Polyphenols from peanut skins and their free radical-scavenging effects

Article abstract of DOI:10.1016/j.phytochem.2004.06.026

Separation of the water-soluble fraction of peanut skins led to the isolation of five proanthocyanidins. Based on the spectroscopic investigation and partial acid catalyzed degradation, their structures were determined to be epicatechin-(2β→O→7, 4β→6)-[epicatechin- (4β→8)]-catechin (1), epicatechin-(2β→O→7, 4β→8) epicatechin-(4β→8)-catechin-(4β→8)- epicatechin (2), and procyanidins B2 (3), B3 (4) and B4 (5). The absolute configuration of the new compounds was determined from their circular dichroism curves and the ¹H NMR spectra of analysis of flavan-3-ols formed by thiolytic degradation of 1 and 2 in the presence of a chiral dirhodium complex (dirhodium tetra-(R)-(trifluoromethyl) phenyl acetate).

Full text of DOI:10.1016/j.phytochem.2004.06.026

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2391–2399
www.elsevier.com/locate/phytochem
Polyphenols from peanut skins and their free
radical-scavenging effects
a,
Hongxiang Lou , Huiqing Yuan , Bin Ma , Dongmei Ren , Mei Ji , Syuichi Oka
b
a
a
a
c,
*
*
a
School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China
b
School of Medicine, Shandong University, Jinan 250012, PR China
c
International Patent Organism Depository (IPOD), AIST, Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan
Received 3 July 2003; received in revised form 3 May 2004
Available online 6 August 2004
Abstract
Separation of the water-soluble fraction of peanut skins led to the isolation of five proanthocyanidins. Based on the spectroscopic
investigation and partial acid catalyzed degradation, their structures were determined to be epicatechin-(2b!O!7, 4b!6)-[epi-
catechin-(4b!8)]-catechin (1), epicatechin-(2b!O!7, 4b!8) epicatechin-(4b!8)-catechin-(4a!8)-epicatechin (2), and procy-
anidins B2 (3), B3 (4) and B4 (5). The absolute configuration of the new compounds was determined from their circular
1
dichroism curves and the H NMR spectra of analysis of flavan-3-ols formed by thiolytic degradation of 1 and 2 in the presence
of a chiral dirhodium complex (dirhodium tetra-(R)–(trifluoromethyl) phenyl acetate).
ꢀ
2004 Elsevier Ltd. All rights reserved.
Keywords: Arachis hypogaea; Leguminosae; Peanut skin; Proanthocyanidins
1
. Introduction
2. Results and discussion
Peanut skins are used to treat chronic haemorrhage
Peanut skins were extracted with boiling water and
the extract was then fractionated by adsorption on
HP-20 resin (Lou et al., 1999). The fraction obtained
by elution with 70% (v/v) aqueous ethanol was then sub-
jected to chromatography using Toyopearl HW-40 and
eluted with aqueous ethanol and acetone. Fractions 11
and 12, obtained by eluting with 50% (v/v) acetone in
water, from Toyopearl HW 40 contained oligomeric
proanthocyanidins as detected by a characteristic orange
coloration with anisaldehyde-sulfuric acid reagent
(Morimoto et al., 1987). When subjected to further sep-
aration on Sephadex LH-20 and preparative HPLC,
compounds 1–5 were obtained.
and bronchitis in Chinese traditional medicine (Jiansu
Xin Medical College, 1977). Six A-type proanthocyani-
dins (Lou et al., 1999) and flavonoids (Lou et al.,
2
001) have been isolated from the water-soluble phe-
nolic fraction of skin of the mature seed of peanut,
Arachis hypogaea. In this study, five oligomeric proanth-
ocyanidins were isolated from the water-soluble fraction
of peanut skins. Among them, two were identified as
new polyphenols based on their spectral data, their
chemical conversion to monomeric flavan-3-ol and their
1
H NMR spectra in the presence of a chiral dirhodium
complex (dirhodium tetra-(R)–(trifluoromethyl)phenyl
acetate, Rh (DTPA) ).
4
Compounds 3 and 4 were identified as procyanidins
B2 and B3 by direct comparison of their R values
2
f
*
obtained by TLC and R values obtained by HPLC
t
Corresponding authors. Tel.: +86-531-838-2373; fax: +86-531-
38-2019.
