 Hz, Arb H-1),
4.79 [1H, d, J=4.5 Hz, H-4 (C)], 5.46 [1H, br s, H-2
(F)], 5.93 [1H, d, J=2 Hz, H-6 (A)], 5.95 [1H, d, J=2
Hz, H-8 (A)], 6.28 [1H, s, H-6 (D)], 6.8–7.8 (11H, pro-
tons of B/E-rings and C6H5CH2S–).
CD (MeOH): [ꢁ]204ꢀ9.1ꢂ103, [ꢁ]273+4.0ꢂ103, [ꢁ]258
+
1.3ꢂ103, [ꢁ]234+5.5ꢂ104, [ꢁ]209+3.4ꢂ104, [ꢁ]234ꢀ5.6
1
ꢂ104. H NMR ꢀ: 2.71 [1H, br, H-4 (I)], 2.84 [1H, br d,
J=15 Hz, H-4 (I)], 3.95 [1H, br s, H-3 (F)], 4.31 [1H, br,
H-3 (I)], 4.48 [1H, d, J=3.5 Hz, H-3 (C)], 4.73 [1H, br s,
11–4 (F)], 4.74 [1H, br s, H-4 (C)], 4.94 [1H, br, H-2 (I)],
5.35 [1H, br s, H-2 (F)], 5.88 [1H, d, J=2 Hz, H-6 (A)],
5.96 [2H, H-6 (G) and H-8 (A)], 6.22 [1H, s, H-6 (D)],
6.6–7.3 (9H, B-ring protons). Data for the galactose
protons are shown in the text. 13C NMR ꢀ: 25.7 [C-4
(C)], 36.3 [C-4 (F)], 61.6 (Gal C-6), 66.0 [C-3 (I)], 69.2
(Gal C-4), 71.6 (Gal C-2), 72.1 [C-3 (C)], 72.7 [C-3 (F)],
73.7 (Gal C-3), 76.0 (Gal C-5), 77.7 [C-2 (F)], 79.0 [C-2
(I)], 95.9 [C-8 (A)], 96.3 [C-6 (D)], 96.9 [C-6 (G)], 97.8
[C-6 (A)], 99.4 [C-2 (C)], 100.1 [C-4a (G)], 101.0 (Gal C-
1), 102.9 [C-8 (D)], 104.2 [C-4a (D)], 105.5 [C-4a (A)],
106.7 [C-8 (G)], 114.8, 115.0, 115.1, 115.6, 115.8, 116.2
[C-20 (B/E/H), C-50 (B/E/H)], 118.9, 119.0, 119.7 [C-60
(B/E/H)], 131.2, 131.4, 131.5 [C-10 (B/E/H)], 144.6–
146.2 [C-30 (B/E/H), C-40 (B/E/H)], 152.3–158.1 [C-5(A/
D/G), C-7 (A/D/G), C-8a (A/D/G)].
3.9. Inhibitory effects of cacao polyphenols on lipid
peroxidation in rat liver microsomes
Spraque–Dawley rats (7 weeks old), which were
obtained from Clea Japan Inc. (Tokyo, Japan), were
kept in a controlled environment at 23 ꢁC and 55%
relative humidity under a 12 h dark–light cycle. Livers
were excised from the rats which were fasted for 15 h
prior to the excision, and were homogenized in 1.15%
KC1 solution (4 fold volume of liver). A microsomal
fraction was obtained as a pellet after centrifugation
ꢁ
(78,000 g for 90 min at 0 C) of the supernatant from
centrifugation (10,000 g for 15 min at 0 ꢁC) of a rat liver
homogenate. A mixture of a suspension of the micro-
some (1.5 mg protein/ml) (50 ml), a solution of the test
compound (50 m1) and a solution (400 ml) containing
Tris–HCl buffer (pH 7.5, 0.05 M), ADP (2 mM),
Fe(NO3)3 (0.12 mM), MgCl2 (7 mM), isocitric acid (2
mM), NADP (0.1 mM) and NADP-isocitrate dehy-
3.7. Acid hydrolysis of compounds 2, 3 and 4
A solution of 2 (1 mg) in 1% HCl (1 ml) in a sealed
tube was heated in an oven set at 97 ꢁC for 40 min. The
resulting solution was extracted with EtOAc, and the
remaining aqueous solution was treated with Diaion
SA-20AP resin to neutralize the solution. Co-HPLC
with the standard sample on a TSK-gel Amide-80 col-
umn [TOSOH], 4.6ꢂ250 mm; mobile phase, acetone–
H2O (1:1); detector, Shodex RI SE-51] showed the pre-
sence of arabinose in the solution. Compounds 3 and 4
were treated in analogous ways, and HPLC analyses of
the reaction mixtures showed the presence of arabinose
and galactose, respectively.
