A-type proanthocyanidins from peanut skins

Article abstract of DOI:10.1016/S0031-9422(98)00736-5

Six A-type proanthocyanidins were isolated from the water-soluble fraction of peanut skins. On the basis of spectral data, reductive cleavage with sodium cyanoborohydride, and chiral HPLC analysis, three new compounds, epicatechin-(2β → O → 7, 4β → 6)-catechin, epicatechin-(2β → O → 7, 4β → 6)-ent-catechin and epicatechin-(2β → O → 7, 4β → 6)-ent- epicatechin were unambiguously identified, together with the three known compounds, proanthocyanidin A-1, proanthocyanidin A-2 and epicatechin-(2β → O → 7, 4β → 8)-ent-epicatechin. ¹³C NMR chemical shift rules to distinguish between [2 → O → 7, 4 → 8] and [2 → O → 7,4 → 6] double- linked heptamethyl ethers of A-type proanthocyanidins are proposed. Bioassay experiments showed that these six compounds possess substantial activity against hyaluronidase.

Full text of DOI:10.1016/S0031-9422(98)00736-5

Phytochemistry 51 (1999) 297±308
A-type proanthocyanidins from peanut skins
Hongxiang Loua, 1, Yoshimitsu Yamazakia, Tsutomu Sasakib, Masaru Uchidab,
Hideoki Tanakaa, Syuichi Okaa,*
aNational Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba 305-8566, Ibaraki,
Japan
bTokiwa Phytochemical Co. Ltd., Kinogo, Sakura 285-0801, Chiba, Japan
Received 21 April 1998; accepted 22 September 1998
Abstract
Six A-type proanthocyanidins were isolated from the water-soluble fraction of peanut skins. On the basis of spectral data,
reductive cleavage with sodium cyanoborohydride, and chiral HPLC analysis, three new compounds, epicatechin-(2b4O47, 4b
4 6)-catechin, epicatechin-(2b 4 O 4 7, 4b 4 6)-ent-catechin and epicatechin-(2b 4 O 4 7, 4b 4 6)-ent-epicatechin were
unambiguously identi®ed, together with the three known compounds, proanthocyanidin A-1, proanthocyanidin A-2 and
epicatechin-(2b4O47, 4b48)-ent-epicatechin. 13C NMR chemical shift rules to distinguish between [24O47, 448] and [24O
47,446] double-linked heptamethyl ethers of A-type proanthocyanidins are proposed. Bioassay experiments showed that these
six compounds possess substantial activity against hyaluronidase. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Arachis hypogaea; Leguminosae; Proanthocyanidin; Hyaluronidase; Inhibition
1. Introduction
2. Results and discussion
Peanut skins were extracted with boiling water with
the resulting extract fractionated by adsorption on
HP-20 resin eluted with water and aqueous acetone.
The fraction obtained by elution with 70% aqueous
acetone exhibited marked hyaluronidase inhibitory ac-
tivity. This active fraction was subjected to sequential
gel chromatography on Toyopearl HW-40 and
Sephadex LH-20, and ®nally gave, following reverse
phase low- and high-pressure liquid chromatographies,
six compounds: compounds 1, 2 and 4 were obtained
as colorless needles, whereas compounds 3, 5 and 6
were white amorphous powders.
Peanut skins are used to treat chronic hemorrhage
and bronchitis in China (Jiangsu Xin Medical College,
1977) and have a high tannin content (Karchesy &
Hemingway, 1986). In the course of our studies on
bioactive natural compounds, the water-soluble frac-
tion of the mature seed skins of peanut Arachis hypo-
gaea L. was found to inhibit hyaluronidase. From this
active fraction, six A-type proanthocyanidins were pur-
i®ed by monitoring its enzymatic inhibition. Among
them, three were identi®ed as new A-type proantho-
cyanidin dimers on the basis of: their spectral data;
their conversion to monomers by reductive cleavage
with sodium cynaoborohydride and from the results of
chiral HPLC analyses.
All six compounds yield an orange coloration on
reaction with anisaldehyde-sulfuric acid reagent
(Morimoto, Nonaka, & Nishioka, 1987) which is
thought to be characteristic of proanthocyanidins. The
1H NMR (CD3OD) spectra of all six compounds clo-
sely resembled each other (Table 1) and the presence
of the isolated AB coupling system at d 4.0±4.5 with
J3,4 =3.4±3.8 Hz was ascribed as a diagnostic feature
* Corresponding author.
1 Permanent address: Faculty of Pharmaceutical Sciences,
Shandong Medical University, Jinan 250012, People's Republic of
China.
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(98)00736-5

298
H. Lou et al. / Phytochemistry 51 (1999) 297±308
of the C-ring protons of A-type proanthocyanidins
(Jacques, Haslam, Bedford, & Greatbanks, 1974). The
meta-substitution pattern, as revealed by the two
coupled doublets and a residual one proton singlet at
around d 6.0, as well as the two AMX coupling sys-
tems in the aromatic region (d 6.5±7.5) were indicative
of a dimeric ¯avanol. This class of compounds was
also con®rmed by the presence of 1 methylene, 13
methines and 16 quarternary carbons in the 13C NMR
spectrum (Table 2), and the molecular weights of 576
amu for each compound, as established by FAB±MS
([M+1]+ at m/z 577).
Compound 1 was identi®ed as proanthocyanidin A-
1 and compound 2 was identi®ed as proanthocyanidin
A-2 by direct comparison of their spectral data with
those of authentic samples. Data for their heptamethyl
ethers also support this conclusion.
The 1H NMR spectrum of 3 resembled that of 2.
The presence of the AB coupling system at d 4.16
(1H, d, J=3.5 Hz, H-3) and d 4.41 (1H, d, J=3.5 Hz,

H. Lou et al. / Phytochemistry 51 (1999) 297±308
299
H-4), the meta-coupled doublets at d 5.89 and 6.07
(each 1H, d, J = 2.3 Hz, ring A), a residual one
aromatic proton singlet at d 6.08 (ring D), and two
AMX systems in the aromatic region (d 6.5±7.5) due
to rings B and E con®rmed the A-type proanthocyani-
din dimeric structure. This doubly linked dimeric
structure was also demonstrated by the one acetal car-
bon at d 100.46 in its 13C NMR spectrum which was
assigned unequivocally by the COLOC experiment.
The 2,3-cis relative con®guration of the lower ¯avanol