E-mail address: louhongxiang@sdu.edu.cn (H. Lou).
with those of authentic samples, and 5 was identified
as procyanidin B4 by direct comparison of its NMR
8
0
031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.06.026

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H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
1
1
spectral data with that of the known compound (Hatano
and Hemingway, 1997) (Scheme 1).
signed to the protons of the A and G rings in H- H
COSY, and this confirmed that another flavan-3-ol unit
is linked to the A-type proanthocyanidin entity through
Compound 1 was obtained as an off-white amor-
phous powder. Its molecular composition was deter-
mined to be C H O Æ2H O based on the results of
00
0
C (I ring) and C (D ring). The long-range correlations
0
4
8
00
0
of H-4 (I ring) at d 4.83 with C at d 111.7, C at d
4
5
36 18
2
8
7
0
0
elemental analysis and the presence of molecular ions
+
at m/z 865 (M +l) in FAB-MS, which revealed that 1
151.7, C at d 152.8 (D ring) and H-2 (F ring) at d
5.07 (1H, d, J=4.3 Hz) with C in the HMBC also con-
9
0
9
0
0
0
is a trimeric flavan-3-ol. The presence of three flavanyl
1
firmed this C ! C linkage (Fig. 1).
4
8
3
units was also indicated by C resonances at d 68.0
The terminal flavanol unit was determined to be 2,3-
00
0
0
0
(
7
C-3), 30.8 (C-4), 82.4 (C-2 ), 68.77 (C-3 ), 26.8 (C-4 ),
00
cis (I ring) from the singlet at d 5.34 (1H, s, 2 -H) and
00
00
00
8.0 (C-2 ), 74.5 (C-3 ), and 37.7 (C-4 ), arising from
the corresponding carbon at d 78.0 (C-2 ), while the
middle unit (the doubly-substituted terminal unit) was
flavanyl heterocyclic rings (rings C, I and F) and one ke-
tal carbon at d 101.8 (C-2) as assigned by an HMBC
experiment. Proof of the existence of an A-type proanth-
deduced to have a 2,3-trans configuration (F ring) from
0
the proton signal at d 5.07 (1H, br. d, J=7.5 Hz, 2 -H) in
1
1
0
13
ocyanidin unit was obtained in the H NMR spectrum,
which showed an isolated AB coupling system at d 4.05
H NMR and d 82.4 (C-2 ) in C NMR. Circular dichr-
oism measurement (Fig. 2) reflected the b orientation of
the flavanyl substitutes, i.e., an R configuration, at both
(
d, J=3.8 Hz, H-3) and 4.34 (d, J=3.8 Hz, H-4) (C ring),
00
while one ketal carbon was seen at d 101.8 (C-2) in the
C NMR spectrum (Jacques et al., 1974). The two fla-
the C-4 (C ring) and C-4 (I ring) positions for a high-
amplitude positive Cotton effect at a diagnostic wave-
length of 240nm (De240 +26.18) (Barrett et al., 1979;
Botha et al., 1981). Treatment of 1 with benzylthiol/ace-
tic acid in ethanol yielded 6, which was identified to be
epicatechin-(2b!O!7, 4b!6)-catechin by direct
1
3
van-3-ol units of this A-type entity in 1 were deduced
0
to be linked through C (C ring) and C (D ring) based
4
6
on the absence of NOE between H-4 and the protons on
0
the E ring or H-2 (F ring) in NOESY (Bilia et al., 1996).
Two sets of meta-coupled protons at d 6.02 (1H, d,
J=2.0 Hz) and 6.09 (1H, d, J=2.0 Hz), and d 6.06
1
comparison of its H NMR spectrum with that previ-
ously reported (Lou et al., 1999), confirming the abso-
lute configuration of the A-type entity. The thioether
(
1H, d, J=1.8 Hz) and 5.63 (1H, d, J=1.8 Hz), were as-
OH
OH
OH
O
HO
OH
HO
O
OH
OH
HO
O
O
OH
HO
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
O
OH
OH
O
O
HO
HO
O
OH
HO
HO
OH
HO
OH
OH
OH
OH
1
2
OH
OH
OH
OH
OH
OH
HO
O
HO
O
HO
O
OH
OH
OH
OH
OH
OH
OH
OH
OH
HO
OH
HO
OH
HO
O
O
O
OH
OH
OH
OH
HO
HO
OH
4
5
3
Scheme 1.

