Proanthocyanidins and a phloroglucinol derivative from Rumex acetosa L.

Article abstract of DOI:10.1016/j.fitote.2009.08.015

From the ethyl acetate soluble fraction of an acetone-water extract of the aerial parts of Rumex acetosa L. (Polygonaceae), a variety of monomeric flavan-3-ols (catechin, epicatechin, epicatechin-3-O-gallate), A- and B-type procyanidins and propelargonidins (15 dimers, 7 trimers, 2 tetramers) were isolated with 5 so far unknown natural products. Dimers: procyanidin B1, B2, B3, B4, B5, B7, A2, epiafzelechin-(4β→8)-epicatechin, epiafzelechin-(4β→8)-epicatechin-3-O-gallate (new natural product), epiafzelechin-(4β→6)-epicatechin-3-O-gallate (new natural product), epiafzelechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate, B2-3′-O-gallate, B2-3,3′-di-O-gallate, B5-3′-O-gallate, and B5-3,3′-di-O-gallate. Trimers: procyanidin C1, epiafzelechin-(4β→8)-epicatechin-(4β→8)-epicatechin (new natural product), epicatechin-(4β→8)-epicatechin-(4β→8)-catechin, cinnamtannin B1, cinnamtannin B1-3-O-gallate (new natural product), tentatively epicatechin-(2β→7, 4β→8)-epiafzelechin-(4α→8)-epicatechin (new natural product), and epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate. Tetramers: procyanidin D1 and parameritannin A1. All compounds were elucidated by ESI-MS, CD spectra, 1D- and 2D-NMR experiments as free phenols or peracetylated derivatives and, in part, after partial acid-catalysed degradation with phloroglucinol. A more abundant proanthocyanidin polymer was also isolated, purified and its chemical composition studied by ¹³C NMR. In addition a so far unknown phloroglucinolglycoside (1-O-β-d-(2,4-dihydroxy-6-methoxyphenyl)-6-O-(4-hydroxy-3,5-dimethoxybenzoyl)-glucopyranoside) was isolated.

Full text of DOI:10.1016/j.fitote.2009.08.015

Fitoterapia 80 (2009) 483–495
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Proanthocyanidins and a phloroglucinol derivative from Rumex acetosa L.
⁎
J. Bicker, F. Petereit, A. Hensel
Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Hittorfstraße 56, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
From the ethyl acetate soluble fraction of an acetone–water extract of the aerial parts of Rumex
acetosa L. (Polygonaceae), a variety of monomericflavan-3-ols(catechin, epicatechin, epicatechin-
3-O-gallate), A- and B-type procyanidins and propelargonidins (15 dimers, 7 trimers, 2 tetramers)
were isolated with 5 so far unknown natural products. Dimers: procyanidin B1, B2, B3, B4, B5, B7,
A2, epiafzelechin-(4β→8)-epicatechin, epiafzelechin-(4β→8)-epicatechin-3-O-gallate (new
natural product), epiafzelechin-(4β→6)-epicatechin-3-O-gallate (new natural product),
epiafzelechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate, B2-3′-O-gallate, B2-3,3′-di-O-
gallate, B5-3′-O-gallate, and B5-3,3′-di-O-gallate. Trimers: procyanidin C1, epiafzelechin-
(4β→8)-epicatechin-(4β→8)-epicatechin (new natural product), epicatechin-(4β→8)-
epicatechin-(4β→8)-catechin, cinnamtannin B1, cinnamtannin B1-3-O-gallate (new natural
product), tentatively epicatechin-(2β→7, 4β→8)-epiafzelechin-(4α→8)-epicatechin (new
natural product), and epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate-(4β→8)-
epicatechin-3-O-gallate.
Received 9 July 2009
Accepted 9 August 2009
Available online 18 August 2009
Keywords:
Rumex acetosa L.
Polygonaceae
Proanthocyanidins
A-type
Phloroglucinolglucoside
Flavan-3-ol
Tetramers: procyanidin D1 and parameritannin A1. All compounds were elucidated by ESI-MS,
CD spectra, 1D- and 2D-NMR experiments as free phenols or peracetylated derivatives and, in
part, after partial acid-catalysed degradation with phloroglucinol.
A more abundant proanthocyanidin polymer was also isolated, purified and its chemical
composition studied by 13C NMR.
In addition
a so far unknown phloroglucinolglycoside (1-O-β-D-(2,4-dihydroxy-6-
methoxyphenyl)-6-O-(4-hydroxy-3,5-dimethoxybenzoyl)-glucopyranoside) was isolated.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The aerial parts have been reported to contain flavonoids
(rutin, hyperoside, quercitrin, quercetin-3-O-glucuronide,
Rumex acetosa L. (Polygonaceae) is a perennial plant
worldwide distributed in areas with temperate climate. The
aerial parts of this so called “sorrel” are used within food
technology and for phytotherapeutic use. Medicinal applica-
tions are related to the tannin content of the material, leading
to adstringent effects which are useful for treatment of
diarrhoe and skin irritations. Modern phytotherapeutic prep-
arations with nationally registrated drug status in Europe
contain extracts of R. acetosa for treatment of acute and
chronic infections of the upper respiratory system [1].
avicularin, vitexin, orientin, isoorientin and their acetyl de-
rivatives) [[2] and literature cited therein], 1,8-dihydro-
xyanthraquinones (chrysophanol and its 8-O-glucoside,
physcion, physcionanthrone, emodin and its 8-O-glucoside,
emodinanthrone, aloeemodin, acetoxyaloeemodin) [3,4]
oxalic acid, flavan-3-ols with catechin and epicatechin [5],
phenolic acids (gallic acid, protocatechuic acid, ferrulic acid,
p-coumaric acid) and higher amounts of polysaccharides from
the rhamnogalacturonan and arabinogalactan type with
immunstimulating and antiphlogistic properties [6].
Despite the fact that the aerial parts of R. acetosa contain
substantial amounts of tannins it seems interesting that no
phytochemical details are published on the respective struc-
tural features.
⁎
Corresponding author. Tel.: +49 251 8333380; fax: +49 251 833841.
E-mail address: ahensel@uni-muenster.de (A. Hensel).
0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2009.08.015

484
J. Bicker et al. / Fitoterapia 80 (2009) 483–495
2. Experimental
2.1. Plant material
performed using a combination of CC on MCI-Gel® CHP-20P
(75–150 μm, Mitsubishi Chemical Industries, Tokyo, Japan,
2.5×50 cm), MPLC on RP18 material (3.6×50 cm, 18–32 μm;
Besta Technik, Wilhelmsfeld, Germany), MLCCC (Ito Multi-
layer Coil Separator Extractor, P.C. Inc. Potoay, Maryland, U.S.
A., 325 ml column, 1.6 mm i.D. at 800 rpm and 1 ml/min flow
rate) with EtOAc–water (1:1, upper phase) as mobile phase,
FCPC (Fast Centrifugal Partition Chromatography) on a CRC
Kromaton system (Kromaton Technologies, Angers, France)
at 1.600 rpm, 25 ml/min flow rate with water–EtOH–hexane–
EtOAc (7:2:1:8, upper phase) as mobile phase, preparative
HPLC on Silica Uptishere Diol, 6 μm, 250×21.2 mm or prep.
TLC.
Dried plant material of R. acetosa L. (Herba Rumicis
acetosae conc., Ch-B.: 43146115) was obtained from Caesar
& Loretz GmbH, Hilden, Germany. Identification was per-
formed by microscopic investigations. A voucher specimen
is retained in the documentation file of the Institute of
Pharmaceutical Biology and Phytochemistry under the code
Rumex 1.
2.2. General experimental procedures
A portion of the above Sephadex-fraction 2 (3.8 g) was
purified by MLCCC and CC on MCI-Gel (20–80% MeOH linear
gradient; system 1) followed by MPLC (20–80% MeOH linear
gradient) to yield 28 (102 mg). A part of fraction 3 (1.7 g)
was fractionated by MLCCC followed by purification on MCI-
Gel (system 1) to yield 1 (36 mg) and 2. Compound 2 was
finally purificated by prep. HPLC (ACN/MeOH/water-gradi-
ent) to yield 26 mg. A portion of fraction 4 (510 mg) was
fractionated using a step gradient on MCI-Gel (20–50%
MeOH, 50% MeOH isocratic, 50–80% MeOH; system 2)
followed by prep. TLC of a peracetylated subfraction of
impure 8 to yield 8a (30 mg).
A part of Sephadex-fraction 5 (1 g) was at first frac-
tionated using MCI-Gel (system 1, MeOH 20%, 2 L, than MeOH
80% 2 L, cleaning with MeOH 100%500 mL) which yielded two
proanthocyanidin containing subfractions (a and b). Subfrac-
tion a contained compounds 4–7 which were isolated as their
peracetates 4a–7a after preparative TLC (KG 60 F254, 0.5 mm
layer, mobile phase toluene:acetone (7:3 V/V) of the perace-
tylated subfraction a. Subfraction b was purified by MPLC
(system 2, with MeOH 20%, 2 L, than MeOH 80%, 2 L, than
cleaning with MeOH 100%, 500 mL) yielding pure compound
3 (204 mg) An additional slightly red spot was observed in an
accompanying MPLC subfraction after spraying with vanillin/
HCl reagent. Complete acetylation of this subfraction and
purification with prep. TLC (KG 60 F254, 0.5 mm layer, mobile
phase toluene:acetone (7:3 V/V) yielded 19a (15 mg).
Parts of Sephadex-fraction 6 (300 mg) were separated by
FCPC (water–EtOH–hexane–EtOAc 7:2:1:8 (upper phase)
followed by peracetylation of all subfractions yielded the
peracetates of 9, 13, 14, 15 and 20 (9a, 14 mg; 13a, 17 mg;
14a, 12 mg; 15a, 14 mg; 20a, 25 mg).
NMR spectra of the peracetylated derivatives were re-
corded in CDCl3 (δ 7.26 and 77.00 ppm) on a Varian Unity
plus 600, a Varian INOVA 500 or a Varian m400 spectrom-
eter. Spectra of free-phenolic compounds were recorded
in MeOD (δ 3.31 and 49.05 ppm) on a Varian m400 spec-
trometer. Assignment of rotameric signals are marked with
HR and CR. MS data were obtained on a Quattro LC mass
spectrometer. CD spectra were measured with a Jasco J-815
CD spectrometer in MeOH. Optical rotations were measured
with a Perkin-Elmer 341 digital polarimeter in MeOH.
Analytic TLC was carried out on silica gel aluminium plates
(0.2 mm, Merck) using ethyl acetate/water/formic acid
(90:5:5) as solvent. Compounds were visualized as red
coloured spots by spaying with vanillin–HCl reagent. Prep-
arative TLC of peracetylated compounds was performed on
silica gel glass plates (0.5 mm, Merck) using toluene/acetone
(7:3) as solvent. Acetylation of compounds was performed
in pyridine/acetic acid anhydride (1:1) at room tempera-
ture for 24 h in the dark. Acid degradation with phloroglu-
cinol was performed according to the method described by
Fletcher et al. [7].
Zone capillary electrophoresis of the carbohydrate part
from 28 was performed on a PACE 50101 Beckmann Coulter
CE (Palo Alto, U.S.A) with 50 mM sodium borate buffer and
4.4 M acetonitrile at pH 10.3 on a capillary with 50 μm i.d.
over 77 cm. Injection 1–5 s, detection 200 nm. Enantioselec-
tive separation of (+) and (−)-catechin with 1 mg test
sample in MeOH/H2O (8:2) in a buffer with 20 mM NaH2PO4,
20 mM Na2HPO4 (pH 7.0), 20 mM γ-hydroxypropylcyclodex-
trin, 100 mM sodiumdodecylsulfat according to Noe and
Freissmuth [8].
Sephadex-fraction 7 (300 mg) was fractionated by FCPC
(H2O/EtOH/hexane/EtOAc 7:2:1:8 (upper phase) to give pure
10 (32 mg), 24 (10 mg), 11 (22 mg), 23 (38 mg) and 21
(18 mg). A subfraction showed next to spots from 23 and 21 a
third slightly red spot on the TLC plate. After peracetylation of
that subfraction followed by preparative TLC (conditions see
above) the peracetate 16a (7 mg) was isolated. A portion of
Sephadex-fraction 8 (1.8 g) was subfractionated by FCPC
(H2O/EtOH/hexane/EtOAc 7:2:1:8 (upper phase). From the
resulting subfractions compounds 17 (80 mg), 12 (520 mg),
23 (560 mg) and compound 26 (15 mg) were isolated in a
pure state. Sephadex-fraction 9 (400 mg) was again fraction-
ated by the above described FCPC system to yield pure 25
(69 mg) and 27 (45 mg). 1.5 g of Sephadex-fraction 11 was
fractionated by FCPC (H2O/MeOH/EtOAc 5:2:5) to give 18
(194 mg) from subfraction 1. Compound 22 was enriched in
2.3. Extraction and isolation
The dried, cut plant material (2.5 kg) was exhaustively
extracted with cold acetone/water (7:3, 15 l, Ultraturrax®).
The combined extracts were evaporated in vacuo, filtered to
remove the precipitated chlorophyll, defatted with petroleum
benzene and freeze-dried to yield the crude extract (252 g).
This extract was partitioned between water and EtOAc. After
removal of solvent, the residues were lyophilized to yield
215 g H2O-soluble fraction (W) and 36 g EtOAc-soluble frac-
tion (E). 35 g of E were fractionated by column chromatog-
raphy over Sephadex® LH-20 (900×55 mm) using stepwise
gradient elution with increasing polarity (ethanol (18 l)-
methanol (14 l)-acetone/water 7:3 (5 l)) to give 13 fractions.
Fractions were monitored by TLC. Further fractionation was