300
H. Lou et al. / Phytochemistry 51 (1999) 297±308
Table 1
1H NMR data (6 ppm) for compounds 1±6 (270 MHz; CD3OD)
Ring
C
H
1
2
3
4
5
6
3
4.07 d (3.5)
4.24 d (3.5)
5. 90 d (2.4)
6.05 d (2.4)
7.13 d (2.4)
6.79 d (8.4)
7.01 dd (8.4, 2.4)
4.74 d (7.8)
4.16 m
4.05 d (3.4)
4.39 d (3.4)
6. 00 d (2.3)
6.07 d (2.3)
7.14 d (2.4)
6.82 d (8.2)
7.07 dd (8.2, 2.4)
4.91 br.s
4.16 d (3.5)
4.41 d (3.5)
5.89 d (2.4)
6.07 d (2.4)
7.15 d (2.4)
6.84 d (8.4)
7.04 dd (8.4, 2.4)
5.02 br.s
4.12 d (3.5)
4.29 d (3.5)
4. 10 d (3.5)
4.28 d (3.5)
6.03 d (2.2)
4.12 d (3.2)
4.30 d (3.2)
6.01 d (2.3)
6.07 d (2.3)
7.16 d (2.2)
6.82 d (8.1)
7.05 dd (8.1, 2.4)
4.80 br.s
4
A
6
6.03 d (2.2)
6.08 d (2.2)
8
6.09 d (2.2)
7.16 d (2.4)
6.81 d (8.4)
B
10
13
14
2'
7.17 d (2.4)
6.82 d (8.4)
7.05 dd (8.4, 2.4)
4.67 d (6.5)
4.05 m
7.06 dd (8.4, 2.4)
4.63 d (7.3)
3.99 m
F
3'
4.23 m
4.23 m
2.87 m
4.16 m
2.87 m
4'a
4'b
6'
2.58 dd (16.5,8.1)
2.81 dd (16.5,5.4)
6.08 s
2.74 dd (17.3, 3.2)
2.95 dd (17.3, 4.6)
6.09 s
2.81 dd (16.5, 5.4)
2.62 dd (16.5, 8.1)
2.91 dd (16.7, 5.4)
2.58 dd (16.7, 7.6)
6.08 s
8'
6.09 s
6.07 s
6.09 s
D
E
10'
13'
14'
6.91 d (2.0)
6.80 d (7.8)
6.80 dd (7.8, 2.0)
7.13 d (2.2)
6.81 d (8.2)
6.99 dd (8.2, 2.2)
7.14 d (2.0)
6.81 d (8.2)
6.95 dd (8.2, 2. 0)
6.80 d (2.2)
6.75 d (8.4)
6.68 dd (8.4, 2.2)
6.79 d (2.2)
6.70 d (8.4)
6.68 dd (8.4, 2.2)
6.93 d (2.2)
6.73 d (8.3)
6.75 dd (8.3, 2.2)
Assignments were made by combination analysis of the 1H±1H Cosy and NOE associations. J values (Hz) are given in parentheses.
moiety was evident from the singlet at d 5.02 (H-2',
ring D).
Methylation of 3 with diazomethane for 24 h at
room temperature yielded hexamethyl ether 9 ([M +
1]+ at m/z 661) as the major product because of the
hinderic e€ects of E ring in terminal units to HO-5 of
A ring. Its methylation when continued for 4 d
a€orded heptamethyl ether 10. The molecular formula
Table 2
13C NMR data (6 ppm) for compounds 1±6 (CD3OD, 67.5 MHz)
Carbon
1
2
3
4
5
6
2
100.34
67.79
29.23
100.21
68.12
29.31
100.46
67.78
29.27
100.57
68.44
29.74
100.61
68.57
29.72
100.57
67.65
29.79
3
4
4a
5
104.06
156.76
98.22
104.29
157.05
98.34
104.11
156.72
98.02
104.22
155.14
96.70
104.23
155.37
97.01
104.27
155.28
96.92
6
7
158.14
96.78
158.16
96.67
158.12
96.53
158.11
96.65
158.23
96.55
158.20
96.65
8
8a
9
154.24
132.30
115.72
146.79
145.65
116.36
119.87
84.49
154.31
132.50a
115.66
146.34b
145.69b
116.08
120.41
81.81
154.17
132.34a
115.75
146.23b
146.23b
116.26
119.90
80.87
154.39
132.23
115.84
146.84
146.19
115.68
119.99
82.51
154.44
132.25a
115.95
146.86b
146.25b
115.68
120.01
82.83
154.41
132.13
115.84
145.86
146.69
115.70
120.01
79.96
10
11
12
13
14
2'
3'
4'
4'a
5'
6'
7'
8'
8'a
9'
10'
11'
12'
13'
14'
68.12
28.96
67.02
29.93
67.18
29.50
67.64
27.88
67.67
28.30
67.13
29.61
103.16
156.15
96.58
102.46
156.65
96.53
101.95
156.63
96.60
103.54
155.25
108.78
152.48
96.65
103.57
155.37
108.76
152.48
96.67
102.78
155.81
108.85
152.93
96.92
152.19
106.80
151.42
130.58
115.77
146.79
146.34
115.72
120.73
152.34
107.25
152.18
131.24a
115.70
146.80b
146.03b
115.97
119.81
152.09
106.97
151.31
131.44a
115.70
145.69b
146.08b
115.25
119.97
151.96
132.19
114.96
146.67
146.27
116.18
119.60
152.03
132.16a
114.98
145.71b
146.25b
116.17
119.89
152.01
132.27
115.25
145.85
145.98
115.93
119.44
Assignments for 1, 4 and 6 were made by HETCOR and COLOC experiments. The corresponding carbons of 2, 3 and 5 were inferred by
a,b
direct comparison. Symbol
in the same column may be interchanged.