H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
2393
OH
OH
B
HO
OH
H
OH
B
HO
H
O
C
G
H
HO
O
C
A
OH
H
OH
K
O
HO
A
O
D
OH
I
OH
OH
OH
OH
OH
O
OH
H
H
OH
H
H
OH
OH
D
H
O
O
OH
OH
H
H
F
H
O
HO
O
HO
HO
H
I
F
H
L
E
G
E
OH
J
OH
HO
OH
HO
OH
OH
OH
1
2
Fig. 1. Long-range correlations between H and C as observed in HMBC.
goflavanoids (Kolodziej et al., 1993). This is a branched
A-type proanthocyanidin with C –C and C –C link-
4
6
4
8
ages to the same A ring of a flavanyl unit (Foo and
Hemingway, 1984) (see Schemes 2 and 3).
+
The FAB-MS of 2 indicated an [M+l] ion at m/z
1153, consistent with a flavan-3-ol tetramer. Although
the occurrence of conformational isomers of 2 makes
1
the H NMR spectrum even more complex, only one
conformer (which account for more than 90%) appeared
to be the major constituent responsible for the possible
hindrance of its structure, as revealed in the 600 MHz
1
H NMR spectrum (recorded in CD OD), and this
3
made it possible to assign its NMR spectroscopic data,
1
the C NMR spectrum of 2, the presence of four flava-
3
nyl units was also indicated by C-3 signals at d 67.0,
67.2, 70.8, and 74.9, and C-4 signals at d 29.0 (d), 38.0
(
d), 38.5 (d) and 23.7 (t). The presence of an A-type pro-
Fig. 2. CD measurement of 1 and 2.
anthocyanidin in the tetrameric chain was evident from
the characteristic ketal carbon resonance at d 100.3 as
determined by HMBC and the typical AB coupling pro-
tons at d 3.59 (1H, d, J=3.5 Hz, H-3) and 4.03 (1H, d,
J=3.5 Hz, H-4) (A ring) in the H– H COSY. This A-
type proanthocyanidin unit was deduced to be at the
top of the structure based on the occurrence of meta
coupling aromatic protons in the A-ring at d 5.96 (1H,
d, 1.8 Hz, H-6) and 5.84 (1H, d, J=1.8 Hz, H-8) which
correlated with the same C-7 (d 156.6) (A ring) as well as
the correlations between H-6 and C-5 (154.4), and H-4
and C-5 in HMBC (Fig. 1). The NOE effects between
(
7), derived from the lower terminal unit in the thiolytic
degradation of 1, was desulfurized with Raney nickel
Morimoto et al., 1987) to give 8. Compound 8 was then
methylated with CH N to form 9, the structure of
1
1
(
2
2
which was finally confirmed to be tetra-O-methyl ether
1
of epicatechin by measurement of its H NMR in the
presence of Rh (DTPA) ). In the presence of
2
4
Rh (DTPA) ), the proton signals of the A- and C-rings
2
4
all shifted to a lower field while the protons of the B-ring
shifted up-field, possibly because the chiral reagent was
closer to the A- and C-rings in the solution. These dia-
0
H-4 and H-2 (d 5.6, 1H, s) (F ring) revealed that the
interflavanoid linkage of this A-type proanthocyanidin
1
stereomeric dispersions due to complexation in the H
0
0
NMR spectrum allowed us to identify epicatechin and
ent-epicatechin subunits, as well as their enantiomeric
ratio, as illustrated in Fig. 3.
Consequently, the structure of 1 was determined to be
epicatechin-(2b!O!7,4b!6)-[epicatechin-(4b!8)]-
catechin according to the nomenclature for A-type oli-
is through C -O-C and C –C (Bilia et al., 1996).
2 4
7
8
The terminal flavan-3-ol had a 2,3-cis configuration (L
ring) as suggested by the coupling constants of the ali-
phatic proton at d 4.92 (1H, s, H-2 ). A carbon resonance
at d 80.2 also confirmed this 2,3-cis configuration. The
remaining flavan-3-ol unit with a 2,3-trans configuration
000

2394
H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
1
Fig. 3. Enantioselectivity of tetramethyl ether of epicatechin by H NMR spectra recorded with CDCL
3
as solvent (Hameed et al., 1998). (a) 1:1
); (b) 1:1 mixture of 9 and 9a in the presence of Rh (DTPA) );
). The signals below 7.0 ppm overlapped with that of the chiral reagent, while no significant
mixture of 9 and the tetra-O-methyl ether of ent-epicatechin (9a) without Rh
c) 2:1 mixture of 9 and 9a in the presence of Rh (DTPA)
2
(DTPA)
4
2
4
(
2
4
chemical shift change was observed for the signals of methoxyl groups above 4.3 ppm.