J. Bicker et al. / Fitoterapia 80 (2009) 483–495
485
subfraction 3 (315 mg). A portion could be purified using
prep. HPLC to yield pure 22 (25 mg).
6.50 [d, J=2.2 Hz, H-6 (A)], 6.68 [s, H-6 (D)], 6.89 [dd, J=2.0/
8.4 Hz, HR-6′ (E)], 6.96 [dd, J=2.0/8.4 Hz, H-6′ (E)], 7.03
[s, HR-6 (D)], 7.06 [d, J=8.4 Hz, H-5′ (E)], 7.09 [d, J=8.6 Hz,
H-3′/5′ (B)], 7.12 [d, J=2.0 Hz, HR-2′ (E)], 7.13 [d, J=2.0 Hz,
H-2′ (E)], 7.44 [d, J=8.6 Hz, H-2′/6′ (B)], 7.58 [2H, s, HR-2″/6″
(G)], 7.67 [2H, s, H-2″/6″ (G)], 13C NMR (CDCl3, 100 MHz): δ
19–22 [–CO–CH3], 26.52 [C-4 (F)], 34.21 [C-4 (C)], 68.74 [C-3
(F)], 71.20 [C-3 (C)], 73.23 [C-2 (C)], 77.17 [C-2 (F)], 107.50
[C-8 (A)], 108.76 [C-6 (A)], 110.57 [C-6 (D)], 111.64 [C-4a
(A)], 111.71 [C-4a (D)], 116.87 [C-8 (D)], 121.42 [C-3′/5′
(B)], 121.83 [C-2′ (E)], 122.12 [C-2″/6″ (G)], 123.12 [C-5′
(E)], 124.57 [C-6′ (E)], 127.27 [C-2′/6′ (B)], 127.63 [C-1″
(G)], 134.24 [C-1′ (E)], 135.22 [C-1′ (B)], 138.88 [C-4″ (G)],
141.75/141.78 [C-3′/4′ (E)], 143.44 [C-3″/5″ (G)], 147.93 [C-
5 (A) and C-7 (D)], 148.97 [C-5 (D)], 149.16 [C-7 (A)], 150.44
[C-4′ (B)], 154.09 [C-8a (D)], 155.45 [C-8a (A)], 163.91
[Carboxyl-C (G)], 167–170 [–CO–CH3].
Preparation of the polymeric fraction was achieved and
defined according to the procedure described by Foo et al.
[34]. The H2O-soluble fraction (W) obtained after extraction
(30 g) was fractionated by CC on Sephadex® LH-20
(900×55 mm) with MeOH–H2O 1:1 (17 l) and MeOH
(4.2 l) until the eluent was colourless; then acetone–H2O
7:3 (5 l) was used for elution to obtain the polymeric fraction
(20.3 g).
2.3.1. Epiafzelechin-(4β→8)-epicatechin (8)
Compound 8 was obtained as epiafzelechin-(4β→8)-
epicatechin-peracetate (8a): [α]2D0 =+46,77° (c=0.62);
ESI-MS: [M+Na]+ m/z 963, 5; [2 M+Na]+ m/z 1902,5;
[V]210 128688, [V]240 23869, 1H NMR (CDCl3, 400 MHz; dup-
lication due to dynamic rotational isomerism; two sets of
signals in the ratio ca 3:1): δ 1.59–2.38 [3H, all s, aliphatic and
phenolic –OAc] δ2.84–3.09 [m, H-4a,b (F)], 4.45 [d, J=1.8 Hz,
H-4 (C)], 4.56 [brs, H-2 (F)], 4.64 [d, J=1.8 Hz, HR-4 (C)], 5.11
[m, H-3 (F)], 5.19 [brs, H-3 (C)], 5.24 [brs, HR-2 (F)], 5.31
[m, HR-3 (C)], 5.39 [brs, HR-2 (C)], 5.52 [m, HR-3 (F)], 5.59
[brs, H-2 (C)], 5.99 [d, J=2.1 Hz, H-8 (A)], 6.24 [d, J=2.1 Hz,
H-6 (A)], 6.58 [s, HR-6 (D)], 6.62 [d, J=2.1 Hz, HR-6 (A)],
6.65 [s, H-6 (D)], 6.77 [d, J=2.1 Hz, HR-8 (A)], 6.89
[dd, J=1.8/8.0 Hz, H-6′ (E)], 7.03 [d, J=1.8 Hz, H-2′ (E)],
7.03 [d, J=8.0 Hz, H-5′ (E)], 7.05 [d, J=8.6 Hz, HR-3′/5′ (B)],
7.09 [d, J=8.6 Hz, H-3′/5′ (B)], 7.38 [d, J=8.6 Hz, HR-2′/6′
(B)], 7.46 [d, J=8.6 Hz, H-2′/6′ (B)], 13C NMR (CDCl3,
100 MHz): δ 19–22 [all s, –CO–CH3], 26.30 [CR-4 (F)], 26.63
[C-4 (F)], 34.15 [C-4 (C)], 34.27 [CR-4 (C)], 66.41 [CR-3 (F)],
66.80 [C-3 (F)], 70.83 [CR-3 (C)], 71.19 [C-3 (C)], 73.97 [C-2
(C)], 74.77 [CR-2 (C)], 77.17 [C-2 (F)], 77.22 [CR-2 (F)], 107.25
[C-8 (A)], 108.19 [CR-8 (A)], 108.60 [C-6 (A)], 108.95 [CR-6
(A)], 109.45 [CR-4a (A) and/or CR-4a (D)], 110.30 [C-6 (D)],
110.88 [CR-6 (D)], 111.62 [C-4a (A) or C-4a (F)], 111.66 [C-4a
(A) or C-4a (F)], 116.81 [C-8 (D)], 117.57 [CR-8 (D)], 121.38
[C-3′/5′ (B)], 122.47 [C-2′ (E)], 122.74 [C-5′ (E)], 125.01 [C-6′
(E)], 127.51 [CR-2′/6′ (B)], 127.56 [C-2′/6′ (B)], 134.32 [CR-1′
(E)], 134.51 [C-1′ (E)], 135.26 [C-1′ (B)], 135.58 [CR-1′ (B)],
141.60 [C-3′ (E)], 141.73 [CR-3′ (E)], 141.92 [C-4′ (E)], 142.07
[CR-4′ (E)], 147.57 [CR-5 or CR-7 (D)], 147.82 [C-5 (A) or
C-7 (D)], 147.92 [C-5 (A) or C-7 (D)], 148.58 [CR-5 or CR-7
(D)], 149.06 [C-5 (D) or C-7 (A)], 149.12 [C-5 (D) or C-7 (A)],
149.80 [CR-5 or CR-7 (A)], 149.92 [CR-5 or CR-7 (A)], 150.37
[C-4′ (B)], 151.81 [CR-4′ (B)], 154.17 [C-8a (D)], 155.17
[CR-8a (A)], 155.33 [CR-8a (D)], 155.58 [C-8a (A)], 168-171
[all s, –CO–CH3].
2.3.3. Epiafzelechin-3-O-gallate-(4β→8)-epicatechin-3-O-
gallate (10)
Compound 10 (15 mg) was peracetylated for analytical
investigation to epiafzelechin-3-O-gallate-(4β→8)-epicate-
chin-3-O-gallate-peracetate (10a, 19 mg): [α]2D0 =−37.80°
(c=0.69); ESI-MS: [M+NH3]+ m/z 1430, 5; [V]206 -40635;
1H NMR (CDCl3, 600 MHz): δ 1.83–2.37 [3H, all s, aliphatic
and phenolic –OAc], 3.06 [m, H-4a,b (F)], 4.45 [d, J=2.4 Hz,
H-4 (C)], 4.75 [brs, H-2 (F)], 5.34 [m, H-3 (F)], 5.50 [m, H-3
(C)], 5.71 [brs, H-2 (C)], 6.16 [d, J=2.4 Hz, H-8 (A)], 6.28
[d, J=2.4 Hz, H-6 (A)], 6.70 [s, H-6 (D)], 6.97 [dd, J=2.1/
8.5 Hz, H-6′ (E)], 7.06 [d, J=8.5 Hz, H-5′ (E)], 7.07
[d, J=8.6 Hz, H-3′/5′ (B)], 7.16 [d, J=2.1 Hz, H-2′ (E)], 7.45
[d, J=8.6 Hz, H-2′/6′ (B)], 7.59 [2H, s, H-2″/6″ (G)], 7.70
[2H, s, H-2″/6″ (H)], 13C NMR (CDCl3, 150 MHz): δ 19–22 [–
CO–CH3], 26.53 [C-4 (F)], 34.38 [C-4 (C)], 68.72 [C-3 (F)], 72.58
[C-3 (C)], 74.43 [C-2 (C)], 77.21 [C-2 (F)], 107.41 [C-8 (A)],
109.06 [C-6 (A)], 110.59 [C-6 (D)], 111.46 [C-4a (A)], 111.76
[C-4a (D)], 116.65 [C-8 (D)], 121.73 [C-3′/5′ (B)], 121.85 [C-2′
(E)], 122.13 [C-2″/6″ (H)], 122.30 [C-2″/6″ (G)], 123.13 [C-5′
(E)], 124.66 [C-6′ (E)], 127.39 [C-1″ (G)], 127.64 [C-1″ (H)],
127.85 [C-2′/6′ (B)], 134.29 [C-1′ (E)], 134.74 [C-1′ (B)], 138.89
[C-4″ (G, H)], 141.78/141.80 [C-3′/4′ (E)], 143.36 [C-3″/5″ (G)],
143.46 [C-3″/5″ (H)], 147.89–147.93 [C-5 (A) and C-7 (D)],
149.06 [C-5 (D)], 149.24 [C-7 (A)], 150.54 [C-4′ (B)], 154.12
[C-8a (D)], 155.36 [C-8a (A)], 162.90 [Carboxyl-C (G)],
163.94 [Carboxyl-C (H)], 173–182 [–CO–CH3].
2.3.4. Epiafzelechin-(4β→6)-epicatechin-3-O-gallate (16)
Compound 16 was obtained as epiafzelechin-(4β→6)-
epicatechin-3-O-gallate-peracetate (16a): [α]2D0 =+18.52°
(c=0.05); ESI-MS: [M+Na]+ m/z 1199.2; [ ]237 84028; 1H
NMR (CDCl3, 600 MHz; 16a displayed at room temperature
extremely broad and overlapping aromatic and heterocyclic
absorptions due to the effect of rotational isomerism): δ 1.64–
2.38 [3H, all s, aliphatic and phenolic –OAc], 2.84–3.10 [m,
2×H-4 (F)], 4.30 [m, H-4 and HR-4 (C)], 5.39 [brs, H-2 (C)],
5.18 [m, H-3 (C)], 5.26 [brs, H-2 (F)], 5.62 [m, H-3 (F)], 6.67
[s, H-8 (D)], 6.74 [d, J=2.0 Hz, H-8 (A)], 6.81 [s, HR-8 (D)],
7.05 [d, J=8.6 Hz, H-3′/5′ (B)], 7.21 [d, J=8.6 Hz, H-2′/6′
(B)], 7.59 [2H, s, HR-2″/6″ (G)], 7.65 [2H, s, H-2″/6″ (G)]; other
signals were not determined with certainty.
2.3.2. Epiafzelechin-(4β→8)-epicatechin-3-O-gallate (9)
Compound 9 was obtained as epiafzelechin-(4β→8)-
epicatechin-3-O-gallate-peracetate (9a): [α]2D0 =+38.96°
(c=0.77); ESI-MS: [M+Na]+ m/z 1199, 5; [V]212 49772,
[V]245 -11783, [V]260 -7172, [V]280 -16251; 1H NMR (CDCl3,
400 MHz; duplication due to dynamic rotational isomerism;
two sets of signals in the ratio ca 5:1): δ 1.70–2.36 [all s,
aliphatic and phenolic –OAc], 3.04 [m, H-4a,b (F)], 4.43
[d, J=2,4 Hz, H-4 (C)], 4.45 [d, J=2,4 Hz, HR-4 (C)], 4.76
[brs, H-2 (F)], 4.82 [brs, HR-2 (F)], 5.25 [m, H-3 (C)], 5.28
[m, HR-3 (F)], 5.31 [m, H-3 (F)], 5.35 [m, HR-3 (C)], 5.59
[brs, H-2 (C)], 5.68 [brs, HR-2 (C)], 6.12 [d, J=2.2 Hz, H-8 (A)],