H. Lou et al. / Phytochemistry 51 (1999) 297±308
301
C37H38O12 was established for 10 by elemental analysis
and FAB±MS ([M+1]+ at m/z 675). In its H NMR
diazomethane generated 21, whose structure was deter-
mined by comparison of its 1H NMR spectrum and
retention time in chiral HPLC on Chiralcel OD col-
umn with those of authentic compound; such HPLC
proved to be an e€ective method for separating the
enantiomers under the given conditions (see Section 3).
The structure of the bi¯avanoid 25 in this experiment
was expected from the reaction. Methylation of it
yielded 26. Their structures were determined by MS,
CD and NMR measurements. Formation of 15 and 27
did not contribute signi®cantly to the structure deter-
mination. Their structure determinations are not dis-
cussed in detail further, only the spectral data are
listed in the experimental section. This straight forward
approach lead to the unambiguous determination of 3
as epicatechin-(2b4O47, 4b48)-ent-epicatechin. This
compound was previously identi®ed in Prunus spinosa
in the form of its heptamethyl diacetate (Antonio
Gonzalez, 1994).
1
(Table 3) spectrum, in addition to six methoxy signals
at d 3.73±3.95, one up®eld methoxyl signal at d 3.14
was assignable to MeO-5 owing to the anisotropic
shielding of E ring in the lower ¯avanol unit, but
absence of this strongly shielded resonance in 9. This
observation not only revealed the structure of 9 as an
HO-5 unchanged methyl derivative but also suggested
that the two ¯avanol units are linked through C-4 and
C-8'. This linkage was further supported by the obser-
vation of NOE associations of H-4 (d 5.01, ring C)
with the protons of the E ring in 10. The NOE associ-
ations of H-3 (d 4.33, ring C) with H-6' (d 6.20, ring
D) and the protons of the B ring de®ned the 3,4-trans
con®guration of the C ring unambiguously (Cronje,
Burger, Brandt, Kolodziej, & Ferreira, 1990) as well as
helped in the assignment of the protons of B and E
rings.
In addition, the two ¯avanol units of A-type
proanthocyanidins must possess either (2a, 4a) or (2b,
4b) double inter¯avanyl bonds. The high-amplitude
positive Cotton e€ect at short wavelengths ([y ]221
+2.3Â106) of the CD spectrum of 3 allowed assign-
ment of the absolute con®guration of C-4 as R (Botha,
Yong, Ferreira, & Roux, 1978; Barrett et al., 1979),
thus decided 2b,4b-con®guration for 3. Taking the
NOE interactions into consideration, this positive
Cotton e€ect de®ned epicatechin as the upper unit
with 2S, 3R and 4R absolute con®gurations.
The absolute con®guration of the lower terminal
unit was deduced to be 2'S and 3'S, that is ent-epicate-
chin according to the nomenclature (Kolodziej et al.,
1993), on the basis of the pronounced NOE e€ects of
MeO-5 (d 3.14)_with H-2' (d 4.94) of 10 (Kolodziej,
Sakar, Burger, Engelshowe, & Ferreira, 1991). In con-
trast, in 8, in which the lower ent-epicatechin (2'S-con-
®guration) unit of 10 is replaced by epicatechin (2'R-
con®guration), the NOE association of MeO-5 (d 3.48)
with H-2' (d 5.01) was apparently absent. This absol-
ute con®guration of 3 was further con®rmed by reduc-
tive cleavage with sodium cyanoborohydride in TFA
(Steynberg et al., 1997) and the formation of 18 and
20 in addition to 15, 25 and 27.
The presence of a doublet at d 4.67 (d, J=6.5 Hz,
H-2', ring F) in the H NMR spectrum of 4 suggested
1
a 2',3'-trans relative con®guration of the lower ¯ava-
nol moiety. In DIFNOE experiments, NOE associ-
ations similar to those of 3 were observed for 4.
However, NOE associations between H-4 and the pro-
tons of ring E were obviously lacking suggesting a [4±
6] linked dimeric structure. The existence of a [24O4
7], rather than [2 4O 45], ether linkage between the
two ¯avanol units was unequivocally deduced for the
NOE associations of H-8' (d 6.09, s, ring D) with H-2'
(d 4.67, d, J=6.5 Hz, ring F) and H-10 (d 7.17, d, J=
2.4 Hz, ring B). This conclusion was also con®rmed by
the NOE association of the methylene signal H2-4'
(ring F) with the methoxy function at d 3.89 (MeO-5',
ring D) and the absence of a high ®eld shift methoxy
signal in its heptamethyl ether 12. Methylation of 4
also yielded one hexamethyl ether 11, its molecular
weight was shown to be 660 (m/z 661, [M +1]+) by
FAB±MS. The absence of the NOE association of the
methylene signal H2-4' with the methoxy function in
11 decided the structure of this hexamethylated deriva-
tive is unchanged at HO-5'.
By a combination of H- and 13C NMR, HETCOR
1
and COLOC spectral analysis (Fig. 1), the signals of H
and C can be de®nitively assigned (Tables 1±4).
Careful examination of the 13C NMR data of the hep-
tamethylated ethers revealed that there are highly diag-
nostic di€erence in C-4'a, 6' and 8' chemical shifts
that make it possible to distinguish between the [24O
47, 446] and [24O47, 448] double linkage struc-
tures. In 12, a [24O47, 446] double linked structure,
the chemical shift of C-4a', 6' and 8' are at ca. d 107,
112 and 100 respectively, whereas in [24O47, 448]
double linked structures the chemical shifts of these
carbons are at about d 102, 92 and 106 respectively. A
methoxy carbon at 60.8 ppm, which is attributable to
Compound 18 was identi®ed as the tetramethyl
ether of ent-catechin on the basis that its 1H NMR
spectrum and retention time in HPLC on Chiralcel
OD column were identical to those of authentic com-
pound obtained by methylation of 16. Formation of
18 was due to the upper monomeric unit of 10 because
the acetal functional group was cleaved in a predomi-
nantly SN2 manner thus lead to inversion of the con-
®guration at C-2 (Steynberg et al., 1997). Compound
20 was identi®ed as 5, 3', 4'-tri-O-methyl-ent-epicate-
1
chin by H NMR and its molecular weight of 332 as
established by mass spectra. Methylation of 20 with