ꢁ
drogenase (0.05 U/ml) was incubated at 37 C for 1 h.
Then, a mixture of 0.8% thiobarbituric acid, 20% ace-
tate buffer (pH 3.5) and 8.1% sodium dodecylsulfate
(75:75:20) (2.0 ml) was added to the reaction mixture,
and heated on a boiling-water bath for 1 h. After the
solution was cooled in water for 5 min, n-butanol was
added, and the mixture was agitated for 20 s. The fluo-
rescence intensity (Ex 515 nm, Em 553 nm) of the upper
layer obtained after centrifugation (3000 rpm, 10 min)
was measured, and the amount of peroxides was esti-
mated using tetramethoxypropane as a standard.
3.8. Degradation of 3 in the presence of toluene-ꢂ-thiol
An ethanol solution (0.75 ml) of compound 3 (3 mg)
in a sealed tube was heated in an oven at 97 ꢁC for 18 h.
Then, the solvent was removed by evaporation, and the
residue was subjected to column chromatogprahy on
Sephadex LH-20 with EtOH as an eluant, to give 4b-
benzylthioether (20) of 3T-O-b-l-arabinosyl-proantho-
cyanidin A2 and (ꢀ)-epicatechin (8). The latter com-
3.10. Inhibitory effects of cacao polyphenols on the
V-70-induced autoxidation of linoleic acid
A solution of V-70 [2, 20-azobis(4-methoxy-2, 4-dime-
thyl-valeronitrile)] (1.6 mM) (Wako) in 20% CH3CN
(0.1 ml) was added to a mixture of 1 mM linoleic acid
(0.1 ml) in phosphate-buffered saline (PBS) solution

T. Hatano et al. / Phytochemistry 59 (2002) 749–758
757
containing 10 mM sodium dodecyl sulfate and a PBS
solution of the test compound (20 ml), and the mixture
was incubated at 37 ꢁC for 1 h. The amount of peroxide
was estimated by measuring the absorbance at 234 nm,
and the inhibitory effects of the test compounds were
expressed as the IC50, the concentration required for
50% inhibition of the formation of peroxides in the
absence of the test compound.
Foo, L.Y., Lu, Y., Howell, A.B., Vorsa, H., 2000. A-Type proantho-
cyanidin trimers from cranberry that inhibit adherence of uro-
pathogenic P-fimbriated Escherichia coli. Journal of Natural
Products 63, 1225–1228.
Fujita, Y., Komagoe, K., Niwa, Y., Uehara, I., Hara, R., Mori, H.,
Okuda, T., Yoshida, T., 1988b. Studies on inhibition mechanism of
autoxidation by tannins and flavonoids. III. Inhibition mechanism
of tannins isolated from medicinal plants and related compounds on
autoxidation of methyl linoleate. Yakugaku Zasshi 108, 528–537 (in
Japanese).