486
J. Bicker et al. / Fitoterapia 80 (2009) 483–495
2.3.4.1. Conversion of proanthocyanidins into anthocyanidins.
2 mg of 16a were refluxed with 5% HCl in EtOH for 1 h. The
reaction mixture was subsequently chromatographed on
cellulose (Cellulose F, 0.1 mm, Merck) in HCO2H–HCl–H2O
(10:1:3) with pelargonidin as ref. substance.
(B)], 7.07 [d, J=8.5 Hz, H-5′ (E)], 7.08 [d, J=8.7 Hz, HR-3′/5′
(B)], 7.12 [dd, J=1.9/8.5 Hz, H-6′ (E)], 7.17 [d, J=1.9 Hz, H-2′
(E)], 7.17 [d, J=8.2 Hz, H-5′ (H)], 7.19 [dd, J=1.9/8.2 Hz, H-6′
(H)], 7.27 [d, J=8.7 Hz, H-2′/6′ (B)], 7.29 [d, J=1.9 Hz, H-2′
(H)], 7.47 [d, J=8.7 Hz, HR-2′/6′ (B)], 13C NMR (CDCl3,
150 MHz): δ 19–23 [–CO–CH3], 26.41 [C-4 (I)], 34.47 [C-4
(C)], 35.07 [C-4 (F)], 66.57 [C-3 (I)], 70.74 [C-3 (C)], 71.31 [C-4
(F)], 74.94 [C-2 (F)], 75.10 [C-2 (C)], 77.03 [C-2 (I)], 108.17
[C-8 (A)], 109.25 [C-6 (A)], 109.97 [C-4a (G)], 110.64 [C-6
(G)], 110.97 [C-6 (D)], 111.68 [C-4a (A)], 112.16 [C-4a (D)],
117.58 [C-8 (G)], 117.82 [C-8 (D)], 121.28 [C-2′ (E)], 121.32
[CR-3′/5′ (B)], 121.43 [C-3′/5′ (B)], 121.66 [C-2′ (H)], 123.07
[C-5′ (E)], 123.24 [C-5′ (H)], 123.96 [C-6′ (E)], 124.09 [C-6′
(H)], 127.46 [C-2′/6′ (B)], 128.11 [CR-2′/6′ (B)], 134.23 [C-1′
(B)], 135.20 [C-1′ (E)], 135.74 [C-1′ (H)], 141.67–142.14 [C-3′/
4′ (E, H)], 147.20 [C-7 (G)], 147.61 [C-7 (D)], 148.51 [C-5 (G)],
148.58 [C-5 (D)], 149.90 [C-5/7 (A)], 150.59 [C-4′ (B)], 151.76/
151.89 [C-8a (D, G)], 155.10 [C-8a (A)], 168–172 [–CO–CH3].
2.3.5. Epicatechin-(4β→6)-epicatechin-3-O-gallate (17)
Compound 17 (20 mg) was peracetylated for analytical
investigation to epicatechin-(4β→6)-epicatechin-3-O-gal-
late-peracetate (17a, 28 mg): [α]2D0 =+22.53° (c=0.71);
ESI-MS: [M+Na]+ m/z 1257.5; [V]238 69716; 1H NMR
(CDCl3, 500 MHz; 17a displayed at room temperature ex-
tremely broad and overlapping aromatic and heterocyclic
absorptions in a ratio ca 1–1.5:1 due to the effect of rotational
isomerism): δ 1.84–2.37 [3H, all s, aliphatic and phenolic –
OAc], 2.90 [dd, J=4.1/17.0 Hz, H-4a (F)], 3.04 [m, H-4b (F)],
4.31 [brs, H-4 (C)] 4.36 [brs, HR-4 (C)], 5.25 [m, H-3 (C)], 5.27
[brs, H-2 (F)], 5.35 [brs, H-2 (C)], 5.60 [m, HR-3 (F)], 5.66
[m, H-3 (F)], 6.60 [d, J=2.3 Hz, H-6 (A) and HR-6 (A)], 6.69
[s, H-8 (D)], 6.73 [d, J=2.3 Hz, HR-8 (A)], 6.75 [d, J=2.3 Hz,
H-8 (A)], 6.83 [s, HR-8 (D)], 7.13 [d, J=8.5 Hz, H-5′ (E)], 7.16
[brs, HR-2′ (B or E), 7.17 [dd, J=2.0/8.5 Hz, H-6′ (E)], 7.19
[d, J=8.5 Hz, HR-5′ (E)], 7.21 [d, J=8.4 Hz, H-5′ (B)], 7.29
[dd, J=1.9/8.4 Hz, H-6′ (B)], 7.31 [d, J=2.0 Hz, H-2′ (E)], 7.35
[d, J=1.9 H-2′ (B)], 7.58 [2H, s, HR-2″/6″ (G)], 7.64 [2H, s, H-
2″/6″ (G)], 13C NMR (CDCl3, 125 MHz): δ 19–22 [–CO–CH3],
26.40 [C-4 (F)], 34.72 [C-4 (C)], 68.12 [C-3 (F)], 68.17 [CR-3 (F)],
70.89 [C-3 (C)], 71.02 [CR-3 (C)], 73.83 [CR-2 (C)], 73.87 [C-2
(C)], 76.86 [C-2 (F)], 107.35 [C-8 (A)], 107.42 [CR-8 (A)], 108.62
[C-6 (A)], 109.71 [CR-8 (D)], δ 110.63 [C-8 (D) and C-4a (A)],
δ 110.89 [CR-4a (D)], δ 110.92 [C-4a (D)], 116.70 [C-6 (D)],
121.88 [C-2′ (B)], 121.94 [CR-2′ (B)], 122.00 [CR-2′ (E)], 122.08
[C-2′ (E)], 122.22 [CR-2″/6″ (G)], 122.32 [C-2″/6″ (G)], 123.14
[CR-5′ (E)], 123.21 [C-5′ (E)], 123.55 [C-5′ (B)], 124.29 [C-6′
(B)], 124.37 [CR-6′ (B)], 124.57 [CR-6′ (E)], 124.62 [C-6′
(E)], 127.68 [C-1″ (G)], 127.77 [CR-1″ (G)], 135.26 [C-1′ (B)],
135.32 [CR-1′ (B)], 135.97 [CR-1′ (E)], 136.02 [C-1′ (E)], 138.99
[CR-4″ (G)], 139.03 [C-4″ (G)], 141.97–142.32 [C-3′/4′ (B, E)],
143.53 [CR-3″/5″ (G)], 143.58 [C-3″/5″ (G)], 149.01 [C-5 (A) or
C-5 (D) and C-7 (D)], 150.26 [C-5 (A) or C-5 (D) and C-7 (A)],
154.01 [C-8a (D)], 155.17 [C-8a (A)], 163.51 [Carboxyl-CR (G)],
163.65 [Carboxyl-C (G)], 167–171 [–CO–CH3].
2.3.7. Epicatechin-(4β→8)-epicatechin-(4β→8)-catechin (21)
Compound 21 (12 mg) was peracetylated for analytical
investigation to epicatechin-(4β→8)-epicatechin-(4β→8)-
catechin-peracetate (21a, 15 mg): [α]2D0 =+113.04° (c=
1.84); ESI-MS: [M+Na]+ m/z 1519.5; [V]212 220364, [V]280
13786, 1H NMR (CDCl3, 500 MHz; duplication due to dynamic
rotational isomerism; two sets of signals in the ratio ca 2:1;
signal set of the minor rotamer was not determined): δ 1.43–
2.38 [3H, all s, aliphatic and phenolic –OAc], 2.74 [dd, J=8.3/
16.7 Hz, H-4a (I)], 3.12 [dd, J=5.6/16.7 Hz, H-4b (I)], 4.63
[brs, H-4 (F)], 4.74 [brs, H-4 (C)], 5.07 [d, J=7.8 Hz, H-2 (I)],
5.16 [m, H-3 (I)], 5.34 [m, H-3 (C) and H-2 (F)], 5.36 [brs, H-2
(C)], 5.41 [m, H-3 (F)], 6.64 [d, J=2.3 Hz, H-6 (A)], 6.69 [s, H-6
(G)], 6.75 [s, H-6 (D)], 6.75 [d, J=2.3 Hz, H-8 (A)], 6.91–7.35
[protons of the rings B, E and H]; 13C NMR (CDCl3, 125 MHz):
δ 19–23 [–CO–CH3], 25.44 [CR-4 (I)], 25.61 [C-4 (I)], 34.43 [C-
4 (C)], 35.12 [C-4 (F)], 61.51 [CR-3 (I)], 68.33 [C-3 (I)], 70.54
[C-3 (C)], 71.01 [C-4 (F)], 74.67 [C-2 (C)], 74.95 [C-2 (F)],
78.09 [CR-2 (I)], 78.19 [C-2 (I)], 108.13 [C-6 (A)], 109.27 [C-
8 (A)], 110.66 [C-6 (G)], 111.08 [C-6 (D)], 111.14 [C-4a (G)],
111.61 [C-4a (D)], 111.74 [C-4a (A)], 117.45/117.51 [C-8 (D,
G)], 120.31–125.43 [C-2′/5′/6′ (B, E, H)], 133.31–136.90 [C-1′
(B, E, H)], 141.47–142.43 [C-3′/4′ (B, E, H)], 147.48 [C-7 (D,
G)], 148.10/148.43 [C-5 (D, G)], 149.89 [C-5/7 (A)], 151.68/
151.78 [C-8a (D, G)], 154.90 [C-8a (A)], 168–172 [–CO–CH3].
2.3.6. Epiafzelechin-(4β→8)-epicatechin-(4β→8)-epicatechin
(19)
Compound 19 was obtained as epiafzelechin-(4β→8)-
2.3.8. Epicatechin-(2β→7, 4β→8)-epicatechin-(4β→8)-
epicatechin (23)
epicatechin-(4β→8)-epicatechin-peracetate (19a): [α]D20
=
+65.57° (c=0.61); ESI-MS: [M+Na]+ m/z 1461.5; [V]210
213439, [V]220 167395, 1H NMR (CDCl3, 600 MHz; duplication
due to dynamic rotational isomerism; two sets of signals in the
ratio ca 2–3:1): δ 1.4–2.38 [3H, all s, aliphatic and phenolic –
OAc], 2.91 [dd, J=n.d./17.7 Hz, HR-4a (I)], 2.96 [dd, J=n.d./
17.7 Hz, H-4a (I)], 3.03 [dd, J=4.5/17.7 Hz, HR-4b (I)], 3.08
[dd, J=4.5/17.7 Hz, H-4b (I)], 4.66/4.69 [brs, HR-4 (C, F)], 4.70
[brs, H-4 (F)], 4.77 [brs, H-4 (C)], 4.98 [m, HR-3 (C)], 5.11
[brs, HR-2 (I)], 5.12 [m, HR-3 (I)], 5.21 [brs, H-2 (I)], 5.35
[m, H-3 (C)], 5.37 [brs, H-2 (C)], 5.39 [brs, H-2 and H-3 (F)],
5.41 [brs, HR-3 (I)], δ 5.47 [m, H-3 (I)], 5.72 [brs, HR-2 (C)],
5.94 [brs, HR-6 or 8 (A)], 6.26 [brs, HR-8 or 6 (A)], 6.61 [s, HR-6
(G)], 6.65 [s, H-6 (G)], 6.65 [d, J=2.2 Hz, H-6 (A)], 6.71 [s, H-6
(D)], 6.76 [d, J=2.2 Hz, H-8 (A)], 7.04 [d, J=8.7 Hz, H-3′/5′
Compound 23 (cinnamtannin B1, 40 mg) was peracety-
lated for analytical investigation to Epicatechin-(2β→7,
4β→8)-epicatechin-(4β→8)-epicatechin-peracetate (23a,
54 mg): [α]2D0 =+32.7° (c=0.06); ESI-MS: [M+H]+ m/z
865.1; [V]232 62021, [V]250 5724, [V]258 -10228, [V]270
38159, [V]270, [V]284;
1H NMR (CDCl3, 600 MHz, duplication
due to dynamic rotational isomerism; two sets of signals
in the ratio ca 1:1): δ 1.46–2.32 [3H, all s, aliphatic and
phenolic –OAc], δ 2.90–3.07 [m, 2×H-4 and 2×HR-4 (I)],
4.30 [d, J=4.1 Hz, H-4 (C)], 4.30 [d, J=2.4 Hz, H-4 (F)], 4.62
[d, HR-4 (C)], 4.63 [d, HR-4 (F)], 4.79 [brs, H-2 (I)], 5.00
[d, J=4.1 Hz, H-3 (C)], 5.01 [d, J=4.1 Hz, HR-3 (C)], 5.20
[m, H-3 (I), HR-2 (I) and HR-3 (F)], 5.36 [dd, J=2.4 and
2.6 Hz, H-3 (F)], 5.42 [brs, HR-2 (F)], 5.53 [m, HR-3 (I)], 5.42