302
H. Lou et al. / Phytochemistry 51 (1999) 297±308

H. Lou et al. / Phytochemistry 51 (1999) 297±308
303
subsequent chiral HPLC experiments. This straightfor-
ward method led to the unambiguous establishment of
the structure of 4 as epicatechin-(2b4O 47, 4b46)-
catechin and that of 5 as epicatechin-(2b4O47, 4b4
6)-ent-catechin.
The lower terminal unit of 6 was identi®ed as 2', 3'-
cis-¯avan-3-ol by the presence of a singlet at d 4.80.
The same strategy as that outlined above also facili-
tated the determination of the inter¯avanol linkages
and the absolute con®gurations for 6. The NOEs
observed with 6, a high-amplitude positive Cotton
e€ect at short wavelengths, and the diagnostic 13C
NMR spectrum of its heptamethyl ether 14 implied
that 6 is also a [24O47, 446] double-linked dimer
with the same con®guration at upper unit as that of 5.
Direct comparison of the 1H NMR spectrum of 14
with that of the heptamethyl ether of proanthocyani-
din A-6 isolated from Aesculus hippocastanum
(Nonaka, Morimoto, Kinjo, Nohara, & Nishioka,
1987) yielded a relative di€erence of signal pattern,
thus suggested that 6 is a C2', C3' stereoisomer of
proanthocyanidin A-6. Accordingly established the
structure of 6 as epicatechin-(2b 4O 47, 4b 46)-ent-
epicatechin. This absolute structure was con®rmed by
the formation of 18 and 20 after reductive cleavage.
Structure determinations of other product were not
performed.
Fig. 1. NOE associations for determination of [247, 446] double
linkage and COLOC experiments for determination of quaternary
carbons. Arrows other than those labeled NOE correspond to
COLOC correlations.
MeO-5', is another diagnostic feature of [24O47, 4
46] structures. As expected from the unchanged HO-
5' in 11, no signal at about d 60 was apparent for this
compound and the chemical shifts of C-4'a (d 102.82),
6' (d 106.21), and 8' (d 96.42) do not di€er markedly
from those of 4.
Hyaluronidase, as
a
mucosplitting enzyme, is
thought to contribute to many biological functions
(Chambers & Zwerfach, 1947; Cameron, Pauling, &
Leibovitz, 1979; Kakegawa, Masatsumoto, & Satoh,
1992). Its inhibition by compounds 1±6 was assayed
by the modi®ed method of Davidson and Aronson
(1969) yielding IC50 values of 0.35, 0.28, 0.23, 0.14,
0.22 and 0.30 mM respectively. The corresponding
value for epigallocatechin gallate was 0.38 mM. No
marked stereostructure-activity relationship was found
in the inhibition.
The expected NOE between H-3 and H-8' con®rmed
the 3,4-trans relative con®guration Fig. 1. The positive
cotton e€ect at short wavelengths in the CD spectrum
of 4 was consistent with the 2S, 3R, and 4R absolute
con®gurations for the C ring.
With the exception of the relative chemical shift
1
di€erence, the H NMR spectrum of 5 is almost indis-
tinguishable from that of 4, indicating a close struc-
tural resemblance. Identical NOE observations and
highly similar chemical shifts at C-4'a, 6' and 8' for
the heptamethyl ether 13 and compound 12 suggested
the [2 4O 47, 4 46] double linkage structure for 5.
The positive Cotton e€ect at short wavelengths in the
CD spectrum of 5 demonstrated 2S, 3R and 4R absol-
ute con®gurations for the upper terminal unit. Thus 4
and 5 were shown to be stereoisomers at C2' and C3'.
The absolute con®guration of the terminal units of 4
and 5 were then determined by the reductive cleavage
method. From the reaction mixture of 12, compound
18 and 23 along with 15, 28 and 29 were obtained by
PLC. Compound 18 was identi®ed by comparison of
its 1H NMR spectrum and retention time in chiral
HPLC with those of the authentic compound. The
same approach identi®ed 23 as 5,3',4'-tri-O-methylca-
techin after its conversion to 24 by methylation and
These A-type proanthocyanidins from peanuts were
determined to be [24O47, 448] or [24O47, 446]
doubly linked dimers of ¯avan-3-ol. They exhibit the
same absolute con®gurations at C2, C3 and C4. The
isomerization at C2' and C3' of the lower terminal
unit and the di€erent inter¯avanol linkage points gave
rise to the di€erent compounds. In the structure deter-
minations, DIFNOE and CD experiments proved
informative with regard to con®gurations. Comparison
of the di€erence in 13C chemical shifts between
unmethylated dimers and their heptamethylated de-
rivatives allow the [24O47, 448] and [24O47, 44
6] linkages in A-type proanthocyanidins to be readily
di€erentiated. Conversion to monomers by reductive
cleavages followed by chiral HPLC analysis, also
proved to be a powerful approach for stereocenters de-
termination in the terminal units. Tannins occurred in
seed skins are thought to possess fungistatic properties

304
H. Lou et al. / Phytochemistry 51 (1999) 297±308
Table 4
13C NMR data (6 ppm) of methylated derivatives 7±14 (CDCl3, 67.5 MHz)
Carbon
7
8
9
10
99.17
11
99.04
12
99.03
13
99.06
14
99.03
2
99.20
67.56
25.94
98.94
67.47
27.63
99.19
66.63
27.53
3
67.24
27.52
66.73
27.54
67.54
28.19
67.47
28.33
67.53
28.41
4
4a
5
103.73
158.75
92.81
103.73
159.00
93.22
102.62
155.36
96.80
103.89
158.72
92.68
103.50
156.27
93.26
103.45
156.18
93.11
103.57
156.22
93.27
103.43
156.85
93.27
6
7
160.05
93.20
159.99
93.47
160.05
94.47
159.85
93.13
160.17
94.69
160.19
93.40
160.10
93.35
160.12
93.35
8
8a
9
153.52
130.60
110.11
149.75
149.75
111.14
119.55
81.22
152.88
130.83a
110.18
149.98b
149.75b
110.97
119.37
78.24
152.32
130.36a
110.04
148.91b
149.57b
111.48
118.45
79.57
153.11
131.17
110.07
149.70
149.18
111.19
119.47
78.70
152.93
130.13a
110.11
149.44b
150.00b
111.30
119.65
81.76
153.29
129.84
110.06
149.82
149.46
111.28
119.56
81.85
153.09
130.09a
109.95
148.85b
149.39b
111.21
119.53
81.76
153.08
130.47a
109.54
148.85b
149.14b
110.79
118.58
78.43
10
11
12
13
14
2'
3'
4'
67.45
27.51
65.44
28.78
66.31
27.71
66.59
28.14
68.35
27.92
68.39
29.09
68.01
27.92
66.23
28.28
4'a
5'
6'
7'
8'
101.81
157.46
91.96
101.74
157.66
92.13
101.22
157.94
93.15
101.25
157.73
92.25
102.82
154.26
106.21
151.46
96.42
107.88
158.43
112.50
151.60
99.94
107.83
158.68
112.61
151.59
99.98
106.36
158.70
112.86
151.62
100.28
154.12
130.51a
109.99
148.92b
149.77b
111.23
119.55
60.89
151.23
106.00
151.23
130.60
109.91
148.97
148.98
110.81
119.17
56.05
151.67
106.66
151.13
130.33a
109.98
149.74b
149.98b
110.83
119.20
56.06
151.12
105.55
149.88
129.30a
109.36
149.57b
149.88b
110.88
119.47
56.10
151.53
106.50
151.01
130.74
109.39
148.73
148.73
110.87
118.38
56.13
8'a
9'
150.43
130.24a
110.04
149.37b
149.01b
110.94
119.89
55.57
154.44
130.38
110.06
148.87
149.61
110.83
120.12
60.86
154.21
130.40a
109.66
149.39b
149.79b
110.81
119.78
60.84
10'
11'
12'
13'
14'
0Me
55.99(Â2)
55.72
55.50
55.99(Â3)
55.41
55.54
56.06
55.99
56.04
55.99
56.01
56.08(Â3)
56.33
56.06
55.97
56.08
55.99(Â3)
55.68
55.45
55.92(Â3)
55.83
55.51
55.97(Â3)
55.81
55.43
55.95
55.65
55.34
55.95
55.61
55.42
55.20
55.29
55.47
55.36
Complete assignments for 7, 10 and 12 were based on HETCOR and COLOC experiments. The corresponding quarternary carbons of other
a,b
compounds were inferred by direct comparison. Symbol
in same column may be interchanged.
(Karchesy & Hemingway, 1986). However these poly-
phenols might also play an important role in prevent-
ing oxidative damage to seeds and to seed oil.
based on the TMS standard. FAB±MS were per-
formed with a JEOL JMX-HX110 mass spectrometer.
CD spectra were recorded on a Jasco J-720 spectropo-
larimeter. Preparative HPLC was performed with a
Shimazu SCL-10A system on an Intersil packed prep-
ODS column (10Â250 mm, GL Science), with a mix-
ture of methanol and 5% THF in H2O (20:80, v/v) as
mobile phase and detection at 280 nm. A Chiralcel
OD column from Diacel Chemical Co. was used for
the identi®cation of enantiomers with hexane and 2-
propanol (9:1, v/v) as eluting solvents. TLC was per-
formed on precoated aluminum sheets (Rp-18 F254, 0.2
mm, Merck) with 0.1 M sodium acetate bu€er (pH
4.1) and acetonitrile (7:3, v/v) with spots detected by
spraying with anisaldehyde and H2SO4 reagents. PLC
on Kieselgel 60 F254 (0.5 mm, Merck) was performed
3. Experimental
Melting points were determined with a Yanaco
micro-melting point apparatus and are uncorrected.
Optical rotations were measured with a Jasco DIP-140
polarimeter. UV and IR spectra were determined with
a Shimazu UV 240 spectrophotometer and a Jasco
FT/IR-7300 spectrometer respectively. 1H- and 13C
NMR spectra were recorded with a JEOL EX270
1
NMR spectrometer (270 MHz for H NMR, 67.5 Hz
for 13C NMR). Chemical shifts were given in d (ppm),

