Flavan-3-ols and procyanidins from the bark of Salix purpurea L.

Article abstract of DOI:10.1691/ph.2007.3.6577

From a commercial aqueous ethanolic extract obtained from the bark of Salix purpurea L. the flavan-3-ols catechin, epicatechin, gallocatechin, catechin-3-O-(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic acid)-ester, the dimeric procyanidins B1, B3 and the trimeric procyanidins epicatechin- (4β→8)-catechin-(4α→8)-catechin and epicatechin- (4β→8)-epicatechin-(4β→8)-catechin were isolated. Structure elucidation was performed by NMR, CD, MS, degradation and optical rotation methods. A fraction containing higher oligomeric procyanidins was investigated by ¹³C NMR. Data indicate an average degree of oligomerization of 4 to 5 flavan-3-ol units with dihydroxylated B-rings and predominance of 2,3-cis-stereochemistry.

Full text of DOI:10.1691/ph.2007.3.6577

ORIGINAL ARTICLES
Institute of Pharmaceutical Biology and Phytochemistry, Westfa¨lische Wilhems-University, Mu¨nster, Germany
Flavan-3-ols and procyanidins from the bark of Salix purpurea L.
G. Ju¨rgenliemk, F. Petereit, A. Nahrstedt
Received April 18, 2006, accepted July 10, 2006
Prof. Dr. Adolf Nahrstedt, Institute of Pharmaceutical Biology and Phytochemistry of the Westf. Wilhelms-
University, Hittorfstr. 56, D-48149 Mu¨nster, Germany.
nahrste@uni-muenster.de
Pharmazie 62: 231–234 (2007)
doi: 10.1691/ph.2007.3.6577
From a commercial aqueous ethanolic extract obtained from the bark of Salix purpurea L. the flavan-
3-ols catechin, epicatechin, gallocatechin, catechin-3-O-(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic
acid)-ester, the dimeric procyanidins B1, B3 and the trimeric procyanidins epicatechin-(4ß!8)-cate-
chin-(4a!8)-catechin and epicatechin-(4ß!8)-epicatechin-(4ß!8)-catechin were isolated. Structure
elucidation was performed by NMR, CD, MS, degradation and optical rotation methods. A fraction
containing higher oligomeric procyanidins was investigated by 13C NMR. Data indicate an average
degree of oligomerization of 4 to 5 flavan-3-ol units with dihydroxylated B-rings and predominance of
2,3-cis-stereochemistry.
1. Introduction
2. Investigations, results and discussion
The bark of Salix species (Salicaceae) is traditionally used
as an analgesic, antipyretic and anti-inflammatory crude
drug material. The European Pharmacopoeia (2005) al-
lows several Salix species, including S. purpurea L., S.
daphnoides Vill. and S. fragilis L. containing not less than
1.5% total salicin. Commercial preparations prepared from
water or ethanol/water extracts of Salicis cortex are used
against rheumatic diseases, fever and headache (Meier and
Meier-Liebi 1998). Though there is some information
about the phytochemistry of the drug material (Meier and
Meier-Liebi 1998), most was foccussed on the derivatives
of salicylic alcohol regarded as active constituent for a
long time. There is less information about other secondary
plant constituents such as the proanthocyanidins (for re-
view see Ferreira et al. 1999) for the three single species