Fujita, Y., Uehara, I., Morimoto, Y., Nakashima, M., Hatano, T.,
Okuda, T., 1988a. Studies on inhibition mechanism of autoxidation
by tannins and flavonoids. II. Inhibition mechanism of caffeetannins
isolated from leaves of Artemisia species on lipoxygenase dependent
lipid peroxidation. Yakugaku Zasshi 108, 129–135 (in Japanese).
Hatano, T., Edamatsu, R., Hiramatsu, M., Mori, A., Fujita, Y., Ysu-
hara, T., Yoshida, T., Okuda, T., 1989. Effects of the interaction of
tannins with co-existing substances. VI. Effects of tannins and rela-
ted polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-
picrylhydrazyl radical. Chemical and Pharmaceutical Bulletin 37,
2016–2021.
3.11. Radical scavenging effects of cacao polyphenols on
the DPPH radical
To a solution of DPPH radical in EtOH (60 mM, 0.5 ml),
a solution of the test polyphenol in MeOH (0.5 ml) was
added, and the reaction mixture was left to stand for 30
min at room temperature. The scavenging activity of each
polyphenol at 1.25, 2.5, 5, 7.5 and 10 mg/ml was estimated
by measuring the absorption of the mixture at 520 nm,
which reflects the amount of DPPH radical remaining in
the solution. The scavenging activity was expressed as
the EC50, the concentration of polyphenol required for
scavenging 50% of DPPH radical in the solution.
Jacques, D., Haslam, E., Bedford, G.R., Greatbanks, D., 1974. Plant
proanthocyanidins. Part II. Proanthocyanidin-A2 and its derivatives.
Journal of the Chemical Society, Perkin Transactions 1, 2663–2671.
Kiatgrajai, P., Wellons, J.D., Gollob, L., White, J.D., 1982. Kinetics
of polymerization of (+)-catechin with formaldehyde. Journal of
Organic Chemistry 47, 2913–2917.
Kolodziej, H., Sakar, M., Burger, J.F.W., Engelshowe, R., Ferreira,
D., 1991. A-Type proanthocyanidins from Prunus spinosa. Phy-
tochemistry 30, 2041–2047.
Acknowledgements
Kondo, K., Kurihara, M., Fukuhara, K., Tanaka, T., Suzuki, T.,
Miyata, N., Toyoda, M., 2000. Conversion of procyanidin B-type
(catechin dimer) to A-type: evidence for abstraction of C-2 hydro-
gen in catechin during radical oxidation. Tetrahedron Letters 41,
485–488.
The Varian VXR500 used in this study is the property
of the SC-NMR Laboratory of Okayama University,
which is gratefully acknowledged.
Lou, H., Yamazaki, Y., Sasaki, T., Uchida, M., Tanaka, H., Oka, S.,
1999. A-Type proanthocyanidins from peanut skins. Phytochem-
istry 51, 297–308.
References
Morimoto, S., Nonaka, G., Nishioka, I., 1986. Tannins and related
compounds. XXXVIII. Isolation and characterization of flavan-3-ol
glucosides and procyanidin oligomers from cassia bark (Cinnamomum
cassia Blume). Chemical and Pharmaceutical Bulletin 34, 632–642.
Nonaka, G., Morimoto, S., Nishioka, I., 1983. Tannins and related
compounds. Part 13. Isolation and structures of trimeric, tetrameric
and pentameric proanthocyanidins from cinnamon. Journal of the
Chemical Society, Perkin Transactions 1, 2139–2145.
Balde, A.M., Pieters, L.A., Gergely, P.A., Kolodziej, H., Claeys, M.,
Vlietinck, A.J., 1991. A-Type proanthocyanidins from stem-bark of
Pavetta owariensis. Phytochemistry 30, 337–342.
Barrett, M.W., Klyne, W., Scopes, P.M., Fletcher, A.C., Porter, L.J.,
Halsam, E., 1979. Plant proanthocyanidins. Part 6. Chiroptical
studies. Part 95. Circular dichroism of procyanidins. Journal of the
Chemical Society, Perkin Transactions 1, 2375–2377.