J. Bicker et al. / Fitoterapia 80 (2009) 483–495
487
[d, J=2.6 Hz, H-2 (F)], 6.25 [s, H-6 (D)], 6.43 [d, J=2.2 Hz,
H-6 (A)], 6.52 [d, J=2.2 Hz, HR-6 (A)], 6.59 [s, HR-6 (D)],
6.59 [s, HR-6 (G)], 6.62 [s, H-6 (G)], 6.72 [d, J=2.2 Hz, H-8 (A)],
6.85 [d, J=2,2 Hz, HR-8 (A)], 7.04 [dd, J=2.0 and 8.5 Hz, H-6′
(E)], 7.05 [dd, J=2.0 and 8.5 Hz, H-6′ (H)], 7.08 [d, J=8.5 Hz,
H-5′ (E)], 7.13–7.14 [m, HR-5′ (E), HR-5′ (H) and HR-6′ (H)],
7.19 [d, J=2.0 Hz, H-2′ (E)], 7.21 [d, J=2.0 Hz, H-2′ (H)],
7.23 [m, HR-2′ (H) and HR-5′ (B)], 7.26 [d, J=2.0 Hz, HR-2′
(E)], 7.27 [d, J=8.5 Hz, HR-5′ (B)], 7.28 [d, J=8.5 Hz, H-5′
(H)], 7.39 [d, J=2.1 Hz, H-2′ (B)], 7.47 [d, J=2.1 Hz, HR-2′
(B)], 7.50 [dd, J=2.1 and 8.6 Hz, H-6′ (B)], 7.57 [dd, J=2.1
and 8.6 Hz, HR-6′ (B)]; 13C NMR (CDCl3, 150 MHz): δ 19.4–
21.2 [–CO–CH3], 26.15 [C-4 (I)], 26.30 [CR-4 (I)], 27.38 [C-4
(C) and CR-4 (C)], 33.44 [C-4 (F)], 33.61 [CR-4 (F)], 66.34 [C-3
(I)], 66.70 [C-3 (C)], 67.90 [CR-3 (C)], 69.73 [CR-3 (F)], 70.47
[C-3 (F)], 75.33 [C-2 (F)] 75.50 [CR-2 (F)], 76.69 [C-2 (I)],
97.78 [CR-2(C)], 98.26 [C-2(C)], 104.17 [CR-6 (D)], 104.77
[C-6 (D)], 106.65 [C-8 (A)], 107.10 [CR-8 (A)], 107.29 [CR-4a
(D)], 108.16 [CR-8 (D)], 108.44 [C-8 (D)], 108.73 [CR-4a (D)],
109.70 [C-6 (A)], 109.77 [CR-6 (A)], 109.92 [CR-4a (G)],
110.26 [C-6 (G)], 110.83 [CR-6 (G)], 110.95 [C-4a (G)],
113.05 [CR-4a (A)], 114.18 [C-4a (A)], 116.67 [C-8 (G)],
117.80 [CR-8 (G)], 121.36 [CR-2′ (H)], 121.73 [C-2′ (H)],
122.08 [CR-5′ (E)], 122.80 [C-2′ (B)], 122.82 [C-5′ (B)], 122.99
[CR-2′ (B) and C-5′ (E)], 123.03 [CR-5′ (B)], 123.21 [C-5′ (H)],
123.31 [CR-5′ (H)], 123.59 [CR-6′ (H)], 123.67 [C-2′ (E)],
124.27 [C-6′ (H)], 124.34 [CR-2′ (E)], 125.26 [CR-6′ (B)],
125.50 [C-6′ (E)], 125.53 [C-6′ (B)], 125.94 [CR-6′ (E)], 134.77
[CR-1′ (E)], 134.82 [C-1′ (H)], 135.29 [CR-1′ (B)], 135.36 [C-1′
(E)], 135.39 [C-1′ (B)], 135.46 [CR-1′ (H)], 141.45–143.02
[C-3′/4′ and CR-3′/4′ (B, E, H)], 148.55 [C-7 (G)], 148.58 [CR-7
(G)], 148.10 and 148.13 [C-5/7 (D) or C-5 (A)], 148.55 [C-5 (G)],
148.58 [CR-5 (G)], 148.77 [CR-5 (A)], 149.45 [C-7 (A)], 149.61
and 144.66 [C-5/7 (D)], 150.17 or 150.20 [C-5/7 or CR-7 (A)],
151.88 and 151.92 [CR-8a (G) or C-8a (D)], 152.20 [CR-8a (D)],
153.36 [C-8a (G)], 153.85 [CR-8a (A)], 154.09 [C-8a (A)], 167.6–
170.51 [–CO–CH3].
123.02 [C-2′ (B and E)], 124.74 [C-5′ (B)], 125.30 [C-6′ (B)],
126.15 [C-6′ (E)], 134.70 [C-1′ (E)], 135.23 [C-1′ (B)],
141.64–143.02 [C-3′/4′ (B and E)], 148.69–150.41 [C-5/7 (A
and D); C-1/3/5 (G)], 152.04 [C-8a (D)], 155.57 [C-8a (A)],
168–172 [–CO–CH3].
2.3.9. Epicatechin-(2β→7, 4β→8)-epiafzelechin-(4α→8)-
epicatechin (24)
Compound 24 (10 mg) was peracetylated for analytical
investigation to epicatechin-(2β→7, 4β→8)-epiafzelechin-
(4α→8)-epicatechin-peracetate (24a, 18 mg): [α]2D0
=
+10.36° (c=1.93); ESI-MS: [M+NH3]+ m/z 1412.4 [M+
Na]+ m/z 1417.6; [V]210 -52052, [V]232 25372, [V]250 1049,
[V]270 14171, [V]280 -7602; 1H NMR (CDCl3, 600 MHz, dup-
lication due to dynamic rotational isomerism; two sets of
signals in the ratio ca 1:1): δ 1.4–2.36 [3H, all s, aliphatic and
phenolic –OAc], 2.90–3.06 [m, H-4a,b and HR-4a,b (I)], 4.29
[d, J=4.8 Hz, H-4 (F)], 4.35 [d, J=4.1 Hz, H-4 (C)], 4.62
[d, J=4.8 Hz, HR-4 (F)], 4.63 [d, J=4.1 Hz, HR-4 (C)], 4.80
[brs, H-2 (I)], 5.00 [d, J=4.1 Hz, H-3 (C)], 5.01 [d, J=4.1 Hz,
HR-3 (C)], 5.18 [brs, HR-2 (I)], 5.18 [m, HR-3 (F)], 5.21 [m, H-3
(I)], 5.39 [m, H-3 (F) and HR-2 (F)], 5.51 [m, HR-3 (I)], 5.71
[d, J=2.3 Hz, H-2 (F)], 6.24 [s, H-6 (D)], 6.45 [d, J=2.2 Hz,
H-6 (A)], 6.55 [d, J=2.2 Hz, HR-6 (A)], 6.59 [s, HR-6 (G)],
6.60 [s, HR-6 (D)], 6.62 [s, H-6 (G)], 6.73 [d, J=2.2 Hz, H-8 (A)],
6.86 [d, J=2.2 Hz, HR-8 (A)], 7.01 [d, J=8.5 Hz, HR-3′/5′ (E)],
7.04 [d, J=8.5 Hz, H-3′/5′ (E)], 7.05 [dd, J=1.9 and 8.4 Hz, H-6′
(H)], 7.21 [d, J=1.9 Hz, H-2′ (H)], 7.23 [d, J=1.9 Hz, HR-2′ (H)],
7.23 [d, J=8.6 Hz, H-5′ (B)], 7.25 [d, J=8.5 Hz, HR-2′/6′ (E)],
7.27 [d, J=8.6 Hz, HR-5′ (B)], 7.29 [d, J=8.4 Hz, H-5′ (H)], 7.40
[d, J=8.5 Hz, H-2′/6′ (E)], 7.41 [d, J=2.1 Hz, H-2′ (B)], 7.48
[d, J=2.1 Hz, HR-2′ (B)], 7.51 [dd, J=2.1 and 8.6 Hz, H-6′ (B)],
7.57 [dd, J=2.1 and 8.6 Hz, HR-6′ (B)], 13C NMR (CDCl3,
150 MHz): δ 19–22 [–COCH3], 26.21 [C-4 (I)], δ 26.31 [CR-4 (I)],
27.36 [C-4 (C)], 27.39 [CR-4 (C)], 33.56 [C-4 (F)], 33.85 [CR-4
(F)], 66.34 [C-3 (I)], 66.40 [CR-3 (I)], 66.73 [C-3 (C)], 67.84 [CR-3
(C)], 70.18 [CR-3 (F)], 70.70 [C-3 (F)], 75.67 [C-2 (F)], 75.91
[CR-2 (F)], 76.69 [C-3 (I)], 97.68 [CR-2 (C)], 98.21 [C-2 (C)],
104.12 [C-6 (D)], 104.71 [CR-6 (D)], 106.80 [C-8 (A)], 107.32
[CR-8 (A)], 107.50 [CR-4a (D)], 108.30 [C-8 (D)], 108.45 [CR-8 (D)],
108.84 [C-4a (D)], 109.65 [C-6 (A)], 109.78 [CR-6 (A)], 109.88
[C-4a (G)], 110.20 [C-6 (G)], 110.83 [CR-6 (G)], 110.86 [CR-4a
(G)], 113.10 [CR-4a (A)], 114.17 [C-4a (A)], 116.74 [C-8 (G)],
117.91 [CR-8 (G)], 121.14 [CR-3′/5′ (E)], 121.28 [C-3′/5′ (E)],
121.35 [CR-2′ (H)], 121.66 [C-2′ (H)], 122.82 [C-2′ (B)],
122.99 [CR-2′ (B)], 123.03 [C-5′ (B)], 123.21 [C-5′ (H)],
123.29 [CR-5′ (H)], 124.23 [C-6′ (H)], 125.25 [CR-6′ (B)],
125.54 [C-6′ (B)], 128.47 [CR-2′/6′ (E)], 128.85 [C-2′/6′ (E)],
133.67 [C-1′ (E)], 134.28 [CR-1′ (E)], 134.784 [C-1′ (H)],
135.33 [C-1′ (B)], 135.41 [CR-1′ (B)], 135.52 [CR-1′ (H)],
141.45–143.23 [C-3′/4′ (B, H)], 147.58 [C-7 (G)], 147.76 [CR-
7 (G)], 148.09 [CR-5 (D)], 148.25 [C-5 (D)], 148.51 [CR-5
(G)], 148.55 [C-5 (G)], 149.58 [C-7 (D)], 150.20 [C-5/7 (A)],
150.78 [CR-4′ (E)], 150.99 [C-1′ (E)], 151.87 [C-8a (G)],
152.11 [CR-8a (D)], 152.40 [C-8a (D)], 153.37 [CR-8a (G)],
153.83 [CR-8a (A)], 154.12 [C-8a (A)], 168–172 [–CO–CH3].
Degradation of 20 mg 23 with 30 mg phloroglucinol
in 2 ml 1% ethanolic HCl yielded epicatechin (2) and 29,
which were purified using a Sephadex® LH-20 column
(25×80 mm) with first 300 ml EtOH, then 300 ml MeOH.
Compound 29 (12 mg) was peracetylated for analytical in-
vestigation to epicatechin-(2β→7, 4β→8)-epicatechin-
(4β→8)-phloroglucinol-peracetate (29a, 14 mg: [α]2D0
=
+106.82° (c=0.44); ESI-MS: [M+Na]+ m/z 1127.5.; [V]210
-34546, [V]230 67077, [V]250 5536, [ ]270 22775, [V]284 -3387;
1H NMR (CDCl3, 400 MHz): δ 1.56–2.33 [3H, all s, aliphatic
and phenolic –OAc], 4.42 [d, J=3.2 Hz, H-4 (F)], 4.60 [d, J=
4.2 Hz, H-4 (C)], 5.01 [d, J=4.2 Hz, H-3 (C)], 5.02 [dd, J=1.6
and 3.2 Hz, H-3 (F)], 5.52 [d, J=1.6 Hz, H-2 (F)], 6.50
[d, J=2.4 Hz, H-6 (A)], 6.55 [s, H-6 (D)], 6.84 [d, J=2.4 Hz,
H-4/6 (G)], 6.85 [d, J=2.4 Hz, H-8 (A)], 6.94 [d, J=2.4 Hz, H-
6/4 (G)], 7.13 [d, J=8.2 Hz, H-5′ (E)], 7.20 [dd, J=2.0
and 8.2 Hz, H-6′ (E)], 7.26 [d, J=2.0 Hz, H-2′ (E)], 7.27
[d, J=8.2 Hz, H-5′ (B)], 7.48 [d, J=2.0 Hz, H-2′ (B)], 7.58
[dd, J=2.0 and 8.2 Hz, H-6′ (B)]; 13C NMR (CDCl3, 100 MHz):
δ 19–21 [–CO–CH3], 27.29 [C-4 (C)], 33.82 [C-4 (F)], 67.76
[C-3 (C)], 70.14 [C-3 (F)], 75.22 [C-2 (F)], 97.75 [C-2 (C)],
104.15 [C-6 (D)], 106.71 [C-4a (F)], 107.20 [C-8 (A)], 109.70
[C-6 (A)], 113.06 [C-4a (A)], 114,39 [C-4/6 (G)], 115.24 [C-6/4
(G)], 118.59 [C-8 (D)], 120.24 [C-2 (G)], 122.82 [C-5′ (E)],
2.3.10. Epicatechin-3-O-gallate-(2β→7, 4β→8)-epicatechin-
(4β→8)-epicatechin (25)
Compound 25 (20 mg) was peracetylated for analytical
investigation to epicatechin-3-O-gallate-(2β→7, 4β→8)-