H. Lou et al. / Phytochemistry 51 (1999) 297±308
305
for preparative purposes. Column chromatography
3.2. Proanthocyanidin A-1 (1)
was performed on Diaion HP-20 (Nippon Rensui),
Sephadex LH-20 (Phamacia Biotech), Toyopearl HW-
40 (®ne grade, Tosoh), Wakogel LP60 C18 and
Wakogel C-300 (Wako Chemical). Sodium cyanoboro-
hydride was obtained from Aldrich. Hyaluronidase
was obtained from bovine testes (790 units/mg solid)
whereas the hyaluronic acid potassium salt came from
human umbilical cord; p-dimethylaminobenzene-alde-
hyde was purchased from Sigma.
Colorless needles (H2O); mp 2808C (decomp); [a]D22
+63.878 (c 1.12, acetone); FAB±MS: m/z 577 [M +
1]+; Anal Calcd for C30H24O12Á2H2O: C 58.82, H
4.57, Found: C 58.35, H 4.43. CD [y ]287 1800, [y]271
22000, [y ]236 +47000sh, [y]221 +73000, [y]208
1
11300; HH NMR (270 MHz; CD3OD) d: Table 1;
13C NMR (67.5 MHz; CD3OD) d: Table 2.
Methylation of each compound was performed with
an excess of CH2N2 in MeOH-Et2O for 1±4 d. After
evaporation of solvent, the methylated derivatives were
puri®ed ®rst by column chromatography on Wakogel
C-300, eluted with benzene±acetone (85:15, v/v), then
by crystallization from a mixture of ethyl acetate and
hexane.
3.3. Heptamethyl ether of proanthocyanidin A-1 (7)
Methylation of 1 (60 mg) under the conditions
described above for 3 d gave, after puri®cation by col-
umn chromatography and crystallization from hexane±
ethyl acetate (3:7, v/v), 7 (32 mg) as a crystalline pow-
der. mp 149±150.68C; FAB±MS: m/z 675 [ M+1]+
;
Anal Calcd for C37H38O12: C 66.06, H 5.39; Found: C
1
66.17, H 5.29; H NMR (270 MHz; CDCl3) d: Table
3; 13C NMR (67.5 MHz, CDCl3) d: Table 4.
3.4. Proanthocyanidin A-2 (2)
3.1. Extraction and isolation of phenolic compounds
Peanut skins (from 279 kg of peanuts) were
extracted twice with boiling water (1000 l), each time
for 2 h. The combined extracts were adsorbed on HP-
20 (400 l), and components were eluted with water and
70% (v/v) aqueous acetone. The eluate with 70%
acetone was concentrated to dryness under reduced
pressure and the residue was then extracted with 95%
(v/v) ethanol (20 l). After evaporation of the solvent
under reduced pressure, the extract (190 g) was further
fractionated by chromatography over Toyopearl HW
40 (bed volume, 1000 ml). Subsequent elution with
mixtures of aqueous ethanol and acetone yielded 12
fractions. Fractions 8 and 10 were obtained with etha-
nol±acetone±water (60:15:25, v/v/v) and 50% (v/v)
acetone respectively, contained mostly phenolic com-
ponents as detected by TLC and showed potent hya-
luronidase inhibitory activities. Fraction 8 (26 g) was
subjected to further chromatography on Sephadex LH
20 (5 Â 60 cm), with elution performed with aqueous
acetone. The fractions eluted with 30% acetone con-
tained polyphenols with ¯avanol moieties, as suggested
by the characteristic orange colorations with anisalde-
hyde±H2SO4 reagents. These were further puri®ed by
repeated chromatography on Wakogel LP60 C18 (3.5
 20 cm) with elution performed with 20% (v/v)
methanol in water. Final puri®cation was achieved by
preparative HPLC on prep-ODS column with a mix-
ture of MeOH±THF±water (20:5:75) as mobile phase.
Compound 1 (960 mg) and 2 (16 mg) were ®nally crys-
tallized as colorless needles from water and compound
3 (300 mg) was obtained as an amorphous powder.
The same procedure was used to purify fraction 10 (18
g) from the Toyopearl HW 40 column to yield com-
pound 4 (340 mg) as colorless needles and compounds
5 (53 mg), 6 (77 mg) as white powders.
Colorless needles (H2O); mp 2738C (decomp); [a]D
+55.638 (c 1.08, acetone); FAB±MS: m/z 577 [M +
1]+; CD [y]287 2500, [y]271 17800, [y]237 +46000sh,
[y]223 +67000, [y ]207 106000; 1H NMR (270 MHz,
CD3OD) d: Table 1; 13C NMR (67.5 MHz, CD3OD)
d: Table 2.
3.5. Heptamethyl ether of proanthocyanidin A-2 (8)
Methylation of 2 (13 mg) as described above for 3
d, puri®cation by column chromatography and crystal-
lization from ethyl acetate yielded 8 (8 mg) as needles.
mp 191±192.68C; FAB±MS: m/z 675 [M + 1]+
;
1H
NMR (270 MHz; CDCl3) d: Table 3; 13C NMR (67.5
MHz; CDCl3) d: Table 4.
3.6. Epicatechin-(2b4O47, 4b48)-ent-epicatechin
(3)
White crystalline powder (H2O); mp 2608C
(decomp); FAB±MS: m/z 577 [M + 1]+; Anal Calcd
for C30H24O12Á2H2O: C 58.82, H 4.57; Found: C
58.73, H 4.63; CD [y ]287 +14200, [y]271 12500, [y]237
1
+43000sh, [y ]221 +238000, [y ]207 202000; H NMR
(270 MHz; CD3OD) d: Table 1; 13C NMR (67.5 MHz;
CD3OD) d: Table 2.
3.7. Hexamethyl ether of epicatechin-(2b4O47, 4b4
8)-ent-epicatechin (9)
Methylation of 3 (60 mg) as described for 24 h, puri-
®cation by column chromatography, and crystalliza-
tion from ethyl acetate±hexane (9:1, v/v)) yielded 9 (40

