Studies on the constituents of bark of Parameria laevigata Moldenke

Article abstract of DOI:10.1248/cpb.49.551

One new trimeric proanthocyanidin, epicatechin-(2β→O→7,4β→6)-epicatechin- (2β→O→7, 4β→8)epicatechin (5) and two new tetrameric proanthocyanidins, epicatechin-(2β→O→7, 4β→8)-[epicatechin-(4β6)]epicatechin-(4β→8)- epicatechin, named as parameritannin A-1 (6), and epicatechin-(2β→O→5, 4β→6)[epicatechin-(2β→O→7, 4β→8)]-epicatechin-(4β→8)-epicatechin, named as parameritannin A-2 (7), have been isolated from the bark of Parameria laevigata Moldenke (Apocynaceae) along with the two known dimers, proanthocyanidin A-2 (1) and proanthocyanidin A-6 (2), and two dimers, cinnamtannin B-1 (3) and aesculitannin B (4). These structures were elucidated by spectral and chemical evidence.

Full text of DOI:10.1248/cpb.49.551

May 2001
Chem. Pharm. Bull. 49(5) 551—557 (2001)
551
1)
Studies on the Constituents of Bark of Parameria laevigata MOLDENKE
Kohei KAMIYA,a,b Chiharu WATANABE,a Hanani ENDANG,c Mansur UMAR,c and Toshiko SATAKE*,a,b
Faculty of Pharmaceutical Sciencesa and High Technology Research Center,b Kobe Gakuin University, Nishi-ku, Kobe
651–2180, Japan and Department of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Indonesia,c
Depok-Jawa Barat, Indonesia. Received October 26, 2000; accepted January 18, 2001
One new trimeric proanthocyanidin, epicatechin-(2b→O→7,4b→6)-epicatechin-(2b→O→7, 4b→8)-epicate-
chin (5) and two new tetrameric proanthocyanidins, epicatechin-(2b→O→7, 4b→8)-[epicatechin-(4b→6)]-epi-
catechin-(4b→8)-epicatechin, named as parameritannin A-1 (6), and epicatechin-(2b→O→5, 4b→6)-[epicate-
chin-(2b→O→7, 4b→8)]-epicatechin-(4b→8)-epicatechin, named as parameritannin A-2 (7), have been isolated
from the bark of Parameria laevigata Moldenke (Apocynaceae) along with the two known dimers, proanthocyani-
din A-2 (1) and proanthocyanidin A-6 (2), and two trimers, cinnamtannin B-1 (3) and aesculitannin B (4). These
structures were elucidated by spectral and chemical evidence.
Key words Parameria laevigata; proanthocyanidin; parameritannin; doubly linked structure; thiolytic degradation; Apocy-
naceae
As a continuation of our studies on Jamu and the medici- trum exhibited the presence of an A-type unit from the AB
nal resources in Indonesia, we have investigated the chemical coupling system at d 3.29 and 4.15 (each d, Jϭ3.5 Hz). This
constituents of the bark of Parameria laevigata Moldenke. P. doubly linked structure was also supported from the one
laevigata (Apocynaceae) is widely distributed from southern ketal carbon signal at d 99.95 in the 13C-resonace. Then,
China to Malaysia and Indonesia. It is one of the drug mate- treatment of 3 with benzylmercaptane/acetic acid in EtOH
rials of traditional folk-medicines, called “Jamu” in Indone- yielded proanthocyanidin A-2 4-benzylthioether (3a) and
sia; it is called “Kayu rapet” and has been traditionally used epicatechin (3b).5) The location of interflavonoid linkage was
1
as an antiulcer and antidiarrhea medicine and to treat deduced to be Cb-4/C-8 based on the comparison of the H-
wounds. In the present paper, we describe the isolation and resonances between 3 and degradation products (3a, b).6)
structural elucidation of seven proanthocyanidins including This absolute configuration was also evident from the posi-
three new compounds from the bark of P. laevigata. The tive Cotton effect in the diagnostic wavelength region of the
AcOEt and n-BuOH-soluble portion, obtained from the CD spectrum.7) Consequently, the structure of 3 was deter-
MeOH extract of the bark of P. laevigata, was subjected to mined as epicatechin-(2b→O→7, 4b→8)-epicatechin-(4b→
repeated column chromatography on SiO2, Sephadex LH-20, 8)-epicatechin (cinnamtannin B-1), previously isolated from
and Rp-18 to afford one new trimeric proanthocyanidin (5) the bark of Cinnamomum zeylanicum.8)
and two new tetrameric proanthocyanidins (6, 7), together
with two known dimeric proanthocyanidins (1, 2) and two 3. Furthermore, the 1H- and 13C-NMR spectra were similar to
known trimeric proanthocyanidins (3, 4). those of 3, suggesting it was also a trimeric proanthocyanidin
Compound 4 gave the same molecular formula as that of
Compound 1 and 2 were obtained as pale yellow amor- possessing one doubly linked structure. However, the signifi-
phous powder. The molecular formula of 1 and 2 were deter- cant differences were observed in the chemical shifts of the
mined by the high-resolution (HR) negative-FAB mass spec- heterocyclic protons and carbons (F-ring). In the 13C-NMR
1
trum to be C30H24O12. The H-NMR spectra of 1 and 2 spectrum, the presence of signals at d 84.48 and 73.87 due to
closely resembled each other (Table1); the presence of the C-2 and C-3 of the F-ring, respectively, indicated a catechin
AB coupling systems due to the C-ring protons of A-type middle unit. This was supported from the proton coupling
proanthocyanidins [at d 4.06 and 4.41 (each d, Jϭ3.4 Hz) in constant (J2,3ϭ9.3 Hz). Besides, the 3,4 trans-relative config-
1 and at d 4.08 and 4.29 (each d, Jϭ3.6 Hz) in 2], meta-cou- uration was also evident from the proton coupling constant
pling doublets [at d 6.01 and 6.07 (each d, Jϭ2.4 Hz) in 1 (J3,4ϭ9.3 Hz). Upon consideration of the above results, 4 was
and d 6.09 and 6.04 (each d, Jϭ2.2 Hz) in 2] and the aro- identified as epicatechin-(2b→O→7, 4b→8)-ent-catechin-
matic singlets [at d 6.10 in 1 and d 6.11 in 2] were observed. (4b→8)-epicatechin (aesculitannin B). This absolute struc-
Furthermore, two ABX coupling systems [(d 6.81—7.15) in ture was supported by the positive Cotton effect in the diag-
1 and (d 6.75—7.17) in 2] of B- and E-ring were observed. nostic wavelength region of the CD spectrum.7) This com-
These findings indicated that they were A-type proantho- pound was first isolated from seed shells of Aesculus hip-
cyanidins composed of two 5,7,3Ј,4Ј-tetrahydroxy flavan-3-ol pocastanum L.4)
units. In the 13C-NMR spectrum, the doubly linked structures
Compound 5 showed a molecular formula of C45H34O18
of 1 and 2 were also evident from the characteristic ketal sig- from the HR-negative-FAB-mass spectrum ([MϪH]Ϫ ion at
nals at d 100.19 in 1 and d 100.55 in 2. From the above data, m/z 861.1649), which was two mass units lower than that of
compound 1 and 2 were identified as proanthocyanidin A- 4. In the NMR spectra, two pairs of AB system [one at d
22,3) and proanthocyanidin A-6,4) respectively by direct com- 4.68 and 3.99 (each d, Jϭ3.1 Hz), and the other at d 4.53 and
1
parison of their spectral data with literature.