488
J. Bicker et al. / Fitoterapia 80 (2009) 483–495
epicatechin-(4β→8)-epicatechin-peracetate (25a, 27 mg):
[α]2D0 = +54.84° (c = 0.93); ESI-MS: [M + NH3]+ m/z
1706.5 [M+Na]+ m/z 1711.3; [V]212 126263, [V]224 76799,
[V]230 83144, [V]253 2325, [V]271 32230, [V]286 -7304, 1H
NMR (CDCl3, 500 MHz): δ 1.56–2.33 [3H, all s, aliphatic
and phenolic –OAc], 2.85–2.97 [m, H-4a,b (I)], 4.22 [d, J=
4.3 Hz, H-4 (C)], 4.35 [d, J=2.8 Hz, H-4 (F)], 4.74 [s, H-2 (I)],
5.08 [m, H-3 (I)], 5.12 [d, J=4.3 Hz, H-2 (C)], 5.21 [m, H-3 (F)],
5.77 [s, H-2 (F)], 6.32 [s, H-6 (D)], 6.57 [d, J=2.2 Hz, H-6 (A)],
6.63 [s, H-6 (G)], 6.71 [d, J=2.2 Hz, H-8 (A)], 7.06 [dd, J=2.0
and 8.5 Hz, H-6′ (H)], 7.12 [d, J=8.5 Hz, H-5′ (H)], 7.12
[d, J=8.3 Hz, H-6 (D)], 7.14 [d, J=8.6 Hz, H-5′ (B)], 7.19
[dd, J=2.0 and 8.3 Hz, H-6′ (E)], 7.21 [s, H-2″/6″ (J)], 7.31
[d, J=2.0 Hz, H-2′ (E)], 7.36 [d, J=2.2 Hz, H-2′ (B)], 7.36
[d, J=1.9 Hz, H-2′ (H)], 7.43 [dd, J=2.2 and 8.6 Hz, H-6′ (B)],
13C NMR (CDCl3, 125 MHz): δ 19–21 [–CO–CH3], 26.06 [C-4 (I)],
27.26 [C-4 (C)], 34.02 [C-4 (F)], 66.41 [C-3 (I)], 68.19 [C-3 (C)],
70.92 [C-3 (F)], 76.69 [C-2 (I)], 76.87 [C-2 (F)], 98.27 [C-2 (C)],
104.76 [C-6 (D)], 106.32 [C-8 (A)], 108.17 [C-8 (D)], 108.65
[C-4a (D)], 110.35 [C-6 (A)], 110.35 [C-6 (G)], 111.04 [C-4a
(G)], 114.06 [C-4a (A)], 117.03 [C-8 (G)], 122.40 [C-2′ (H)],
122.59 [C-2′ (B)], 122.79 [C-2″/6″ (J)], 122.82–123.34 [C-5′
(B, E, H)], 123.18 [C-2′ (E)], 123.31 [C-6′ (H)], 125.46 [C-6′
(B)], 125.56 [C-5′ (E)], 127.63 [C-1″ (J)], 135.01 [C-1′ (H)],
135.19 [C-1′ (B)], 135.65 [C-1′ (E)], 138.80 [C-4″ (J)], 141.69–
142.91 [C-3′/4′ (B, E, H)], 143.06 [C-3″/5″ (J)], 147.77 [C-5
or C-7 (G)], 147.81 [C-5 (A)], 148.10 [C-5 (D)], 148.79 [C-5 or
C-7 (G)], 149.30 [C-7 (A)], 149.41 [C-7 (D)], 152.44/153.50
[C-8a (D, G)], 154.24 [C-8a (A)], 162.18 [Carboxyl-C (J)],
166.15–170.44 [–CO–CH3].
(B)], 122.75–123.18 [C-5′ (B, H, K)], 123.25 [C-5′ (E) and C-6′
(H)], 124.43 [C-2′ (K)], 125.27 [C-6′ (B) or C-6′ (E)], 125.35
[C-6′ (E) or C-6′ (B)], 126.02 [C-6′ (K)], 134.51–135.16 [C-1′
(B, E, H)], 136.91 [C-1′ (K)], 141.40–143.14 [C-3′/4′ (B, E, H,
K)], 147.42 [C-7 (G)], 147.77 [C-7 (D)], 148.68 [C-5 (G)],
148.94 [C-5 (A)], 149.01 [C-5 (D)], 149.80 [C-7 (A)], 150.18
[C-7 or C-5 (J)], 150.24 [C-5 or C-7 (J)], 151.53 [C-8a (D)],
151.81 [C-8a (G)], 153.44 [C-8a (A)], 154.41 [C-8a (J)], 168–
172 [–CO–CH3].
2.3.12. 1-O-β-D-(2,4-dihydroxy-6-methoxyphenyl)-6-O-(4-
hydroxy-3,5-dimethoxybenzoyl)-glucopyranoside (28)
ESI-MS: [M+Na]+ m/z 521.2; m/z 1018.8 [2 M+Na]+
;
1H NMR (MeOD, 400 MHz): δ 3.39–3.48 [m, H-4, H-3 and H-
2], 3.65 [m, H-5], 3.69 [3H, s, –OCH3], 3.88 [2×3H, s, –OCH3],
4.38 [dd, J=7.1 and 12.0 Hz, H-6a], 4.58 [d, J=7.5 Hz, H-1],
4.69 [dd, J=2.0 and 12.0 Hz, H-6b], 5.90 [d, J=2.7 Hz, H-3′],
5.96 [d, J=2.7 Hz, H-5′], 7.33 [2H, s, H-2″/6″]; 13C NMR
(MeOD, 100 MHz): δ 56.51 [–OCH3], 56.95 [2×–OCH3], 65.27
[C-6], 71.84 [C-4], 75.25 [C-2], 76.21 [C-5], 77.56 [C-3],
93.15 [C-5′], 96.84 [C-3′], 107.61 [C-1], 108.41 [C-2″/6″],
121.29 [C-1″], 128.84 [C-1′], 142.09 [C-4″], 148.95 [C-3″/5″],
152.29 [C-2′], 154.73 [C-6′], 156.40 [C-4′], 167.98 [C-7″].
2.3.13. Epicatechin-(2β→7, 4β→8)-epicatechin-(4β→8)-
phloroglucinol (29)
Degradation of 20 mg 23 with 30 mg phloroglucinol
in 2 ml 1% ethanolic HCl yielded epicatechin (2) and 29,
which were purified using a Sephadex® LH-20 column
(25×80 mm) with first 300 ml EtOH, then 300 ml MeOH.
Compound 29 was peracetylated for analytical investigation
to epicatechin-(2β→7, 4β→8)-epicatechin-(4β→8)-phlor-
oglucinol-peracetate (29a): [α]2D0 =+106.82° (c=0.44);
ESI-MS: [M+Na]+ m/z 1127.5.; [V]210 -34546, [V]230
67077, [V]250 5536, [V]270 22775, [V]284 -3387; 1H NMR
(CDCl3, 400 MHz): δ 1.26–2.33 [m, aliphatic and aromatic
OAc], 4.42 [d, J=3,2 Hz, H-4 (F)], 4.60 [d, J=4,2 Hz, H-4 (C)],
5.01 [d, J=4,2 Hz, H-3 (C)], 5.02 [dd, J=1,6 and 3,2 Hz, H-3
(F)], 5.52 [d, J=1,6 Hz, H-2 (F)], 6.50 [d, J=2,4 Hz, H-6 (A)],
6.55 [s, H-6 (D)], 6.84 [d, J=2.4 Hz, H-4/6 (G)], 6.85
[d, J=2,4 Hz, H-8 (A)], 6.94 [d, J=2.4 Hz, H-6/4 (G)], 7.13
[d, J=8.2 Hz, H-5′ (E)], 7.20 [dd, J=2.0 and 8,2 Hz, H-6′ (E)],
7.26 [d, J=2.0 Hz, H-2′ (E)], 7.27 [d, J=8.2 Hz, H-5′ (B)],
7.48 [d, J=2.0 Hz, H-2′ (B)], 7.58 [dd, J=2,0 and 8,0 Hz, H-6′
(B)]; 13C NMR (CDCl3, 100 MHz): δ 27.29 [C-4 (C)], 33.82
[C-4 (F)], 67.76 [C-3 (C)], 70.14 [C-3 (F)], 75.22 [C-2 (F)],
97,75 [C-2 (C)], 104.15 [C-6 (D)], 106.71 [C-4a (F)], 107.20
[C-8 (A)], 109.70 [C-6 (A)], 113.06 [C-4a (A)], 114,39 [C-4/6
(G)], 115.24 [C-6/4 (G)], 118.59 [C-8 (D)], 120.24 [C-2 (G)],
122.82 [C-5′ (E)], 123.02 [C-2′ (B and E)], 124.74 [C-5′ (B)],
125.30 [C-6′ (B)], 126.15 [C-6′ (E)], δ 134.70 [C-1′ (E)],
δ 135.23 [C-1′ (B)], δ 141.64-143.02 [C-3′/4′ (B and E)],
148.69–150.41 [C-5/7 (A and D); C-1/3/5 (G)], 152.04 [C-8a
(D)]; 155.57 [C-8a (A)].
2.3.11. Epicatechin-(2β→7, 4β→8)-[epicatechin-(4β→6)]-
epicatechin-(4β→8)-epicatechin (27)
Compound 27 (parameritannin A1, 30 mg) was peracety-
lated for analytical investigation to epicatechin-(2β→7,4β→8)-
[epicatechin-(4β→6)]-epicatechin-(4β→8)-epicatechin-perace-
tate (27a, 44 mg): [α]D20=+71.07° (c=0.16); ESI-MS: [M+
Na]+ m/z 1973.2; [ ]230 140162, [ ]255 15355, [ ]275 35441; 1H
NMR (CDCl3, 600 MHz): δ 1.48–2.35 [3H, all s, aliphatic and
phenolic –OAc], 2.94–3.12 [m, H-4a,b (I)], 4.37 [brs, H-4 (F)],
4.63 [brs, H-4 (L)], 4.73 [d, J=4.2 Hz, H-4 (C)], 5.09 [brs, H-2 (I)],
5.11 [d, J=4.2 Hz, H-3 (C)], 5.13 [m, H-3 (F)], 5.45 [brs, H-2 (F)],
5.46 [m, H-3 (I)], 5.54 [m, H-3 (L)], 5.67 [brs, H-2 (L)], 6.50
[s, H-6 (G)], 6.57 [d, J=2.2 Hz, H-6 or H-8 (A)], 6.62
[d, J=2.3 Hz, H-6 or H-8 (J)], 6.72 [d, J=2.3 Hz, H-8 or H-6
(J)], 6.80 [d, J=2.2 Hz, H-8 or H-6 (A)], 7.07 [dd, J=2.0 and
8.4 Hz, H-6′ (H)], 7.09 [d, J=2.0 Hz, H-2′ (H)], 7.11
[d, J=8.4 Hz, H-5′ (H)], 7.13 [d, J=8.4 Hz, H-5′ (K)], 7.14
[d, J=8.4 Hz, H-5′ (B)], 7.19 [d, J=8.4 Hz, H-5′ (E)], 7.26
[dd, J=2.0 and 8.4 Hz, H-6′ (K)], 7.29 [d, J=2.0 Hz, H-2′ (K)],
7.43 [2×d, J=2.0 Hz, H-2′ (B) and (E)], 7.50 [dd, J=2.0 and
8.4 Hz, H-6′ (E)], δ 7.55 [dd, J=2.0 and 8.4 Hz, H-6′ (B)]; 13
C
NMR (CDCl3, 125 MHz): δ 19–21 [–CO–CH3], 26.33 [C-4 (I)],
27.67 [C-4 (C)], 32.67 [C-4 (L)], 33.36 [C-4 (F)], 66.42 [C-3
(I)], 67.91 [C-3 (C)], 69.72 [C-3 (F)], 69.85 [C-3 (L)], 74.15
[C-2 (L)], 75.29 [C-2 (F)], 76.76 [C-2 (I)], 98.88 [C-2 (C)],
106.64 [C-6 or C-8 (A)], 107.20 [C-6 or C-8 (J)], 107.68 [C-8 or C-6
(J)], 108.79 [C-8 (D)], 109.08 [C-4a (D)], 109.23 [C-6 (D)], 110.24
[C-8 or C-6 (A)], 110.34 [C-4a (G)], 111.04 [C-6 (G)], 113.52
[C-4a (A)], 114.09 [C-4a (J)], 118.32 [C-8 (G)], 121.53 [C-2′
(H)], 122.01 [C-2′ (B) or C-2′ (E)], 122.62 [C-2′ (E) or C-2′
2.3.14. Epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-
gallate-phloroglucinol (30)
Degradation of 18 mg 22 with 30 mg phloroglucinol in
2 ml 1% ethanolic HCl yielded 30, and 12 which were purified
using a Sephadex® LH-20 column (25×80 mm) with first
300 ml EtOH, then 300 ml MeOH.