306
H. Lou et al. / Phytochemistry 51 (1999) 297±308
+
1
mg) as colorless needles. mp 167±1688C; FAB±MS: m/
z 661 [M+1] ; H NMR (270 MHz; CDCl3) d: Table
3; 13C NMR (67.5 MHz; CDCl3) d: Table 4.
was con®rmed by FAB±MS: 333 [M+1] , H NMR
(270 MHz; CDCl3) d: 7.06 (1H, d, J=2.4 Hz, H-10),
7.05 (1H, dd, J=8.0, 2.4 Hz, H-14), 6.92 (1H, d, J=
8.0 Hz, H-13), 6.12 (1H, d, J=2.4 Hz, H-8), 6.07 (1H,
d, J=2.4 Hz, H-6), 4.96 (1H, s, H-2), 4.28 (1H, m, H-
3), 3.91 (3H, s, OMe), 3.89 (3H, s, OMe), 3.79 (3H, s,
OMe), 2.90 (2H, m, H2-4). Methylation of 20 with
CH2N2 in MeOH±Et2O at 108C for 24 h, after evap-
oration of the solvent, yielded 21. FAB±MS: 347 [M+
+
1
3.8. Heptamethyl ether of epicatechin-(2b4O47, 4b4
8)-ent-epicatechin (10)
Methylation of 3 (120 mg) as described above for 4
d yielded 10 (86 mg) as colorless needles after crystalli-
zation from ethyl acetate. mp 156±1578C; FAB±MS:
m/z 675 [M + 1]+; 1H NMR (270 MHz; CDCl3) d:
Table 3; 13C NMR (67.5 MHz; CDCl3) d: Table 4.
+
1
1] ; H NMR (270 MHz; CDCl3) d: 7.07 (1H, d, J=
2.4 Hz, H-10), 7.05 (1H, dd, J = 8.0, 2.4 Hz, H-14),
6.92 (1H, d, J=8.0 Hz, H-13), 6.20 (1H, d, J=2.4 Hz,
H-8), 6.12 (1H, d, J=2.4 Hz, H-6), 4.97 (1H, s, H-2),
4.26 (1H, m, H-3), 3.93 (3H, s, OMe),_3.90 (3H, s,
OMe), 3.80 (3H, s, OMe), 3.78 (3H, s, OMe), 2.92 (m,
H2-4). Chiral HPLC under the conditions described
above for 18 di€erentiated 21 (Rt=49.1 min) from its
enantiomer (Rt=58.2 min).
3.9. Reductive cleavage of 10
Sodium cyanoborohydride (40 mg) was added in
portions over 30 min to a solution of 10 (46 mg) in
TFA under He. After stirring for 1 h, the reaction was
quenched by addition of H2O (20 ml) and the pH was
adjusted to ca. 7 with 3% (w/v) NaHCO3. The mixture
was then extracted three times with ethyl acetate (50,
30 and 30 ml), and the combined extract was stirred
for 15 min with 3 drops of a solution of tetrabutylam-
monium ¯uoride in THF. After drying of the solution
over Na2SO4 and evaporation of the solvent, the mix-
ture (32 mg) was separated by PLC with benzene±
acetone (8:2, v/v) to give four bands: 1 (Rf =0.8, 2.1
mg). 2 (Rf =0.6, 1.6 mg), 3 (Rf =0.43, 6.2 mg), and 4
(Rf =0.32, 3.2 mg). Band 4 was identi®ed as the start-
ing material 10.
Band 3-3, an amorphous powder, was characterized
to be 3',4',5,6-tetra-O-methyl-ent-catechin-[4b 4 8]-
3',4',5-tri-O-methyl-ent-epicatechin (25). FAB±MS: m/
+
1
z 677 [M+1] ; H NMR (270 MHz; CDCl3) d: 7.16
(1H, d, J=1.8 Hz, H-10), 7.14 (1H, dd, J=8.1, 1.8
Hz, H-14), 6.92 (1H, d, J=8.1 Hz, H-13), 6.76 (1H, d,
J = 7.8 Hz, H-13'), 6.73 (1H, d, J = 1.8 Hz, H-10'),
6.59 (1H, dd, J=7.8, 1.8 Hz, H-14'), 6.29 (1H, s, H-
6'), 5.83 (d, J=2.6 Hz, H-8), 5.68 (d, J=2.6 Hz, H-6),
5.13 (1H, s, H-2'), 5.12 (d, J=4.6 Hz, H-2), 4.44 (s,
H-4), 4.27 (m, H-3), 4.09 (1H, m. H-3'), 3.95 (3H, s,
OMe), 3.90 (3H, s, OMe), 3.88 (3H, s, OMe), 3.86
(3H, s, OMe), 3.80(3H, s, OMe), 3.68 (3H, s, OMe),
3.49 (3H, s, OMe), 2.90 (2H, m, H2-4'). Methylation
of 25 with CH2N2 in MeOH±Et2O at 108C for 24 h
yielded, after evaporation of the solvent, 26. A white
amorphous powder; FAB±MS: m/z: 691 [M+1]+; CD
[y]286 +1200, [y]273 8500, [y]213 +112000, [y]200
Band 1 was identi®ed as 3,5-dimethoxyphenol (15)
by its molecular weight of 154 (M+) and 1H NMR
spectrum (270 MHz; CDCl3) d: 6.02 (2H, d, J = 8.3
Hz), 6.08 (1H, t, J=8.3 Hz).
Band 2, which crystallized as needles from ethyl
acetate, was shown to be 3',4',5,7-tetra-O-methyl-ent-
catechin (18). mp 123.5±1258C; FAB±MS: m/z 347 [M
1
+
1
32000; H NMR (270 MHz; CDCl3) d: 7.31 (1H, d, J
+1] ; H NMR (270 MHz; CDCl3) d: 7.00 (1H, dd, J
=8.2, 2.3 Hz, H-14), 6.97 (1H, d, J=2.3 Hz, H-10),
6.90 (1H, d, J=8.2 Hz, H-13), 6.14 (1H, d, J=2.1 Hz,
H-8), 6.11 (1H, d, J=2.1 Hz, H-6), 4.66 (1H, d, J=8.4
Hz, H-2), 4.07 (1H, m, H-3), 3.06 (1H, dd, J=16.2,
5.4 Hz, Ha-4), 2.57 (1H, dd, J=16.2, 8.9 Hz, Hb-4),
3.75 (3H, s, OMe), 3.80 (3H, s, OMe), 3.89 (6H, s, 2Â
OMe); Rt=14.3 min in chiral HPLC on Chiralcel OD
column (5Â250 mm) with hexane-2-propanol (9:1, v/v)
as mobile phase, a ¯ow rate of 1 ml/min, and detection
at 280 nm. It behaved in a manner identical to that of
the authentic specimen. The Rt of its enantiomer was
32.2 min.
Further separation of band 3 on Rp-18 F254 TLC
with methanol±THF±H2O (7:1:3, v/v/v) yielded three
bands: 3-1 (Rf =0.52, 1.8 mg), 3-2 (Rf =0.44, 1.1 mg)
and 3-3 (Rf =0.33, 2.1 mg). Band 3-1, a white amor-
phous powder, was identi®ed as 3',4',5-tri-O-methyl-
ent-epicatechin 20. Its formula composition C18H20O6
=1.8 Hz, H-10), 7.12 (1H, dd, J=7.6, 1.8 Hz, H-14),
6.88 (1H, d, J=7.6 Hz, H-13), 6.76 (1H, d, J=1.8 Hz,
H-10'), 6.75 (1H, d, J=7.8 Hz, H-13'), 6.60 (1H, dd, J
=7.8, 1.8 Hz, H-14'), 6.30 (1H, s, H-6'), 5.91 (1H, d,
J=2.1 Hz, H-8), 5.68 (1H, d, J=2.1 Hz, H-6), 5.14
(1H, d, J=4.6 Hz, H-2), 4.93(1H, s, H-2'), 4.26 (1H, s,
H-4), 4.23 (1H, m, H-3), 4.10 (1H, m. H-3'), 3.92 (3H,
s, OMe), 3.90 (9H, s, OMe), 3.80(3H, s, OMe), 3.64
(3H, s, OMe), 3.52 (3H, s, OMe), 3.39 (3H, s, OMe),
2.88 (2H, m, H2-4').
Band 3-2 was characterized to be 9-methoxy-2,6-
bis(3,4-dimethoxyphenyl)-2,3-cis-6,7-cis-3,4,7,8-tetrahy-
dro-2H,6H-pyrano[2,3-f]chromene (27). A white pow-
+
1
der; FAB±MS: m/z 525 [M+1] ; H NMR (270 MHz;
CDCl3) d: 6.9±7.3 (6H) for the protons of ring B and
E, 6.24 (1H, s, H-8), 4.99 (2H, s, H-2 and H-6), 4.38
(1H, m, H-3), 4.35 (1H, m, H-7), 2.92±3.11 (4H, m,
H2-4 and H2-8 at rings G and F), 3.80 (3H, s, OMe),