4.10 (each d, Jϭ3.1 Hz) in H resonance] and two ketal sig-
Compound 3 showed a molecular formula of C45H36O18 nals [at d 99.99 and 100.72 in 13C resonance] were observed.
from the HR-negative-FAB mass spectrum, which was as- These findings suggested that 5 was a trimeric proantho-
sumed to be a trimeric proanthocyanidin. The 1H-NMR spec- cyanidin possessing two doubly linked structures. Further-
To whom correspondence should be addressed. e-mail: satake@pharm.kobegakuin.ac.jp
© 2001 Pharmaceutical Society of Japan

552
Vol. 49, No. 5
Fig. 1. Chemical Structures of Compounds 1—7
from the HR-negative-FAB-mass spectrum ([MϪH]Ϫ ion at
m/z 1151.2450), which was consistent with tetrameric proan-
thocyanidin possessing one doubly linked interflavonoid link-
age. The 13C-NMR spectrum indicated the presence of four
flavan-3-ol units from the signals at d 66.84, 67.40, 71.41
and 72.44, attributable to C-3 of each flavan-3-ol unit, and
one ketal carbon signal at d 100.17 and three carbon signals
at d 76.61, 78.73 and 80.18 due to the C-2 of each flavan-3-
ol unit. To determine the structure and the sequence of each
flavan-3-ol unit, acid catalyzed thiolytic degradation was at-
tempted.5) Treatment of 6 with benzylmercaptane/acetic acid
in EtOH yielded the trimeric proanthocyanidin (6a), epicate-
chin (6c) and two thioethers (6b and 6d) (Chart 1). The
trimeric proanthocyanidin (6a) was identified as 3, and 6b
and 6c were identified as 3a and 3b, which were degradation
products of 3, respectively, and further, 6d was identified as
epicatechin 4-benzylthioether from the spectroscopic data.
This fact indicated that 6 was a coupling product of 3 and
epicatechin. From these results, the two alternative structures
were considered for 6; for one structure it was considered
that epicatechin was attached to C-6 (D-ring) of 3, and for
the other structure it was considered that epicatechin was at-
tached to C-6 (G-ring) of 3. To clarify the position of an in-
terflavonoid linkage between 3 and epicatechin, the correla-
tion spectroscopy via long-range coupling (COLOC) experi-
ment of a nonadecaacetyl derivative (6e) derived from 6 was
attempted and completely assigned all carbons and protons
of 6e. In the COLOC experiment (Fig. 3) of 6e, the position
of the interflavonoid linkage between 3 and epicatechin was
confirmed as C-4/C-6 from the correlations between the pro-
ton signal at d 4.64 (H-4, L-ring) of the remaining epicate-
1
more, the H-NMR spectrum indicated the presence of three
ABX coupling systems (d 6.78—7.42), a pair of meta-cou-
pled protons [d 6.02 and 6.07 (each, d, Jϭ2.4 Hz)], and two
aromatic singlets (d 6.06 and 6.16). By a combination of
1H–1H and H–13C shift correlation spectroscopy (COSY),
1
and the hetero nuclear multiple-bond correlation spec-
troscopy (HMBC) (Fig. 2), a planar structure was established
and the signals of proton and carbon can be definitively as-
signed (Table 2). In the H- and 13C-NMR spectral data, the
1
signal patterns of the lower unit and the heterocyclic F-ring
of 5 were quite close to those of the lower unit and the hete-
rocyclic C-ring of 1, suggesting that the middle and lower
units were the same structurally as 1. Moreover, the upper
unit was determined to be epicatechin from the C-3 carbon
signal at d 68.66 (C-ring). The interflavonoid linkage be-
tween the upper and middle unit was confirmed to be C-4/C-
6 on the basis of the carbon chemical shift for C-6 of the D-
ring (d 108.93). As to the C-6 and C-8 chemical shifts of the
extending unit of A-type proanthocyanidin, the [2b→O→7,
4b→8]-interflavonoid linkage (ca. d 107.0 for C-8) are dis-
tinguished from the [2b→O→7, 4b→6]-interflavonoid link-
age (ca. d 108.8 for C-6).3) Moreover, the positive sign of the
Cotton effect in the diagnostic wavelength region (220—
240 nm) of the CD spectrum indicated b orientation of C-4
flavan-3-ol substituents.7) Thus, the structure of 5 was de-
signed as epicatechin-(2b→O→7, 4b→6)-epicatechin-(2b→
O→7, 4b→8)-epicatechin. As for the trimeric proantho-
cyanidin possessing two doubly bonded interflavonoid link-
ages like compound 5, aesculitannin C and D,4) and pavetan-
nin B7 and B89) have so far been found.