J. Bicker et al. / Fitoterapia 80 (2009) 483–495
489
Compound 30 (8 mg) was peracetylated for analytical
investigation to epicatechin-3-O-gallate-(4β→8)-epicatechin-
3-O-gallat-phloroglucinol-peracetate (30a, 11 mg): the spec-
troscopic values (1H NMR, MS, CD) were identical with pub-
lished data [9].
(4β→8)-catechin, 4) B2 (epicatechin-(4β→8)-epicatechin,
5), B3 (catechin-(4α→8)-catechin, 6), B4 (catechin-(4α→8)-
epicatechin, 7), A2 (epicatechin-(2β→7, 4β→8)-epicatechin,
15), B5 (epicatechin-(4β→6)-epicatechin, 14) and B7 (epi-
catechin-(4β→6)-catechin, 15) by comparison of the spec-
troscopic data (1H NMR-, ESI-MS- and CD spectra, optical
rotation) of the peracetylated derivatives 4a–7a and 13a–15a
with published data [11,16,17,33]. Extensive investigation of
the proton chemical shifts in comparison with the values of
peracetylated synthetic procyanidin diastereoisomers [18]
showed that ent-catechin is not a “lower” part of these
procyanidins, because such dimers will show significant shifts
within the heterocyclic and A-ring protons. Further dimeric
proanthocyanidins (Fig. 2) are epiafzelechin-(4β→8)-epica-
techin (8), epiafzelechin-3-O-gallate-(4β→8)-epicatechin-3-
O-gallate (10), epicatechin-(4β→8)-epicatechin-3-O-gallate
(11), epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gal-
late (12), epicatechin-(4β→6)-epicatechin-3-O-gallate (17)
and epicatechin-3-O-gallate-(4β→6)-epicatechin-3-O-gal-
late (18). The spectroscopic data of the peracetates 11a, 12a
and 18a correlated well with published values [16,19–25].
Due to the lack of reference data for the peracetylated com-
pounds 8a, 10a and 17a we here report the complete NMR
data set for these derivatives in detail.
3. Results and discussion
The total tannin content of a water extract from the dried
aerial parts of R. acetosa L. was determined with 3.6% by the
hide powder method of Pharmacopoeia Europea calculated as
pyrogallol. For a detailed investigation of the tannins a crude
acetone–water (7:3) extract of the aerial parts of R. acetosa
was partitioned between ethyl acetate (EtOAc) and water to
yield fractions enriched with flavan-3-ols and low molecular
weight proanthocyanidins (EtOAc extract), and oligomeric
proanthocyanidins of higher molecular weight (water ex-
tract), respectively. TLC studies with different staining
methods of both extracts indicated the presence of pro-
anthocyanidins and the absence of hydrolysable tannins. The
EtOAc-soluble fraction was fractionated subsequently on
Sephadex® LH-20 with ethanol, methanol and an acetone–
water mixture. Fractions obtained were further purified using
multilayer countercurrent chromatography (MLCCC), fast
centrifugal partition chromatography (FCPC), low pressure
chromatography on MCI® gel CHP20P, MPLC on RP-18
material or preparative TLC of the respective peracetylated
derivatives to afford compounds 1 to 28 (Figs. 1–4). Structure
elucidation was performed by 1D- and 2D-NMR as free
phenols or peracetylated derivatives, circular dichroism (CD),
optical rotation [α]2D0, ESI-MS experiments and, in part, by
partial acid-catalysed degradation with phloroglucinol. With-
in the structural investigations of the highly complex R.
acetosa proanthocyanidins the native, free-phenolic com-
pounds had to be analysed, but in some cases a peracetylation
of complex fractions, of isolated free-phenolic compounds
and of degradation products of complex oligomeric proantho-
cyanidins was necessary for effective isolation and unambig-
uous structural elucidation.
Compounds 1–3 (Fig. 1) were identified by the spectro-
scopic data for both, the free-phenolic compounds and their
peracetates 1a–3a as catechin, epicatechin and epicatechin-3-
O-gallate, respectively [10–12]. The decreased positive Cotton
effect at 235 nm in the CD spectrum and the comparatively
low optical rotation+16.4° (c 0.14, MeOH) of compound 1a
indicated the presence of a mixture of (2R, 3 S)-catechin
and (2 S, 3R)-ent-catechin. Quantitative CE-analysis of 1
confirmed the presence of both, catechin and ent-catechin
in a 60:40 ratio. The presence of both enantiomers in one
plant is in many cases not investigated in detail [13]
especially if the biosynthetic pathway towards (+)-catechin
and (−)-epicatechin is considered as the main stream.
The values for the optical rotation as well as the CD spectra
of compounds 2a and 3a were consistent with literature data
[14,15] and to those of authentic reference samples. Thus,
compounds 2 and 3 were confirmed as epicatechin and
epicatechin-3-O-gallate.
Known trimeric proanthocyanidins from R. acetosa were
transferred after isolation to the respective peracetates
and subsequently identified as the peracetates 20a and 21a of
procyanidin C1 (epicatechin-(4β→8)-epicatechin-(4β→8)-epi-
catechin, 20) and epicatechin-(4β→8)-epicatechin-(4β →8)-
catechin (21) [17,23,26] (Fig. 2).
Cinnamtannin B1 (epicatechin-(2β→7, 4β→8)-epicate-
chin-(4β→8)-epicatechin, 23) was characterized as free-
phenolic compound 23 and as its peracetate 23a. (Fig. 3). The
NMR data of 23 were consistent with literature data
published for cinnamtannin B1 [27–29]. However, the data
for the peracetylated compound 23a were consistent with the
published values of the corresponding derivative of pavetan-
nin B1 (epicatechin-(2β→7, 4β→8)-epicatechin-(4β→8)-
ent-epicatechin) instead of cinnamtannin B1 [30]. In contrast,
the NMR spectrum of 23a was superimposable with those of
the peracetate derivative of an authentic reference compound
isolated from Laurus nobilis (unpublished results). In order to
determine unambiguously the absolute configuration of the
terminal flavan-3-ol, 23 was subjected to a controlled acid-
catalysed degradation in the presence of phloroglucinol [7].
From the reaction mixture epicatechin (2) was identified
(NMR, CD, [α]2D0) after peracetylation (2a) as a major
degradation product and therefore 2 must be the ‘bottom’
flavan-3-ol unit (Fig. 1). Also epicatechin-(2β→7, 4β→8)-
epicatechin-(4β→2)-phloroglucinol (29) (Fig. 3) was isolat-
ed from the reaction mixture and identified as its peracetate
(29a) in comparison with the spectroscopic data of the
same derivative performed as phloroglucinol cleavage prod-
uct of cinnamtannin B1 from L. nobilis (unpublished results).
Thus, compound 23 was confirmed to be cinnamtannin B1
and the published data [30] for the peracetylated derivative
has to be revised.
Compounds 4–7 and 13–15 (Figs.
1
and 2) were
Furthermore, the trimer epicatechin-3-O-gallate-(4β→8)-
epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate
(22) was isolated (Fig. 2). This compound as well as the
respective peracetate derivative 22a showed very complex
transferred after isolation of the free penolic compounds to
the respective peracetates and subsequently identified as the
peracetates of the dimeric procyanidins B1 (epicatechin-