accepted by the European Pharmacopeia, or such data
were elaborated from undefined Salix species (Kolodziej
1990). However, for correct characterization and quality
control of the drug material, the phytochemical data of the
dried bark material of each distinct species should be
known.
It was recently shown that the salicylic alcohol derivatives
alone cannot be responsible for the overall efficacy of the
crude drug (Schmidt et al. 2001) which makes it valuable
to collect more knowledge about the compounds present
in well defined Salix species which are used for crude
drug collection. Because proanthocyanidins show anti-in-
flammatory activity in vivo and in vitro (Cos et al. 2003),
this group of compounds was investigated in a commer-
cial extract from the bark of S. purpurea L., one of the
sources of Salicis cortex of the European Pharmacopoeia
(2005), prepared by 70% ethanol. Except catechin, all
compounds reported here were isolated for the first time
from the bark of S. purpurea.
A 70% ethanolic extract from Salicis purpureae cortex was
used for the isolation of flavan-3-ols. Partition between
ethyl-acetate and water gave fractions enriched with low
molecular weight flavan-3-ols and oligomeric proanthocya-
nidins of higher m.w. respectively. Chromatography of both
fractions on Sephadex1LH20 and further purification using
LPLC on MCI1 gel, MPLC on RP-18 and/or HPLC on RP-
18 material yielded compounds 1–8. Structure elucidation
was performed using different spectroscopic techniques,
especially NMR, MS, optical rotation and CD.
ESI-MS data, the typical NMR resonances and optical ro-
tation data identified the monomeric flavan-3-ols as gallo-
catechin (1), catechin (2) and epicatechin (3). Their spec-
troscopic data were identical to those of authentic samples
of our collection and published data (Balde´ et al. 1991;
1
Foo et al. 2000). The H NMR of (4) in the aromatic re-
gion showed an AB-spin-system of H-6 and H-8 and an
AMX-spin-system of the B-ring protons which were simi-
3
lar to those of catechin. The coupling constant of J(H-2/H-3)
of 6.5 Hz indicates a trans-configuration of the hetero-
cyclic protons H-2 and H-3. Additionally, two olefinic
protons (d ¼ 5.55 ppm and 5.97 ppm) and a multiplett
counting for four aliphatic protons (d ¼ 2.24 ppm to
2.52 ppm) were detected. The downfield shift of H-3
(d ¼ 5.24 ppm) in comparison to catechin (d (H-3)
¼ 3.99 ppm) indicates an acyl substitution at the C-3
hydroxyl. The ESI-MS and ESI-MS-MS data suggested
that a catechin-like flavan-3-ol was acylated with a cyclo-
hexene-on-ol-carboxylic acid ([M ꢀ H]ꢀ m/z ¼ 427.4;
[M – acid moiety]ꢀ m/z ¼ 289.3), a typical acyl moiety
that occurs in polyphenols of Salix species. This was cor-
roborated by the two olefinic protons (d ¼ 5.54 and
5.97 ppm) and the 4 methylene group protons (d ¼ 2.23
to 2.53 ppm) whose registered spectrum was in accor-
dance with a simulated one. With its negative optical ro-
tation compound 4 was finally identified as catechin-3-O-
Pharmazie 62 (2007) 3
231