Compound 6 showed a molecular formula of C60H48O24

May 2001
553
Table 1. 1H- and 13C-NMR Spectral Data for Dimeric proanthocyanidins 1
and 2 in CD3OD
proton signal at d 4.25 (C-4, C-ring) was correlated with
quaternary oxygenated carbon signals at d 145.54 and
150.27 (C-7 and C-9, D-ring), and the other at d 4.01 (C-4,
L-ring) was correlated with carbon signals d 149.71 and
145.54 (C-5 and C-7, D-ring). These results indicated that
two flavan-3-ol units possessing two doubly linked structures
were linked to the D-ring. The remaining GHI unit was an
epicatechin from the broad singlet proton signal at d 4.40.
Further correlations were observed by COLOC analysis be-
tween the methine proton signal at d 4.37 (H-4, F-ring) and
quaternary aromatic carbon signals at d 155.85, 109.00 and
155.59 (C-7, 8 and 9, G-ring). Furthermore, in the 13C-NMR
spectrum, carbon signals of the upper and lower units and the
heterocyclic F-ring of 7 were similar to those of 3, suggest-
ing that the sequence of the ABC–DEF–GHI unit of 7 was
the same structure as that of 3. Moreover, 7 showed a positive
Cotton effect in the diagnostic wavelength region (220—
240 nm), indicating b-orientation of each interflavonoid link-
age.7) This result was also supported from the presence of an
upfield shifted H-6Ј (K-ring) proton signal at d 5.94 by
the magnetic anisotropic effect of the aromatic G-ring.6)
Upon consideration of the above results, compound 7 was
determined as epicatechin-(2b→O→5, 4b→6)-[epicatechin-
(2b→O→7, 4b→8)]-epicatechin-(4b→8)-epicatechin (named
as parameritannin A-2).
1
2
Ring
No.
13C
1H
13C
1H
Upper unit
C
C-2 100.19
100.55
C-3 68.07 4.06 (d, 3.4)
C-4 29.26 4.41 (d, 3.4)
C-5 157.00
67.61 4.08 (d, 3.6)
29.70 4.29 (d, 3.6)
155.32
A
C-6 98.33 6.01 (d, 2.4)
C-7 158.12
96.96 6.09 (d, 2.2)
158.12
C-8 96.65 6.07 (d, 2.4)
C-9 154.24
96.62 6.04 (d, 2.2)
154.41
C-10 104.28
104.25
B
C-1Ј 132.46
132.22
C-2Ј 115.68 7.14 (d, 2.2)
C-3Ј 145.65
115.80 7.17 (d, 2.1)
145.74
C-4Ј 146.76
146.79
C-5Ј 116.06 6.81 (d, 8.3)
115.67 6.83 (d, 8.3)
C-6Ј 119.79 7.02 (dd, 8.3, 2.2) 119.96 7.05 (dd, 8.3, 2.1)
Lower unit
F
C-2 81.77 4.92 (br s)
C-3 66.97 4.24 (m)
79.84 4.78 (br s)
67.40 4.14 (m)
C-4 29.89 2.95 (dd, 17.2, 4.9) 29.54 2.90 (dd, 17.0, 4.5)
2.76 (dd, 17.2, 2.3) 2.79 (dd, 17.0, 3.0)
D
E
C-5 156.59
C-6 96.53 6.10 (s)
C-7 152.29
C-8 107.23
C-9 152.12
155.71
108.83
152.78
96.96 6.11 (s)
151.73
The proanthocyanidins possessing a branched chain like 6
and 7 were reported at first from natural sources.
C-10 102.45
C-1Ј 131.19
103.07
132.07
Experimental
General Procedures and Plant Material Optical rotations were mea-
sured using a Jasco DIP-1000 digital polarimeter. CD spectra were recorded
on a Jasco J-725 spectrometer. HR-FAB-MS were performed with a JEOL
JMS-BU 20 spectrometer. IR and UV spectra were measured on a Shimadzu
FT-IR 8300 infrared spectrometer and a Hitachi U-3000 spectrometer, re-
spectively. The NMR spectra were recorded in CD3OD or CDCl3, on a
Bruker DPX-400 instrument. TLC was performed on Merck precoated TLC
plates (Kieselgel 60F254, Rp-18F254). Column chromatography was con-
ducted with Kieselgel 60 (70—230 Mesh, Merck) and Sephadex LH-20
(Pharmacia). Medium pressure liquid chromatography (MPLC, micro pump
KP-7, Kusano Scientific Co., Tokyo) was carried out on a CIG column
[ODS (C-18)]. The bark of P. laevigata was purchased from JAMU factory
in Jakarta and the plant was identified by Dr. Asmanizar, University of In-
donesia. The herbarium specimen has been deposited at the Botanical Mu-
seum of Kobe Gakuin University.
C-2Ј 115.96 7.15 (d, 2.1)
C-3Ј 145.99
C-4Ј 146.30
C-5Ј 115.64 6.81 (d, 8.2)
C-6Ј 120.40 6.98 (d, 8.2, 2.1)
115.25 6.95 (d, 1.7)
145.64
145.89
115.91 6.75 (d, 7.8)
119.39 6.77 (dd, 7.8, 1.7)
All assignments are based on the HH-COSY, CH-COSY and COLOC spectral data.