490
J. Bicker et al. / Fitoterapia 80 (2009) 483–495
Fig. 1. Structural features of proanthocyandidins 1 to 12 isolated from R. acetosa and the respective peracetate derivatives produced from the free-phenolic
compounds.
Fig. 2. Structural features of compounds 13 to 22 isolated from R. acetosa and the respective peracetate derivatives produced from the free-phenolic compounds.

J. Bicker et al. / Fitoterapia 80 (2009) 483–495
491
spectra in ambient and low temperature (243 K) NMR
experiments [44] so that a complete assignment of signals
failed. Therefore 22 was hydrolyzed by acid cleavage in the
present of phloroglucinol in analogy to the experiments
performed with compound 23. The resulting cleavage pro-
ducts were identified (NMR, MS, CD, [α]2D0) as the correspon-
ding peracetate derivatives 12a and 30a of epicatechin-3-O-
gallate-(4β→8)-epicatechin-3-O-gallate (12) and epicate-
chin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate-(4β→2)-
phloroglucinol (30). Thus, the structure of 22 was deduced
from these cleavage products.
Tetrameric proanthocyanidins were identified (Fig. 3) as
their peracetates 26a and 27a with the structural features of
procyanidin D1 (epicatechin-(4β→8)-epicatechin-(4β→8)-
epicatechin-(4β→8)-epicatechin, 26) and parameritannin A1
(epicatechin-(2β→7,4β→8)-(epicatechin-(4β→6))-epicate-
chin-(4β→8)-epicatechin, 27) in comparison with literature
data [27,31]. Structure elucidation of the later peracetate
(27a) was also confirmed by the comparison of its spectro-
scopic data with a reference sample isolated from L. nobilis
(unpublished results).
A more detailed description is made on the structural
features of compounds 9, 16, 19, 24, 25 and 28 which were
found to the best of our knowledge to be new natural products.
Compound 9 was characterized after acetylation as its
peracetate 9a. The ESI-MS pseudomolecular ion ([M+Na]+
m/z 1199.5) of 9a indicated the presence of a monogalloy-
lated dimeric proanthocyanidin with an (epi)catechin and an
(epi)afzelechin unit. 1H NMR in CHCl3 revealed its close
structural resemblance to that of the corresponding deriva-
tive of epiafzelechin-(4β→8)-epicatechin (8a). To prove the
4→8 interflavan linkage in 9a indirect shift parameters were
used. The chemical shift of the A-ring protons (δ 6.12 and
6.50 ppm; [32]) and the strong dominance of one rotamer
(ca 3–5:1; [7]) correlated well with published data for 4→8
linked proanthocyanidins. In contrast to compound 8a,
signals in 9a for H-2 (F) and H-3 (F) were shifted downfield
(ca Δδ 0.2 ppm), probably due to the substitution of the
hydroxyl group at C-3 (F) with gallic acid. Unfortunately, no
direct proof for the point of attachment was visible in the
HMBC spectrum due to the lack of a correlation between the
carboxyl carbon and the respective H-3 proton of the “upper”
Fig. 3. Structural features of compounds 23 to 27, 29 and 30 isolated from R. acetosa and the respective peracetate derivatives produced from the free-phenolic
compounds; key correlations in the 2D-NMR (HMBC) are marked with arrows for 24a and 25a.

492
J. Bicker et al. / Fitoterapia 80 (2009) 483–495
Fig. 3 (continued).
or “bottom” flavan-3-ol unit. However, further evidence for
the point of acylation was deduced by the 1H NMR spectra of
peracetylated dimeric proanthocyanidins from the observa-
tion that the chemical shift of the two-proton singlet of the
gallic acid moiety obviously depends on where the esterifi-
cation has taken place: protons of a galloyl moiety located at
the C-3 hydroxyl of the “bottom”-units are found to have
resonances at δ7.64–7.72 ppm, while substitution at C-3 of
the “upper”-units are monitored at δ7.50–7.60 ppm [43], see
also compounds 10–12, 17, 18). Therefore, the two-proton
Fig. 4. Structure of 1-O-β-D-(2,4-dihydroxy-6-methoxyphenyl)-6-O-(4-hydroxy-3,5-dimethoxybenzoyl)-glucopyranoside 28; key correlations in the 2D-NMR
(HMBC) are marked with arrows.