OH
OH
OH
OH
HO
O
OMe
HO
O
S
OMe
O
OH
MeO
O
OH
OH
CH N
2
2
Raney Ni
PhCH SH
+
2
OH
1
HO
O
OH
H
OMe
OH
9
OH
OH
7
6
Scheme 2. Thiolytic degradation of 1.
was determined based on the chemical shifts of the pro-
tons at d 3.93 (1H, brd, J=6.6 Hz, H-2 ), a unique
up-field chemical shift due to steric interactions with
as 10, 11 and 12. The desulfurization of 11 yielded 13,
which was identified to be proanthocyanidin A-2, as pre-
viously reported (Lou et al., 1999). Compound 12, when
desulfurized and subjected to methylation with CH N ,
00
00
linked flavanyl units, 4.41 (1H, d, J=6.6 Hz, H-3 ) and
4
2
2
0
0
1
.43 (1H, br.s, H-4 ) (I ring) in H NMR, and the carbon
was converted to 15. The structure of 15 was unambig-
0
00
13
0
resonance at d 84.0 (C-2 ) in C NMR. The interflavanyl
linkages were determined to be C AC and C AC ,
uously identified to be the 3 ,4 ,5,7-tetramethyl ether of
0
00
00
000
8
1
catechin by measurement of its H NMR spectra in
4
8
4
respectively, based on the presence of long-range correla-
00
the presence of Rh (DTPA) (Fig. 4). Moreover, the
4
2
0
00
00
tions of H-4 with C (d 109.7) and C (d 151.6), H-2 with
CD spectrum of 2 revealed two positive Cotton effects
at 243 nm (De 5.22) and 236 nm (De 5.39), indicating
8
9
0
0
00
000
000
000
9
C , as well as H-4 with C (d 108.7) and C (d 154.4),
9
8
000
0
and H-2 with C , respectively.
an b-orientation of the 4 -flavanyl substituent (Botha
9
The thiolytic degradation of 2 led to the formation of
and 8, which were identified to be procyanidin B4 and
epicatechin, respectively, by direct comparison, as well
et al., 1981). Accordingly, the structure of 2 was deter-
mined to be epicatechin-(2b!O!7, 4b!8)-epicate-
chin-(4b!8)-catechin-(4a!8)-epicatechin.
5

H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
2395
Scheme 3. Thiolytic degradation of 2.
Oligomeric proanthocyanidins have been shown to
have various biologically significant effects (Lou et al.,
IR. spectra were taken using a Shimadzu UV 240 spec-
trophotometer and a Nicolet Nexus FT/IR-470 spectro-
1
13
2
000). Similar A-type proanthocyanidins in cranberry
photometer, respectively. H NMR and C NMR were
measured using a Brucker Avance 600 NMR spectrom-
prevent the adherence of P-fimbriated Escherichia coli
isolates from the urinary tract to cellular surfaces and
have been used as an effective therapeutic agent in the
treatment and prevention of urinary tract infections
1
eter (600 MHz for H NMR and 150 MHz for
13
C
NMR). Chemical shifts are given in d (ppm), based on
TMS. FAB-MS were measured with a JEOL JM-
HX110 mass spectrometer. CD was recorded with a
JASCO J-15 spectro-polarimeter. Preparative HPLC
was performed using a Waters 600-996 on a Prepak Car-
tridge 25·100 ODS column (Waters) with a mixture of
methanol and water as a mobile phase and detection at
280 nm. The flow rate of the mobile phase was 3.5 ml/
min. TLC was performed on precoated aluminum sheets
(
Foo et al., 2000). In this paper, the scavenging activities
of compounds 1–5 toward the DPPH (1,1-diphenyl-2-
picrylhydrazyl) radical were determined (Table 3). The
IC50 values of 1–5 were about 1.0 lM. These results
showed that the proanthocyanidins in peanut skins have
free radical-scavenging effects, which could protect the
seed fatty residue from oxidation.
(Rp-18 F254, 0.2 mm, Merck) with CH OH–H O (5:5).