Coupling patterns and coupling constants (J) in Hz are given in parentheses.
chin and carbon signals at 148.95 (C-5) and 147.71 (C-7) in
the D-ring. The CD spectrum of 6 revealed a high-amplitude
positive Cotton effect in the diagnostic wavelength region
(220—240 nm), reflecting b-orientation of 4-flavanyl sub-
stituents.7) Accordingly, the structure of 6 was characterized
as epicatechin-(2b→O→7, 4b→8)-[epicatechin-(4b→6)]-
epicatechin-(4b→6)-epicatechin (named as parameritannin
A-1).
Compound 7 showed a molecular formula of C60H46O24
from the HR-negative-FAB mass spectrum ([MϪH]Ϫ ion at
m/z 1149.2290), which was 2 mass units lower than that of 6.
Furthermore, the presence of the H resonances of two AB
coupling systems [one at d 3.45 and 4.25 (each, d, Jϭ
3.5 Hz), and the other at d 3.83 and 4.05 (each, d, Jϭ3.6 Hz)]
Extraction and Isolation The dried bark of P. laevigata (4.7 kg) was
extracted with hot MeOH (7h, 12 lϫ7). The solvent was evaporated off
under reduced pressure to yield MeOH extract (522 g). The MeOH extract
was suspended in a MeOH–H2O (1 : 3 l) mixture and was extracted succes-
sively with CHCl3, AcOEt, and n-BuOH (each 4 lϫ3 times). Each solvent
was evaporated off under reduced pressure to yield CHCl3 (89 g), AcOEt
(65 g), n-BuOH (239 g), and H2O (123 g) extract. The AcOEt extract was
chromatographed on silica gel using a gradient of AcOEt, MeOH and H2O
system to give catechin-containing fractions. These fractions were subjected
repeatedly to Sephadex LH-20 column chromatography using MeOH–H2O
(2 : 1) and to SiO2 column chromatography using CHCl3–MeOH (4 : 1) and
finally were purified by CC on Rp-18 using MeOH–H2O (1 : 3) to give com-
1
and 13C resonances of two ketal carbons at d 100.80 and pounds 1 (150 mg), 2 (35 mg), 3 (3000 mg), 4 (35 mg), and 5 (60 mg). The
n-BuOH extract was chromatographed on silica gel using a gradient of
100.23 were indicated, suggesting it to be a tetrameric proan-
AcOEt, MeOH and H2O system to give catechin-containing fractions. These
fractions were subjected repeatedly to Sephadex LH-20 column chromatog-
raphy using MeOH–H2O (2 : 1) and to SiO2 column chromatography using
CHCl3–MeOH (4 : 1) and finally were purified by CC on Rp-18 using
thocyanidin possessing two doubly linked structures. In the
13C-NMR spectrum, the presence of four flavan-3-ol units
were indicated by the signals at d 66.43, 72.41, 67.57, and
MeCN–H2O (1 : 7) to give compounds 6 (800 mg) and 7 (30 mg).
67.96, each attributable to the C-3 in the heterocyclic ring
(C-, F-, I-, and L-ring), respectively. By the combination of
the HH- and CH-COSY and COLOC spectral analysis, all
carbon and proton signals could be definitively assigned
(Table 3). In the COLOC spectrum (Fig. 4), one AB doublet
Proanthocyanidin A-2 (1) Pale yellow amorphous powder; [a]D21
ϩ58.9° (MeOH, cϭ0.75); HR-negative-FAB-MS: m/z [MϪH]Ϫ 575.1227
(Calcd for C30H23O12: 575.1189); CD: [q]241 ϩ27300, [q]222 ϩ62600, [q]206
KBr
max
Ϫ168400, [q]196 Ϫ10500, [q]188 Ϫ32400sh; IR n
cmϪ1: 3219, 1616,
MeOH
1522, 1448, 1286, 1069; UV l
nm (log e): 207 (4.90), 225sh (4.58),
max

554
237sh (4.32), 280 (4.09); H- and 13C-NMR (400 MHz, CD3OD): see Table and 13C-NMR (400 MHz, CD3OD): see Table 2.
Vol. 49, No. 5
1
1.
Thiolytic Degradation of 3 Compound 3 (200 mg) was treated with
benzylmercaptane (1.3 ml) and acetic acid (2 ml) in ethanol (8 ml) under re-
flux for 40 h. The reaction mixture was chromatographed on Sephadex LH-
Proanthocyanidin A-6 (2) Pale yellow amorphous powder; [a]D21
ϩ17.3° (MeOH, cϭ1.68); HR-negative-FAB-MS: m/z [MϪH]Ϫ 575.1189
(Calcd for C30H23O12: 575.1189); CD: [q]247 ϩ49700, [q]237 ϩ27200sh, 20 using MeOH to give proanthocyanidin A-2 4-benzylthioether (3a)
cmϪ1
:
(45 mg) and epicatechin (3b) (6 mg).
KBr
[q]228 ϩ5900, [q]220 ϩ27200, [q]202 Ϫ81000, [q]188 ϩ19400; IR n
max
MeOH
3275, 1609, 1522, 1437, 1287, 1054; UV l
nm (log e): 223 (4.60),
Proanthocyanidin A-2 4-benzylthioether (3a) Colorless amorphous
max
280sh (4.05); 1H- and 13C-NMR (400 MHz, CD3OD): see Table 1.