H. Lou et al. / Phytochemistry 51 (1999) 297±308
307
3.91 (3H, s, OMe), 3.92 (3H, s, OMe), 3.95 (3H, s,
OMe), 3.96 (9H, s, OMe).
methylation gave 24, a white powder, MS: m/z 347 [M
+
1
+1] ; H NMR (270 MHz; CDCl3) d: same as 18. Rt
was 32.2 min on chiral HPLC under the same con-
ditions as those used for separation of 18.
3.10. Epicatechin-(2b4O47, 4b46)-catechin (4)
Band 4-1 was identi®ed as 3',4',5,7-tetra-O-methyl-
epicatechin-[2b 4 1,4b 4 2]-3-O-methyl-4-[2-hydroxy-3-
Colorless needles (H2O); mp 271±2738C (decomp);
FAB±MS: m/z 577 [M + 1]+; [a]D +10.128 (c 1.05,
acetone); Anal Calcd for C30H24O12ÁH2O: C 60.06, H
4.27; Found: C 59.15, H 4.17; CD [y ]284 3300, [y ]273
11000, [y]47 +48000, [y]220 +29000, [y]208 150000.
1H NMR (270 MHz; CD3OD) d: Table 1; 13C NMR
(67.5 MHz; CD3OD) d: Table 2.
(3,4-dihydroxyphenyl)propyl]-phloroglucinol (28),
white powder, C37H40O12. FAB±MS: m/z 677 [M +
a
+
1
1] . H NMR (270 MHz; CDCl3) d: 7.35 (1H, dd, J=
8.4, 1.8 Hz, H-14), 7.26 (1H, d, J=1.8 Hz, H-10), 6.94
(1H, d, J=8.2 Hz, H-13), 6.74±6.83 (3H, ring E), 6.42
(1H, s, H-8'), 6.37 (1H, d, J=2.0 Hz, H-8), 6.21 (1H,
d, J=2.0 Hz, H-6), 4.82 (1H, d, J=3.5 Hz, H-4), 4.26
(1H, m, H-3), 4.18 (1H, m, H-3'), 3.03 (1H, dd, J=
16.5, 4.6 Hz, Hb-4'), 2.94 (2H, m, H2-2'), 2.92 (1H, dd,
J = 16.5, 7.8 Hz, Ha-4'), 3.78±3.93 (21H, 7Â OMe).
Band 4-2 was identi®ed as 5-methoxy-2,8-bis-{3,4-
dimethoxyphenyl)-2,3-cis-7,8-trans-3,4,6,7-tetrahydro-
2H,8H-pyrano[2,3-f]-chromene (29), a white powder,
FAB±MS: m/z 525 [M + 1]+; 1H NMR (270 MHz;
CDCl3) d: 6.65±7.05 (6H, ring B and E), 6.08 (1H, s,
H-9), 4.65 (1H, s, H-2), 4.60 (1H, d, J=8.4 Hz, H-8),
3.65±3.9 (15H, 5Â OMe), 2.6±3.2 (4H, H2-4 and H2-
6).
3.11. Hexamethyl ether of epicatechin-(2b4O47, 4b
46)-catechin (11)
Methylation of 4 (96 mg) as described for 12 h
a€orded 11 (82 mg). Colorless needles, mp 196±1988C
(ethyl acetate); C36H36O12; FAB±MS: m/z 661 [M +
1]+; 1H NMR (270 MHz; CDCl3) d: Table 3; 13C
NMR (67.5 MHz; CDCl3) d: Table 4.
3.12. Heptamethyl ether of epicatechin-(2b4O47, 4b
46)-catechin (12)
Continued methylation of 11 (60 mg) with CH2N2
in methanol for additional 48 h yielded, after puri®-
cation by column chromatography, 12 (56 mg). A crys-
talline powder, mp 152±1538C (ethyl acetate);
C37H38O12; FAB±MS: m/z 675 [M+1]+; Anal Calcd
for C37H38O12: C 65.87, H 5.64; Found: C 65.72, H
5.68; 1H NMR (270 MHz; CDCl3) d: Table 3; 13C
NMR (67.5 MHz; CDCl3) d: Table 4.
3.14. Epicatechin-(2b4O47, 4b46)-ent-catechin (5)
White amorphous powder (H2O), mp 2628C
(decomp). FAB±MS: m/z 577 [M + 1]+; Anal Calcd
for C30H24O12. 2H2O: C 58.82, H 4.57; Found: C
58.76; H 4.70; CD [y ]283 +9800, [y]273 8900, [y]247
1
+62000, [y ]220 +58000, [y ]208 +90000; H NMR (270
MHz; CD3OD) d: Table 1; 13C NMR (67.5 MHz;
CD3OD) d: Table 2.
3.13. Reductive cleavage of 12
The same procedure as used for the reductive clea-
vage of 10 was employed for the reductive conversion
of 12 (32 mg). The reaction mixture was then separ-
ated by PLC with benzene±acetone (8:2, v/v) to give 5
bands: Band 1 (Rf =0.8, 1.8 mg), band 2 (Rf =0.6, 1.6
mg), band 3 (Rf =0.36, 1.4 mg), band 4 (Rf =0.27, 3.6
mg) and band 5 (Rf =0.15, 3.2 mg) that was identi®ed
as the starting material 12. Band 4 was further separ-
ated on Rp-18 PLC with MeOH±H2O (7:3, v/v) to
give two bands: 4-1 (Rf =0.56, 2.0 mg) and 4-2 (Rf =
0.47, 1.2 mg). Band 1 was characterized as 15 and
3.15. Heptamethyl ether of epicatechin-
(2b4O47, 4b46)-ent-catechin (13)
Methylation of 5 (30 mg) as described for 3 d
yielded 13 (17 mg) as an amorphous powder from
ethyl acetate and hexane; mp 151±1538C. FAB±MS:
m/z 675 [M + 1]+; 1H NMR (270 MHz; CDCl3) d:
Table 3; 13C NMR (67.5 MHz; CDCl3) d: Table 4.
3.16. Epicatechin-(4b46, 2b4O47)-ent-epicatechin
(6)
1
band 2 as 18 by H NMR and chiral HPLC analysis,
respectively, as described above. Band 3 was identi®ed
as 3'4'5-tri-O-methyl-catechin (23). FAB±MS: m/z 333
[M + 1]+; 1H NMR (270 MHz; CD3OD) d: 67±7.0
(3H, protons at ring B), 6.02 (1H, d, J=2.4 Hz, H-7),
5.94 (1H, d, J=2.4 Hz, H-6), 4.63 (1H, d, J=8.6 Hz,
H-2), 4.00 (1H, m, H-3), 3.90 (3H, s, OMe), 3.87 (3H,
s, OMe), 3.79 (3H, s, OMe), 2.83 (1H, dd, J=17, 5.6
Hz, Hb-2), 2.48 (1H, dd, J = 17, 6.0 Hz, Ha-2). Its
White amorphous powder (H2O±MeOH), mp 2808C
(decomp); FAB±MS: m/z 577 [M + 1]+; Anal Calcd
for C30H24O12Á2H2O: C 58.82, H 4.57; Found: C
58.35, H 4.43; CD [y]285 +8400, [y]273 12000, [y]248
+68000, [y]220 +66000, [y]208 +224000; 1H NMR
(270 MHz; CD3OD) d: Table 1; 13C NMR (67.5 MHz;
CD3OD) d: Table 2.