3 2
Column chromatography was carried out using Diaion
HP 20 (Nippon Rensui Co.), Sephadex LH-20 (Pharma-
cia Biotech, Sweden), Toyopearl HW40 (fine grade from
Tosoh, Japan), MCI CH 20P (Mitsubishi Chemical
Co.), and silica gel 300 (Qingdao Chemical Co., Ltd.).
Rh (DTPA) was generously provided by Prof. Helmut
3
. Experimental
3
.1. General experimental procedures
Melting points were determined on a Yanaco micro-
melting point apparatus and are uncorrected. UV and
2
4
Duddeck of Hannover University, Germany.

2396
H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
1
Fig. 4. Enantioselectivity of tetramethyl ether of catechin by H NMR spectra recorded with CDCL
3
as solvent: (a) 1:1 mixture of 15 and the tetra-
(DTPA) ); (c) 2:1 mixture of 15 and
). The signals below 7.0 ppm overlapped with that of the chiral reagent while no significant chemical shift change
for the signals of methoxyl groups above 4.0 ppm.
O-methyl ether of ent-catechin (15a) without Rh
5a in the presence of Rh (DTPA)
2
(DTPA)
4
); (b) 1:1 mixture of 15 and 15a in the presence of Rh
2
4
1
2
4
3
.2. Plant material
3.4. Epicatechin-(2b!O!7, 4b!6)-[epicatechin-
(
4b! 8)]-catechin (1)
A. hypogaea L. was taxonomically identified by Dr.
Masaru Uchida of Tokiwa Phytochemical Co., Ltd.,
Chiba, Japan. A voucher specimen was deposited at
the company under registration number AH-962001.
Peanut skins were collected in December 1996.
Off-white amorphous powder; m.p. 272 ꢁC (decomp).
[a] +86.2 (c 0.3, acetone). UV (MeOH) k : 282nm.
D
max
+
FAB-MS m/z: 865 (M +1), Anal. Calcd. for
C H O Æ2H O: C, 60.00; H, 4.47. Found: C, 60.09;
4
5
36 18
2
1
H, 4.89. CD: De240 +26.18, De H
NMR (600 MHz, CD OD) and C NMR (150 MHz,
ꢀ0.524. For
276
3
1
3
3
.3. Extraction and purification
CD OD) data see in Table 1.
3
The extraction procedure is the same as that in our
previous report (Lou et al., 1999). Fractions 11 and 12
obtained by eluting with 50% acetone in water from a
Toyopearl HW 40 column contained oligomeric pro-
anthocyanidins as detected by UV measurement and
TLC orange coloration with anisaldehyde-sulfuric acid
reagent (Jacques et al., 1974). Fraction 11 (4.8 g) was
further subjected to column chromatography using
Sephadex LH-20 (50 g, 35·3.0 cm) and eluted with
3.5. Thiolytic degradation 1
A mixture of 1 (32 mg), benzylthiol (1.2 ml) and ace-
tic acid (1.2 ml) in ethanol (5 ml) was refluxed for 5 h
with stirring under a nitrogen atmosphere. The reaction
mixture, when concentrated in vacuo to give an oily res-
idue, was subjected to chromatography with Sephadex
LH-20 using 70% methanol in water as a mobile phase.
Compounds 6 (8 mg) and 7 (11 mg) were obtained.
5
0% methanol (1.2 l) (each 100 ml portion was collected
as one eluant). The eluants, which have the same TLC
coloration, were mixed together to give Fr.11-1 (1.1 g,
from eluants 3–6) and Fr.11-2 (0.6 g, from eluants 8–
3.6. Epicatechin-(2b!O!7, 4b!6)-catechin (6)
White needles (MeOH); mp. 272 ꢁC (decomp), FAB-
1
0). Further separation of Fr.11-1 by preparative HPLC
+
1
afforded compounds 1 (46 mg) and 2 (53 mg). The same
method was used to separate Fr. 11-2 to give com-
pounds 3 (88 mg), 4 (22 mg) and 5 (11 mg).
MS: m/z: 577 (M +l). H NMR (600 MHz, CDC1 )
3
1
and C NMR (150 MHz, CDC1 ) data were the same
3
3
as those previously reported (Lou et al., 1999).