powder; [a]D21 ϩ96.6° (MeOH, cϭ1.68); H-NMR (400 MHz, CD3OD) d:
1
Cinnamtannin B-1 (3) Pale yellow amorphous powder; [a]D21 ϩ69.2° 4.06 [1H, d, Jϭ3.3 Hz, H-3 (C)], 4.38 [1H, d, Jϭ3.3 Hz, H-4 (C)], 6.01 [1H,
(MeOH, cϭ0.99); HR-negative-FAB-MS: m/z [MϪH]Ϫ 863.1824 (Calcd for d, Jϭ1.7 Hz, H-6 (A)], 6.08 [1H, d, Jϭ1.7 Hz, H-8 (A)], 7.17 [1H, d,
C45H35O18: 863.1822); CD: [q]242 ϩ105600sh, [q]230 ϩ152800, [q]221
Jϭ2.0 Hz, H-2Ј (B)], 6.83 [1H, d, Jϭ8.2 Hz, H-5Ј (B)], 6.95 [1H, dd, Jϭ8.2,
2.0 Hz, H-6Ј (B)], 5.29 [1H, br s, H-2 (F)], 3.92 [1H, br d, Jϭ2.0 Hz, H-3
(F)], 4.05 [1H, br d, Jϭ2.0 Hz, H-4 (F)], 6.11 [1H, s, H-6 (D)], 7.13 [1H, d,
Jϭ2.1 Hz, H-2Ј (E)], 6.81 [1H, d, Jϭ8.3 Hz, H-5Ј (E)], 7.02 [1H, dd, Jϭ8.3,
2.1 Hz, H-6Ј (E)], 3.97 [2H, s, S-CH2], 7.40 [2H, br d, Jϭ7.3 Hz, H-2Љ, 6Љ
(Benzyl SH)], 7.29 [2H, br t, Jϭ7.3 Hz, H-3Љ, 5Љ (Benzyl SH)], 7.20 [1H,
br t, Jϭ7.3 Hz, H-4Љ (Benzyl SH)]; 13C-NMR (400 MHz, CD3OD) d: 100.10
[C-2 (C)], 68.03 [C-3 (C)], 29.25 [C-4 (C)], 157.10 [C-5 (A)], 98.28 [C-6
(A)], 158.23 [C-7 (A)], 96.60 [C-8 (A)], 154.12 [C-9 (A)], 103.91 [C-10
(A)], 132.32 [C-1Ј (B)], 116.14 [C-2Ј (B)], 145.94 [C-3Ј (B)], 146.73 [C-4Ј
(B)], 116.07 [C-5Ј (B)], 120.52 [C-6Ј (B)], 77.43 [C-2 (F)], 71.22 [C-3 (F)],
44.31 [C-4 (F)], 157.45 [C-5 (D)], 96.97 [C-6 (D)], 153.72 [C-7 (D)],
107.11 [C-8 (D)], 152.01 [C-9 (D)], 102.48 [C-10 (D)], 130.95 [C-1Ј (E)],
115.55 [C-2Ј (E)], 145.61 [C-3Ј (E)], 146.35 [C-4Ј (E)], 115.63 [C-5Ј (E)],
119.74 [C-6Ј (E)], 38.04 (CH2S), [140.14, 130.03, 129.47, and 127.97 (C-1,
C-2, 6, C-3, 5, and C-4 of benzyl SH ring)].
cmϪ1: 3275, 1609,
KBr
ϩ97000sh, [q]208 Ϫ270000, [q]199 ϩ79800; IR n
max
1522, 1437, 1287, 1054; UV lMmaexOH nm (log e): 223 (4.60), 280sh (4.05); 1H-
Epicatechin (3b) Pale yellow amorphous powder; [a]D21 Ϫ53.8°
(MeOH, cϭ1.03); 1H-NMR (400 MHz, CD3OD) d: 4.82 (1H, br s, H-2),
4.19 (1H, m, H-3), 2.87 (1H, dd, Jϭ16.8, 4.6 Hz, H-4), 2.73 (1H, dd,
Jϭ16.8, 2.9 Hz, H-4), 5.94 (1H, d, Jϭ2.3 Hz, H-6), 5.97 (1H, d, Jϭ2.3 Hz,
H-8), 6.98 (1H, d, Jϭ1.9 Hz, H-2Ј), 6.76 (1H, d, Jϭ8.4 Hz, H-5Ј), 6.81 (1H,
dd, Jϭ8.4, 1.9 Hz, H-6Ј); 13C-NMR (400 MHz, CD3OD) d: 79.88 (C-2),
67.49 (C-3), 29.26 (C-4), 158.00 (C-5), 96.38 (C-6), 157.67 (C-7), 95.88 (C-
8), 157.37 (C-9), 100.06 (C-10), 132.28 (C-1Ј), 115.32 (C-2Ј), 145.78 (C-
3Ј), 145.95 (C-4Ј), 115.88 (C-5Ј), 119.39 (C-6Ј).
Aesculitannin B (4) Pale yellow amorphous powder; [a]D21 ϩ125.3°
(MeOH, cϭ1.92); HR-negative-FAB-MS: m/z [MϪH]Ϫ 863.1800 (Calcd for
Fig. 2. HMBC Correlations of Compound 5
Chart 1. Thiolytic Degradation of Compound 6

May 2001
555
Table 2. 1H- and 13C-NMR Spectral Data for Trimeric Proanthocyanidins 3, 4 and 5 in CD3OD
3
4
5
Ring
No.