308
H. Lou et al. / Phytochemistry 51 (1999) 297±308
3.19. Heptamethyl ether of epicatechin-
(2b4O47,4b46)-ent-epicatechin (14)
Acknowledgements
Financial support for this research project was pro-
vided by the Creative and Fundamental R&D
Program from Japan Small Business Corporation. We
thank Dr. K. Obuchi for assistance with NMR spec-
tral measurements, Professor T. Tanaka of Nagasaki
University for proanthocyanidin A-1 and A-2,
Professor S. Morimoto of Kyushu University for the
1H NMR spectrum of heptamethyl ether of proantho-
cyanidin A-6.
Methylation of 6 (36 mg) as described for 4 d
yielded 14 (21 mg) as an amorphous powder from
ethyl acetate and hexane; mp 143±1458C; FAB±MS:
m/z 675 [M + 1]+; 1H NMR (270 MHz; CDCl3) d:
Table 3; 13C NMR (67.5 MHz; CDCl3) d: Table 4.
3.18. Reductive cleavage of 14
The same procedure as that used for the cleavage of
10 was used for the cleavage of 14 (18 mg) yielded 17
(2.1 mg) and 18 (1.2 mg). Compound 17 can be con-
verted to 18 by further methylation. Their structures
were determined by direct comparison as described
above.
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control A585 sample A585
Inhibition ꢀ%† ˆ
 100%
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