J. Bicker et al. / Fitoterapia 80 (2009) 483–495
493
resonance at δ 7.64 ppm in 9a indicated “bottom” acyl
substitution of 9. Within HMBC experiments the correlation
of H-2 (C) to the C-2′/6′ signals of the monohydroxylated B
ring provide the “upper”-unit as epiafzelechin. HMBC cor-
relations of protons H-6 (D) with C-7 (D), C-5 (D), C-8 (D)
and C-4a (D) indicated again the existence of a 4→8 linkage.
The relative 2,3-cis configuration of the flavan-3-ol units was
obvious from the small coupling constants of all heterocyclic
protons (J≤2 Hz). The 4R configuration was deduced from
the strong positive Cotton effect within 200–240 nm in the CD
spectrum of 9a [11]. Thus, 9 was deduced to be epiafzelechin-
(4β→8)-epicatechin-3-O-gallate, a new natural product.
Compound 19, after peracetylation (19a) showed a pseu-
domolecular ion [M+Na]+ at m/z 1461.5 indicating the
presence of two (epi)catechin and one (epi)afzelechin flavan-
3-ol unit. The small coupling constants of the heterocyclic
protons (J≤2 Hz) in the 1H NMR spectrum of 19a indicates
the relative 2,3-cis configuration of all the flavan-3-ol units.
Within HMBC experiments the signals of H-2 (C), H-2 (F) and
H-2 (I) coupled to the respective C-1′ signals of the aromatic
rings B, E and H. Long-range connectivities (HMBC) between
the H-3′/5′ (B) protons and the carbon C-1′ (B) determined
the “upper”-unit to be epiafzelechin. The comparison of the
1H NMR spectrum of 19a with that of procyanidin C1 (20a)
proves the strong resemblance except for an AA′BB′ spin-
system in 19a instead of an AMX-spin-system in 20a. The
high amplitude positive Cotton effect in the 200–240 nm
region of the CD spectrum indicated the configuration at both
interflavan linkages to be 4R. Consequently, the structure
of 19 was deduced to be epiafzelechin-(4β→8)-epicatechin-
(4β→8)-epicatechin, a new natural product.
The ESI-MS of compound 24 after acetylation in form of its
peracetate 24a gave a pseudomolecular ion [M+Na]+ at m/z
1417.6 indicating the presence of a trimeric A-type pro-
cyanidin composed of two (epi)catechin units and one (epi)
afzelechin unit. 1D- and 2D-NMR spectra of 24a were similar
to those obtained from cinnamtannin B1 (23a). The HMBC
spectrum of 24a is given in Fig. 3 and displayed for the proton
H-4 (C), which could be assigned to the A-type-linked flavan-
3-ol, and H-8 (A) a coupling to the carbon C-8a (A) of the
“upper” unit. This shows that the additional A-type linkage is
located between the “upper” and the “middle” flavan-3-ol.
The presence of all 4→8 linkages was proven by the coupling
of H-6 (D/G) to the respective signals of C-5 (D/G) and C-7 (D/
G). H-2 (F) shows a 3J-coupling to the C-2′/6′ of the
epiafzelechin unit which means that the 1,4-disubstituted
aromatic ring is connected to the “middle” flavan-3-ol unit.
The coupling constants of the proton signals of the “middle”
unit (J3,4 =4,8 Hz; J2,3 =2,2 Hz) are to high for a typical 2,3-
cis-3,4-trans-configuration, but to low for a 2,3-trans-3,4-
trans-configuration. According to Schlepp et al. [35] such
coupling constants are typical for 4→8 α-linked 2,3-cis-
flavan-3-ols. To investigate the exact orientation of the inter-
flavan linkage 1D ROESY-NMR experiments were performed
and compared with data obtained from at position C-4 (F) β-
configurated cinnamtannin B1 (23a). Irradiation of H-2 (I)
from the cinnamtannin B1 resulted in signals from H-6 (D),
H-3 (C) and H-2 (F). In contrast to that no signals of these
protons were observed in case of irradiation of H-2 (I) from
24a. This indicates α-orientation of the interflavan linkage.
The CD spectrum of compound 24a showed a decreased
positive Cotton effect between 200 and 240 nm compared to
the CD spectrum of compound 23a. This is another hint for
the alpha-orientation of the interflavan linkage. However, an
ent-configuration of the epiafzelechin unit cannot be exclud-
ed. Thus, the structure of 24 can tentatively be described as
epicatechin-(2β→7, 4β→8)-epiafzelechin-(4α→8)-epicate-
chin, a new natural product.
Compound 16 was characterized after acetylation as its
peracetate 16a. The ESI-MS pseudomolecular peak m/z
1199.2 ([M+Na]+) for 16a indicated the presence of a
monogalloylated dimeric proanthocyanidin with an (epi)
catechin and an (epi)afzelechin unit.
Due to broadening signals caused by of rotational isom-
erism the 1H NMR spectrum of 16a at ambient temperature
and the similarity to the spectrum of 17a indicated the
presence of a 4→6 linkage [7]. The downfield shift of both
the A-ring proton resonance (δ 6.74 ppm, H-8) and the H-2
(F) (δ 5.26 ppm) confirmed the 4→6 interflavanoid linkage
[32,33]. The substitution of gallic acid to the “bottom” unit
was shown again by the typical resonance for the two-proton
singlet at δ 7.64 ppm as argued above (see compound 9).
Unfortunately the yield of 16a was to low to perform 13C NMR
and 2D NMR experiments. To clarify the sequence of the two
flavan-3-ol units, oxidative cleavage of the C–C interflavan
linkage under acidic conditions was performed. Under the
conditions of this anthocyanidin reaction the C–C interflavan
linkages will be cleaved and coloured anthocyanidium
cations will be released, which can be identified easily after
TLC separation against the respective reference compounds.
During this experiment the resulting cleavage product from
the free-phenolic dimer 16 was identified by TLC and
respective reference compounds to be pelargonidin, origi-
nating from the former “upper” flavan-3-ol unit. The high
amplitude positive Cotton effects at low wavelength in the CD
spectrum of 16a confirmed the absolute configuration as 4R.
Thus, the structure of 16 was deduced to be epiafzelechin-
(4β→6)-epicatechin-3-O-gallate, also a new natural product.
Compound 25 was characterized after acetylation as its
peracetate 25a. The ESI-MS pseudomolecular ion m/z 1711.3
[M+Na]+ agreed with a monogalloylated trimeric A-type
procyanidin. Within HMBC experiments (Fig. 3) the signal of
H-4 (C) (δ 4.21 ppm), which correlates to a flavan-3-ol unit
with A-type linkage, coupled to the C-4a of the “upper” unit.
The same carbon connectivity was due for H-8 (A). This
means that the ether bridging system must be located be-
tween the “upper” and “middle” flavan-3-ol units.
The 4→8 linkage of all three flavan-3-ols was deduced by
the coupling of signals from H-6 (D/G) to C-5 and C-7 (D/G).
The galloylation of the “upper” flavan-3-ol unit was evident
from the typical signal at δ 7.21 ppm and by the 3J-coupling
of H-2″/6″ (J) and H-3 (C) to the carboxyl group of the gallic
acid moiety. The course of the CD spectrum and the optical
rotation values were comparable to those measured for 23a.
For that identical stereochemical properties of 25 and
cinnamtannin B1 (23) can be deduced, leading to the struc-
tural features for 25 to be epicatechin-3-O-gallate-(2β→7,
4β→8)-epicatechin-(4β→8)-epicatechin. So far, galloylated
A-type proanthocyanidins have not been described before.
A polymeric procyanidin fraction was obtained from the
aqueous phase of the water–EtOAc partition after elution
from Sephadex® LH20 according [34] an average degree of

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J. Bicker et al. / Fitoterapia 80 (2009) 483–495
polymerisation with 7 to 8 flavan-3-ol units [19,26,38] by
using the ratio of the signals of C-3 of the terminal unit at δ
67 ppm and those of the C-3 carbons of the extender flavan-3-
ol units at δ 73 ppm. The dominance (ca 5:1) of procyanidin
residues over the propelargonidin units was deduced by the
intensity of the respective signals of C-3′ and C-4′ resonances
of the 1,3,4-trisubstituted B rings (δ 145 ppm) and the C-4′ of
the 1,4-substituted analogues at δ 157 ppm. Flavan-3-ol
residues within the polymer chain were mainly 2,3-cis
configurated (typical signal of C-2 at δ 76 ppm) [36,39]. A
signal indicating the presence of 2,3-trans units at δ 79 ppm
was below the limit of quantitation. The presence of gallate
units was obvious by the carbon chemical shift at δ 110, 122
and 139 ppm as well as the carbonyl carbon chemical shift at
δ 166 ppm [37].
Compound 28 was initially regarded to be a proanthocya-
nidin or flavan-3-ol-glycoside due to its red coloured spot on
TLC after vanillin/HCl spray detection and its typical UVmax
around 274 nm. However, the elution before monomeric
flavan-3-ols from the Sephadex® LH-20 column and identical
chromatographic behaviour were observed for phlorogluci-
nolglycosides [40]. The 1H NMR in MeOD indicated signals
typical for carbohydrate residues (δ 3.39–4.69 ppm), also
typical for an aromatic two-proton singlet (δ 7.33 ppm) and a
methoxy substitution pattern (δ 3.69 and δ 3.88 ppm). Within
the HMBC spectrum long-range correlations (3 J) between
the H-6 protons and a carboxylic carbon (C-7″) and the
anomeric proton H-1 to the C-1′ of a further aromatic ring
system showed that the carbohydrate part is located between
two aromatic systems (Fig. 2). Complete HMBC assignments
and NOE experiments indicated one aromatic system to
be 2,4-dihydroxy-6-methoxyphenol and the other one to be
4-hydroxy-3,5-dimethoxy-carboxylic acid (syringic acid).
Identity of the carbohydrate part of the molecule as D-glucose
was determined by capillary zone electrophoresis after
hydrolysis [8] against the respective reference compounds.
β-configuration of the glycosidic linkage was verified by the
large coupling constant 3JH-1/H-2 =7.5 Hz.
interesting that in the B-ring monohydroxylated flavan-3-ol
unit (epiafzelechin) are, except for compound 24, always
found as the “upper” flavan-3-ol unit. On the other side
epiazelechin was not found as flavan-3-ol. These findings
have to be discussed that in the B-ring monohydroxylated
precursors are more effectively converted into the biosyn-
thesis of oligomeric proanthocyanidins.
Spencer et al. [42] recently investigated the proanthocya-
nidin pattern of Rumex obtusifolius. The proanthocyanidin
pattern of this species and R. acetosa seem to be similar
concerning A- and B-type linked procyanidins. Even no
compounds containing epiafzelechin subunits were found,
the published 13C-spectrum of the polymeric fraction of R.
obtusifolius seems to be almost identical to the one recorded
for R. acetosa. In conclusion, the proanthocyanidin pattern of
the two Rumex species are closely related and the existence of
propelargonidins also in R. obtusifolius may be supposed.
Acknowledgements
The authors thank Dr. K. Bergander, Dr. H. Lahl and Ms M.
Heim, Münster for recording NMR spectra. Thanks are also
due to Dr. H. Luftmann and Mr T. Meiners, Münster for MS
measurements, to Mr M. Klaes for providing assistance with
measuring CD values and Ms U. Liefländer-Wulf and Ms B.
Quandt for skilful technical assistance.
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