13C
1H
13C
1H
13C
1H
Upper unit
C
C-2
C-3
C-4
99.95
67.17
28.86
100.11
67.05
28.91
99.99
68.66
29.78
3.29 (d, 3.5)
4.15 (d, 3.5)
3.31 (d, 3.5)
3.95 (d, 3.5)
3.99 (d, 3.1)
4.68 (d, 3.1)
A
C-5
C-6
C-7
C-8
156.74
98.33
157.80
96.60
156.49
97.86
157.95
96.48
158.14
97.86
157.80
96.43
5.97 (d, 2.4)
6.02 (d, 2.4)
5.85 (d, 2.3)
6.00 (d, 2.3)
6.02 (d, 2.4)
6.07 (d, 2.4)
C-9
154.14
104.96
132.44
115.75
145.46
146.59
116.18
119.90
153.93
104.06
132.24
115.68
145.21
145.50
115.98
119.82
154.36
103.70
132.70
115.42
146.04
146.72
115.65
119.63
C-10
C-1Ј
C-2Ј
C-3Ј
C-4Ј
C-5Ј
C-6Ј
B
7.03 (d, 2.0)
7.02 (d, 2.1)
7.09 (d, 2.2)
6.84 (d, 8.1)
6.86 (dd, 8.1, 2.0)
6.83 (d, 8.3)
6.86 (dd, 8.3, 2.1)
6.78 (d, 8.3)
6.98 (dd, 8.3, 2.2)
Middle unit
F
C-2
C-3
C-4
78.86
72.55
38.27
5.70 (br s)
4.13 (br d, 1.4)
4.56 (br s)
84.48
73.87
39.04
4.62 (d, 9.3)
4.57 (t, 9.3)
4.51 (d, 9.3)
100.72
68.09
29.83
4.10 (d, 3.1)
4.53 (d, 3.1)
D
C-5
C-6
C-7
C-8
155.76
96.09
155.37
97.21
149.07
108.93
155.98
98.47
153.36
105.95
132.52
115.52
145.63
146.89
115.74
120.24
5.80 (s)
5.80 (s)
151.08
106.42
151.78
106.73
131.76
116.72
145.89
146.27
115.79
121.36
151.20
106.86
152.21
108.93
131.06
116.52
146.10
146.68
116.34
121.14
6.16 (s)
C-9
C-10
C-1Ј
C-2Ј
C-3Ј
C-4Ј
C-5Ј
C-6Ј
E
7.32 (d, 2.0)
7.20 (d, 2.0)
7.42 (d, 2.1)
6.82 (d, 8.2)
7.19 (dd, 8.2, 2.0)
6.90 (d, 8.2)
7.15 (dd, 8.2, 2.0)
6.88 (d, 8.9)
7.41 (dd, 8.9, 2.1)
Lower unit
I
C-2
C-3
C-4
80.27
65.71
29.84
4.38 (br s)
3.85 (br t, 3.8)
2.83 (m)
79.61
67.61
30.07
4.37 (br s)
81.40
67.10
30.01
4.93 (br s)
4.26 (m)
2.96 (dd, 17.2, 4.8)
2.81 (dd, 17.2, 1.8)
4.07 (br d, 3.5)
2.87 (dd, 17.0, 4.8)
2.77 (br d, 17.0)
G
H
C-5
C-6
C-7
C-8
155.99
96.51
156.19
96.57
156.64
96.23
6.10 (s)
6.08 (s)
6.06 (s)
155.53
108.85
155.78
100.08
133.17
115.48
145.31
145.74
116.03
119.45
156.16
108.55
155.26
100.97
132.92
115.27
145.87
146.65
115.68
119.26
152.68
107.75
151.76
102.46
131.52
115.67
145.65
146.65
116.07
120.02
C-9
C-10
C-1Ј
C-2Ј
C-3Ј
C-4Ј
C-5Ј
C-6Ј
6.82 (d, 1.7)
6.98 (d, 1.8)
7.17 (d, 2.0)
6.76 (d, 8.2)
6.72 (dd, 8.2, 1.7)
6.80 (d, 8.3)
6.88 (dd, 8.3, 1.8)
6.82 (d, 8.2)
7.02 (dd, 8.2, 2.0)
All assignments are based on the HH-COSY, CH-COSY and COLOC spectral data. Coupling patterns and coupling constants (J) in Hz are given in parentheses.
KBr
C45H35O18: 863.1822); CD: [q]241 ϩ151900sh, [q]232 ϩ203400, [q]224 ϩ146400, [q]211 Ϫ218500, [q]198 ϩ114200; IR n
cmϪ1: 3204, 1607,
max
KBr
max
MeOH
max
1
ϩ149800sh, [q]208 Ϫ193600, [q]200 ϩ51100, [q]189 Ϫ79800; IR n
cmϪ1
:
1518, 1449, 1283, 1067; UV l
nm (log e): 218 (5.00), 281 (4.24); H-
MeOH
max
3244, 1609, 1522, 1447, 1285, 1076; UV l
nm (log e): 208 (5.07),
NMR (400 MHz, CD3OD): d 4.0—5.7 (H-2 of both extending and terminat-
234sh (4.67), 281 (4.09); H- and 13C-NMR (400 MHz, CD3OD): see Table ing units), d 3.3—4.1 (H-3 of both extending and terminating units), d
1
2.
2.7—2.9 (H-4 of terminating unit), d 4.2—4.5 (H-4 of extending unit), d
5.8—6.2 (H-6 and -8 of A, G and J-ring), d 6.6—7.3 (H-2Ј, 5Ј and 6Ј of B,
Epicatechin-(2b→O→7, 4b→6)-epicatechin-(2b→O→7, 4b→8)-epi-
catechin (5) Pale yellow amorphous powder; [a]D21 ϩ184.9° (MeOH, E, H and K-ring); 13C-NMR (400 MHz, CD3OD) d: 100.17, 78.73, 80.18
cϭ1.08); HR-negative-FAB-MS: m/z [MϪH]Ϫ 861.1649 (Calcd for and 76.61 (C-2 of C, F, I and L-ring, respectively), 66.84, 72.44, 67.40 and
C45H33O18: 863.1666); CD: [q]227 ϩ474200, [q]208 Ϫ461400, [q]196 71.41 (C-3 of C, F, I and L-ring, respectively), 28.90, 38.38, 29.72 and 37.64
KBr
max
MeOH
max
Ϫ113700; IR n
cmϪ1: 3291, 1622, 1522, 1437, 1286, 1069; UV l
(C-3 of C, F, I and L-ring, respectively), 98.34, 107.77, 96.68 and 96.84 (C-6
nm (log e): 213 (4.88), 226sh (4.80), 280 (4.09); 1H- and 13C-NMR of A, D, G and J-ring), 96.55, 106.94, 108.84 and 96.17 (C-8 of A, D, G and
(400 MHz, CD3OD): see Table 2.