H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
2397
Table 1
NMR spectroscopic data for compound 1
Carbons
Correlated H
H coupled with C
H coupled with H
No
d
C
d
H
2
3
4
5
6
7
8
9
101.8
68.4
30.8
H-3, H-4, H-12
H-4
H-3
4.05, d (3. 8)
4.34, d (3.8)
H-4
H-3
155.9
97.0
H-3, H-7
6.09, d (2.0)
6.02, d (2.0)
H-8
H-6
158.9
97.6
H-6, H-8
155.7
105.9
132.0
114.8
146.5
146.6
117.0
119.8
82.4
H-8
H-3, H-4
H-4, H-12, H-14
1
1
1
1
1
1
1
2
3
4
0
1
2
3
4
5
6.92, d (1.8)
H-16
a
a
H-12
H-15
H-16
H-15
6.69, d (8.2)
H-16
H-12, H-15
6
6.93, d (8.2, 1.8)
5.07, d (7. 5)
4.20, m
0
0
0
0
0
0
0
0
0
H-3 , H-12
H-2 , Ha-4
H-3
H-4 , H2
0
0
0
68.8
26.8
0
2.76, dd (16.5, 4.0), H-a
H-3 , H-2
H-3
2
.57, dd (16.5, 4.6), H-b
0
0
0
0
0
0
5
6
7
8
9
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
151.9
109.6
151.0
111.7
152.8
103.8
133.5
116.9
146.3*
146.5*
117.1
121.1
78.0
H-4
0
H-6
0
H-8
0
0
0
0
0
0
0
r
H-2 , H-3
0
0
1
2
3
4
5
6
0
0
H-2 , H-12
0
6.88, d (2.0)
H-16
0
0
0
0
H-12
H-15
H-16
0
0
6.78, d (8.2)
6.13, dd (8.2, 2.0)
5.34, s
H-16
H-12 , H-15
0
H-15
00
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
00
00
H-3 , H-12
00
H-3
H-2 , H-4
00
00
74.5
37.7
3.78, br. s
4.83, br. s
H-2 , H-4
00
00
H-4
H-4 , H-6
H-3
00
00
00
158.6
97.6
00
H-6 , H-8
00
158.2
97.2
5. 63, d, (2.2)
6.04, d (2.2)
H-8
00
00
H-8 , H-4
00
158.7
105.2
134.0
116.7
145.5
146.9
116.8
121.2
H-6
0
0
0
0
0
0
0
0
00
00
0
1
2
3
4
5
6
H-4 , H-3
H-2 , H-12
0
0
0
0
0
0
00
00
00
00
6.94, d (2.0)
H-2
H-12
H-16
00
00
00
00
H-15
H-16
00
6.69, d (8.2)
6.57, dd (8.2, 2.0)
H-16
H-12 , H-15
00
00
H-15
a
Data are interchangeable; data in parentheses are coupling constants.
0
0
3
.7. 3 ,4 ,5,7-Tetra-O-methyl epicatechin (9)
added and the mixture was then stirred in an ice bath
for 12 h. The methyl ether was formed,purified using sil-
ica gel (6 g) chromatography, and eluted with benzene-
acetone (85:15). Compound 9 (5 mg) was obtained as
a white powder, mp 132–135 ꢁC (acetone), FAB-MS
Compound 7, a white amorphous powder, was iden-
tified to be epicatechin-4-benzylthioether by measure-
+
ment of its FAB-MS, m/z: 413 (M +1), in accordance
+
1
with the formula C H O S. The thioether 7 (8 mg),
2
m/z: 347 (M +1). Its H NMR data were the same as
2
20
6
0
0
dissolved in 1.5 ml of ethanol–acetic acid (10:1), was
desulfurized with Raney nickel at 50 ꢁC for 30 min.
After the catalyst was removed by filtration, the reaction
mixture was concentrated to dryness. The residue was
dissolved in 1 ml MeOH, excess CH N in Et O was
those of the 3 ,4 ,5,7-tetra-O-methyl ether of epicatech
in (Fig. 3(a)). In the presence of the chiral reagent
1
Rh (DTPA) , 9 was determined by measuring its H
2
4
0
0
NMR spectrum using with its enantiomer 3 ,4 ,5,7-tetra-
O-methyl ether of ent-epicatechin (9a) (Figs. 3(b),(c)).