Botha, J.J., Young, D.A., Ferreira, D., Roux, D.G., 1981. Synthesis
of condensed tannins. Part 1. Stereoselective and stereospecific
syntheses of optically pure 4-arylflavan-3-ols, and assessment of
their absolute stereochemistry at C-4 by means of circular dichro-
ism. Journal of the Chemical Society, Perkin Transactions 1, 1213–
1219.
Nonaka, G., Morimoto, S., Kinjo, J., Nohara, T., Nishioka, I., 1987.
Tannins and related compounds. L. Structures of proanthocyanidin
A-1 and related compounds. Chemical and Pharmaceutical Bulletin
35, 149–155.
Okuda, T., Kimura, Y., Yoshida, T., Hatano, T., Okuda, H., Arichi,
S., 1983. Studies on the activities of tannins and related compounds
from medicinal plants and drugs. I. Inhibitory effects on lipid per-
oxidation in mitochondria and microsomes of liver. Chemical and
Pharmaceutical Bulletin 31, 1625–1631.
Burger, J.F.W., Kolodziej, H., Hemingway, R.W., Steynberg, J.P.,
Young, D.A., Ferreira, D., 1990. Oligomeric flavanoids. Part 15.
Base-catalyzed pyran rearrangements of procyanidin B-2, and evi-
dence for the oxidative transformation of B- to A-type procyani-
dins. Tetrahedron 46, 5733–5740.
Osakabe, N., Natsume, M., Adachi, T., Yamagishi, M., Hirano, R.,
Takizawa, T., Itakura, H., Kondo, K., 2000. Effects of cacao liquor
polyphenols on the susceptibility of low-density lipoprotein to oxi-
dation in hypercholesterolemic rabbits. Journal of Atherosclerosis
and Thrombosis 7, 164–168.
Cronje, A., Burger, J.F.W., Brandt, E.V., Kolodziej, H., Ferreira, D.,
1990. Assessment of 3,4-trans and 3,4-cis relative configurations in
the A-series of (4,8)-linked proanthocyanidins. Tetrahedron Letters
31, 3789–3792.
Porter, L.J., Ma, Z., Chan, B.G., 1991. Cacao procyanidins: major
flavanoids and identification of some minor metabolites. Phy-
tochemistry 30, 1657–1663.
Cronje, A., Steynberg, J.P., Brandt, E.V., Young, D.A., Ferreira, D.,
1993. Oligomeric flavanoids. Part 16. Novel prorobinetinidins and
the first A-type proanthocyanidin with a 5-deoxy A- and a 3,4-cis-C-
ring from the maiden investigation of commercial wattle bark
extract. Journal of the Chemical Society, Perkin Transactions 1,
2467–2477.
Sanbongi, C., Osakabe, N., Natsume, M., Takizawa, T., Gomi, S.,
Osawa, T., 1998. Antioxidative polyphenols isolated from Theo-
broma cacao. Journal of Agriculture and Food Chemistry 46, 454–
457.

758
T. Hatano et al. / Phytochemistry 59 (2002) 749–758
Thompson, R.S., Jacques, D., Haslam, E., Tanner, R.J.N., 1972. Plant
proanthocyanidins. Part I. Introduction; the isolation, structure and
distribution in nature of plant procyanidins. Journal of the Chemi-
cal Society, Perkin Transactions 1, 1387–1399.
goe, K., Fujita, Y., Okuda, T., 1989. Studies on inhibition mechan-
ism of autoxidation by tannins and flavonoids. V. Radical-
cavenging effects of tannins and related polyphenols on 1,1-diphe-
nyl-2-picrylhydrazyl radical. Chemical and Pharmaceutical Bulletin
37, 1919–1921.
Yoshida, T., Mori, K., Hatano, T., Okumura, T., Uehara, I., Koma-