J-ring), 104.92, 107.24, 100.01 and 99.41 (C-10 of A, D, G and J-ring),
Parameritannin A-1 (6) Pale yellow amorphous powder; [a]D21 ϩ50.1° 115.47, 115.82, 115.93, 116.00, 116.15, 116.17, 116.24 and 116.76 (C-2Ј
(MeOH, cϭ1.33); HR-negative-FAB-MS: m/z [MϪH]Ϫ 1151.2450 (Calcd and 5Ј of B, E, H and K-ring), 119.28, 119.95, 120.69 and 121.43 (C-6Ј of
for C60H47O24: 1151.2456); CD: [q]243 ϩ135600, [q]237 ϩ152800, [q]233 B, E, H and K-ring), 131.663, 131.75, 132.32 and 132.85 (C-1Ј of B, E, H

556
Vol. 49, No. 5
Fig. 4. COLOC Correlations of Compound 7
Fig. 3. COLOC Correlations of Compound 6e
Table 3. 1H- and 13C-NMR Spectral Data for Tetrameric Proanthocyanidins 6e in CDCl3 and 7 in CD3OD
6e
7
6e
7
Ring
No.
Ring
No.
13C
1H
13C
1H
13C
1H
13C
1H
Upper unit
C
Lower unit
I
C-2
C-3
C-4
98.82
67.84 5.12 (d, 4.2)
27.61 4.74 (d, 4.2)
100.80
66.43 3.45 (d, 3.5)
28.86 4.25 (d, 3.5)
C-2
C-3
C-4
76.79 5.10 (br s)
66.36 5.46 (m)
26.26 2.98 (m)
80.56 4.40 (br s)
67.57 3.85 (m)
29.97 2.94 (dd, 17.4, 4.5)
2.88 (dd, 17.2, 1.6)
156.32
97.41 6.09 (s)
155.85
109.00
A
B
C-5 148.87
C-6 110.2
C-7 150.13
C-8 106.59 6.80 (d, 2.2)
C-9 153.38
157.76
G
H
C-5 148.63
C-6 110.98 6.51 (s)
C-7 147.36
C-8 118.24
C-9 151.75
6.58 (d, 2.2)
98.69 5.92 (d, 2.4)
157.90b)
96.78 6.04 (d, 2.4)
153.66
104.19
155.59
C-10 113.46
C-1Ј 134.44
C-10 110.29
C-1Ј 135.12
100.20c)
131.24
132.89
C-2Ј 122.57 7.43 (d, 2.0)
115.62 7.12 (d, 2.2)
145.68
147.01
C-2Ј 121.47 7.10 (d, 1.8)
115.29 6.81 (d, 1.9)
145.74
145.45
C-3Ј 141.35a)
C-3Ј 142.02a)
C-4Ј 141.63a)
C-4Ј 142.47a)
C-5Ј 125.05 7.14 (d, 8.5)
116.11 6.87 (d, 8.4)
C-5Ј 122.70 7.12 (d, 8.4)
116.02 6.78 (d, 8.2)
C-6Ј 125.29 7.50 (dd, 8.5, 2.0) 119.77 6.94 (dd, 8.4, 2.2)
C-6Ј 123.13 7.07 (dd, 8.4, 1.8) 119.10 6.67 (dd, 8.2, 1.9)
Middle unit
F
Branched unit
L
C-2
C-3
C-4
75.24 5.45 (br s)
69.66 5.14 (br t, 1.7)
33.30 4.37 (br s)
78.78 5.59 (br s)
72.14 4.09 (br d, 1.6)
38.81 4.50 (br t, 1.6)
149.71
C-2
C-3
C-4
74.09 5.67 (br s)
69.80 5.54 (br t, 1.7)
32.61 4.64 (br s)
100.23c)
67.96 3.80 (d, 3.6)
29.45 4.01 (d, 3.6)
156.20
D
C-5 148.95
J
C-5 150.18
C-6 114.02
C-7 147.71
107.04
145.54
C-6 107.65 6.72 (d, 2.2)
C-7 149.76
98.42 5.81 (d, 2.4)
157.87b)
C-8 108.73
C-9 151.48
108.08
150.27
C-8 107.15 6.62 (d, 2.2)
C-9 154.35
96.57 5.93 (d, 2.4)
154.06
C-10 109.01
107.64
C-10 109.19
103.87
E
C-1Ј 134.56
131.52
K
C-1Ј 136.84
131.85
C-2Ј 124.38 7.29 (d, 2.0)
116.66 7.29 (d, 2.0)
145.81
146.26
C-2Ј 121.95 7.43 (d, 2.0)
116.54 6.92 (d, 2.2)
144.73
146.19
C-3Ј 141.65a)
C-3Ј 142.50a)
C-4Ј 141.72a)
C-4Ј 143.09a)
C-5Ј 122.77 7.13 (d, 8.4)
116.11 6.80 (d, 8.3)
C-5Ј 123.20 7.19 (d, 8.6)
115.96 6.50 (d, 8.3)
C-6Ј 125.97 7.26 (dd, 8.4, 2.0) 121.29 7.17 (dd, 8.3, 2.0)
C-6Ј 125.21 7.56 (dd, 8.6, 2.0) 119.60 5.94 (dd, 8.3, 2.2)
All assignments are based on the HH-COSY, CH-COSY and COLOC spectral data. Coupling patterns and coupling constants (J) in Hz are given in parentheses. Symbol a—c)
in each column may be interchanged.
and K-ring), 145.44, 145.56, 145.80, 145.90, 146.30, 146.32, 146.37 and
146.74 (C-3Ј and 4Ј of B, E, H and K-ring), 148.44, 150.30, 150.90, 154.24,
155.47, 155.58, 156.08, 156.73, 157.89, 157.93, 159.31 and 159.44 (C-5, 7
and 9 of A, D, G and J-ring).
Thiolytic degradation of 6 Compound 6 (100 mg) was treated with
benzylmercaptane (1.3 ml) and acetic acid (2 ml) in ethanol (8 ml) under re-
flux for 45 h. The reaction mixture was chromatographed on Sephadex LH-
20 using MeOH to give cinnamtannin B-1 (6aϭ3, 6.4 mg), epicatechin 4-
benzylthioether (6d, 21.8 mg), proanthocyanidin A2-4-benzylthioether
(6bϭ3a, 10.7 mg) and epicatechin (6cϭ3b, 7.4 mg).