2
2
2

2398
H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
Table 2
NMR spectroscopic data for compound 2
Carbons
d
H
Carbons
No
d
H
No
d
C
d
C
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
7
8
9
100.3
67.2
29.1
2
3
4
5
6
7
8
9
84.0
74.9
38.5
156.0*
98.5
155.7*
109.7
151.6
98.6
3.93, d (6.6)
4.41, d (6.6)
4.40, s
3. 59, d (3.5)
4.03, d (3.8)
154.4
97.8
156.6
5.98, d (2.0)
5.84, d (2.0)
6.10, s
98.6
154.9*
105.4
132.6
116.0
146.8**
146.6**
115.8
120.3
77.7
00
0
0
0
00
00
0
0
0
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
0
1
2
3
4
5
10
11
12
13
14
15
16
133.9
116.0
145.7
146.0
116.4
120.4
80.2
6.89, d (1.8)
7.11, d (1.8)
0
0
0
0
6.87, d (8.0)
6.72, dd (8.0, 1.8)
5.78, s
6.76, d (8.0)
6.72, dd (8.0, 1.8)
4.92, s
6
0
000
000
000
2
3
4
0
0
0
0
0
0
0
70.8
38.0
3.68, s
4.46, s
67.0
23.7
4.03, m
2.53, br. d (16.2)
2.08, dd (16.2,3.6)
157.8
96.2
000
000
000
000
000
157.3*
95.6
5.82, s
5
153.3
111.7
151.6
106.7
131.2
117.0
146.0
145.8
115.0
121.5
6
6.02, s
7
155.8
109.7
154.4
100.7
132.0
112.9
145.8
145.7
116.6
118.2
8
0
0
0
0
0
0
0
0
1
2
3
4
5
6
9
000
000
000
000
000
000
10
11
12
13
14
15
7.21, br. s
5.91, d(2.1)
6.85, d (8.2)
6.81, br. d (8.2)
6.40, d (8.2)
5.39, dd (8.2, 2.1)
0
00
1
6
Data with the same ‘‘ ’’ or ‘‘**’’ are interchangeable; data in parentheses are coupling constants.
*
Table 3
Scavenging effect of 1–5 on DPPH radicals
Compound
1
2
3
4
5
EGG
IC50 lM
1.21± 0.11
1.32± 0.16
0.96± 0.09
1.11± 0.15
1.02± 0.09
1.13± 0.08
EGG, epigallocatechin gallate; used as a positive control
3
.8. Epicatechin-(2b!O!7, 4b!8) epicatechin-
4a!8)-catechin-(4a!8)-epicatechin (2)
mixture was concentrated in vacuo, and the obtained
residue was subjected to preparative Rp-18 TLC (0.5
mm, Merck) with CH OH–THF–H O) (7:1:3), to give
(
3
2
Off-white amorphous powder, m.p. 260 ꢁC (decomp).
compounds 5 (3 mg), 8 (3 mg), 10 (2 mg), 11 (6 mg)
and 12 (9 mg). Compound 5 was identified by direct
comparison with an authentic sample. Compound 8
was converted by methylation to 9, which was identified
by the method described above. The FAB-MS of 10 at
m/z: 987 and 11 at m/z: 699 were in accordance with
the formulas C H O S and C H O S, respectively.
[
a] +27.6 (c 0.3, acetone). UV (MeOH) k : 282 nm.
D max
+
FAB-MS m/z: 1153 (M +l), Anal. Calcd. for
C H O Æ3H O: C, 59.70; H, 4.38. Found: C, 59.62;
6
0
48 24
2
H, 4.39. CD: De
H NMR (600 MHz, CD OD) d and C NMR (150
5.39, De243 +5.22, De
ꢀ3.27. For
2
36
271
13
1
3
MHz, CD OD) d data see in Table 2.
3
52 42 18
37 30 12
3
.9. Thiolytic degradation of 2
3.10. Desulfurization of 11
A mixture of 1 (42 mg), benzylthiol (1.5 ml) and ace-
tic acid (1.5 ml) in 5 ml of ethanol was refluxed for 1 h
with stirring under a nitrogen atmosphere. The reaction
Compound 11 (5 mg), dissolved in 1.0 ml of EtOH–
AcOH (10:1), was desulfurized with Raney nickel at 50
ꢁC for 30 min. After the catalyst was removed by filtra-

H. Lou et al. / Phytochemistry 65 (2004) 2391–2399
2399
tion, the reaction mixture was concentrated to dryness.
After purification on Sephadex LH-20 (20 ml bed vol-
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This work was funded by grants from the National
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