Epicatechin 4-benzylthioether (6d) Colorless amorphous powder;
1
[a]D21 Ϫ22.7° (MeOH, cϭ1.09); H-NMR (400 MHz, CD3OD) d: 7.41 (2H,
dd, Jϭ7.4, 1.3 Hz, H-2Љ, 6Љ), 7.30 (2H, t, Jϭ7.4 Hz, H-3Љ, 5Љ), 7.21 (1H, tt,
Jϭ7.4, 1.3 Hz, H-4Љ), 6.93 (1H, d, Jϭ2.0 Hz, H-2Ј), 6.75 (1H, d, Jϭ8.1 Hz,

May 2001
557
for Kobe Gakuin University Joint Research (B). The authors are grateful to
Dr. K. Hori, Akita Research Institute of Food and Brewing, for measure-
ments of HR-FAB-MS, and Misses T. Yano and E. Atobe, Kobe Gakuin Uni-
versity, for their cooperation in experiments.
H-5Ј), 6.68 (1H, dd, Jϭ8.1, 2.0 Hz, H-6Ј), 5.96 (1H, d, Jϭ2.3 Hz, H-6), 5.89
(1H, d, Jϭ2.3 Hz, H-8), 5.22 (br s, H-2), 4.05 (1H, d, Jϭ2.4 Hz, H-4), 3.96
(2H, s, S-CH2), 3.85 (1H, br d, Jϭ2.4 Hz, H-3); 13C-NMR (400 MHz,
CD3OD) d: 75.58 (C-2), 71.65 (C-3), 43.93 (C-4), 159.10 (C-5), 96.83 (C-
6), 158.90 (C-7), 95.76 (C-8), 157.29 (C-9), 100.19 (C-10), 132.08 (C-1Ј),
115.27 (C-2Ј), 145.75 (C-3Ј), 145.97 (C-4Ј), 115.92 (C-5Ј), 119.20 (C-6Ј),
140.44 (C-1Љ), 130.03 (C-2Љ, 6Љ), 129.50 (C-3Љ, 5Љ), 127.92 (C-4Љ), 37.92 (S-
CH2).
References
1) Part 4 in the series “Studies on Jamu and the Medicinal Resources in
Indonesia.” Part 1: Phytochemistry, 50, 303—306 (1999), Part 2:
Chem. Pharm. Bull., 47, 1444—1447 (1999), Part 3: Phytochemistry,
in press.
2) Mayer W., Goll L., Arndt E. V., Mannschreck A., Tetrahedron Lett.,
1966, 429—435.
Acetylation of 6 Compound 6 (200 mg) was acetylated with Ac2O–
pyridine and the product was purified by column chromatography on
Sephadex LH-20 using CHCl3–MeOH (2 : 1) to yield nonadecaacetate 6e
1
(200 mg). 6e: Colorless amorphous powder; H- and 13C-NMR (400 MHz,
3) Lou H., Yamazaki Y., Sasaki T., Uchida M., Tanaka H., Oka S., Phyto-
chemistry, 51, 297—308 (1999).
4) Morimoto S., Nonaka G., Nishioka I., Chem. Pharm. Bull., 35, 4717—
4729 (1987).
5) Thompson R. S., Jacques D., Haslam E., Tanner R. J. N., J. Chem. Soc.
Perkin Trans. I, 1972, 1387—1399.
6) Tanaka N., Orii R., Ogasa K., Wada H., Murakami T., Saiki Y., Chen
C., Chem. Pharm. Bull., 39, 55—59 (1991).
7) Botha J. J., Young D. A., Ferreira D., Roux D. G., J. Chem. Soc. Perkin
Trans. I, 1981, 1213—1219.
8) Nonaka G., Morimoto S., Nishioka I., J. Chem. Soc. Perkin Trans. I,
1983, 2139—2145.
9) Balde A. M., Bruyne T. D., Pieters L., Kolodziej H., Berghe D. V.,
Claeys M., Vlietinck A., Phytochemistry, 38, 719—723 (1995).
CDCl3): see Table 3, methyl protons of –COCH3 d: 2.30, 2.29, 2.28, 2.27
(ϫ2), 2.26 (ϫ2), 2.25, 2.23 (ϫ2), 2.20, 2.04, 2.01, 1.80, 1.75, 1.74, 1.71,
1.55, 1.48 (each 3H, s), carbonyl carbons of –COCH3 d: 170.13, 170.03,
169.81, 169.27, 169.02, 168.49, 168.46, 168.34, 168.33, 168.16, 168.07,
168.05, 167.93, 167.89, 167.88, 167.85, 167.74, 167.49, 167.16, methyl car-
bons of –COCH3 d: 21.13, 21.04, 20.93, 20.75, 20.69, 20.60, 20.58, 20.56,
20.54 (ϫ2), 20.52, 20.36, 20.32, 20.29, 20.25, 20.16, 20.01, 19.92, 19.40.
Parameritannin A-2 (7) Pale yellow amorphous powder; HR-negative-
FAB-MS: m/z [MϪH]Ϫ 1149.2290 (Calcd for C60H45O24: 1149.2300); CD:
[q]255 Ϫ22700, [q]241 ϩ116700, [q]230 ϩ64000, [q]220 ϩ101300, [q]207
KBr
Ϫ264900, [q]197 ϩ61800; IR n
cmϪ1: 3369, 1614, 1522, 1447, 1286,
max
MeOH
1
1065; UV l
nm (log e): 212 (4.97), 231sh (4.79), 280 (4.16); H- and
max
13C-NMR (400 MHz, CD3OD): see Table 3.
Acknowledgments This work was supported in part by a Grant-in-Aid