ORIGINAL ARTICLES
(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic acid)-ester
Compound 8 gave an [M ꢀ H]ꢀ ion peak at m/z ¼ 865 by
ESI-MS indicating a procyanidin skeleton composed of
three catechin/epicatechin moieties. Cleavage according to
Thompson et al. (1972) yielded catechin and procyanidin
B1. The latter was identified as its peracetate by NMR
and optical rotation in comparison with reference material
and published data (Kolodziej 1990). Thus, the “middle”
and “bottom” units could be identified as epicatechin-
using 2D NMR experiments (COSY, HSQC, HMBC) and
by comparison with published data (Hsu et al. 1985).
Comparison of the optical rotation of 4 with our own va-
lue tentatively indicates the 2,3-trans-configuration of ca-
techin; however, the hitherto unknown configuration of the
acyl moiety could not be established (Hsu et al. 1985).
Compound 4 was first described as a constituent of the
bark of S. sieboldiana (Hsu et al. 1985).
1
(4ß!8)-catechin. The H NMR spectrum at ambient tem-
perature of the peracetylated compound 8a showed differ-
ent sets of rotameric signals so that the complete analysis
of resonances failed. Low-temperature NMR measurement
at ꢀ20 ꢁC (253 K) in the free phenolic state (8) gave a
sufficiently resolved spectrum without or with only weak
conformational rotamers. The NMR data sets were in ac-
cordance with those reported in the literature (Shoji et al.
2003) and identify compound 8 as epicatechin-(4ß!8)-
epicatechin-(4ß!8)-catechin.
5´
OH
6´
8
8a
HO
O
OH
2´
6
4a
O
OH
2´´
7´´
4
3´´
4´´
OH
1´´
6´´
O
O
5´´
Catechin-3-O-(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic acid)-ester (4)
OR
B
2
The procyanidins B1 [epicatechin-(4ß!8)-catechin] (5)
and B3 [catechin-(4a!8)-catechin] (6) were the only di-
meric proanthocyanidins which could be detected in the
extract. After peracetylation, their structures were con-
firmed by different spectroscopic methods (ESI-MS,
NMR, CD, optical rotation) in comparison with published
values of their peracetylated derivatives (Weinges et al.
1968; Kolodziej 1986).
RO
8a O
C
OR
E
A
2'
3
6
OR
4a
OR
OR
4
RO
RO
8
O
F
D
OR
4
OR
OR
RO
RO
The molecular mass ([M þ Na]þ m/z ¼ 1519) and
1H NMR data of the peracetylated compound 7 (7a) indi-
cated a trimeric flavan-3-ol constitution with catechol-type
B-rings. Bonding positions of interflavanoid linkages were
H
8
O
I
2
G
3
OR
4
3
RO
elucidated by HMBC experiments. J cross peaks of H-4
Epicatechin-(4ß!8)-epicatechin-(4ß!8)-catechin (8, R ¼ H; 8a, R ¼ Ac)
(C and F) to the respective C-8a signals together with the
connectivities of all 2D-NMR experiments clearly indi-
cated C-4 to C-8 linkages. While the coupling constants
of the “upper” unit proved a H-2/H-3 cis-configuration
(3J(H-2/H-3) ¼ brs), the “middle” unit was trans-configurated
(3J(H-2/H-3) ¼ 10.4 Hz). The upfield “g-shift” of the “upper”
unit of carbon C-2 (d ¼ 73.77 ppm) resulted from a qua-
siaxial orientation of the flavan-3-ol unit at C-4 (Fletcher
et al. 1977). In contrast, the relative 2,3-trans-stereochem-
istry of the “bottom” unit cannot be determined with cer-
tainty by the small coupling constants of the H-2/H-3 (I)
protons (3J(H-2/H-3) ¼ 3.1 Hz). However, the observed small
value can be explained with conformational changes of
ring I of which the catechol substituent is mainly in an
axial position (Balas et al. 1995). Thus, in accordance
with its optical rotation value identical to that published
(Foo and Karchesy 1989), compound 7 could be identified
as epicatechin-(4ß!8)-catechin-(4a!8)-catechin.
Although isolation, purification and structure elucidation
of oligomeric proanthocyanidins higher than tetrameric
flavan-3-ols is difficult or even impossible today concern-
ing their full stereochemistry, some fundamental informa-
tion about the flavan-3-ol composition of higher oligomers
can be obtained by 13C NMR spectroscopy. The spectrum
of an oligomeric fraction of Salicis purpureae cortex ex-
tract (Fig.) was prepared and analysed according to the
literature (Newman et al. 1987; Eberhardt and Young
1994). The ratio of cis : trans isomers was estimated by
integration of the signals at d ¼ 77 ppm and 83.5 ppm.
The 5 : 1 ratio indicates that flavan-3-ols with a relative
2,3-cis configuration predominate in the bark of S. purpur-
ea. By integration of the C-3 resonances of the extender
units (d ¼ 73 ppm) and the resonances of C-3 of all term-
inal units (d ¼ 68 ppm), the average degree of oligomeri-
zation could be estimated to be 4 to 5 flavan-3-ol units.
Notable is the absence of carbonyl- or carboxyl carbons at
ca. d ¼ 200 ppm and 170 ppm, respectively, indicating
that there is no acyl substitution in the set of oligomers.
Furthermore, no evidence was found for the occurrence of
proanthocyanidins with trihydroxylated B-rings by careful
inspection of the expected B-ring carbon region (ca. 107–
112 ppm) of prodelphinidin-type constituents.
OR
6'
B
2
RO
8a O
C
OR
A
2'
3
"upper"
6
OR
4a
OR
OR
4
RO
RO
E
8
O
F
"middle"
D
In summary, this is the first report on the isolation and
structure elucidation of monomeric and oligomeric flavan-
3-ols [except catechin (Pearl and Darling 1970)] from the
bark of Salix purpurea. Additional structure analogues
with another hydroxylation pattern than 3,4-dihydroxyla-
tion in the B-ring were not observed for flavanol oligo-
mers. It could also be shown that the higher oligomeric
proanthocyanidin spectrum of S. purpurea consists mainly
OR
4
8
OR
RO
RO
H
O
I
2
"bottom"
OR
G
3
OR
4
RO
Epicatechin-(4ß!8)-catechin-(4a!8)-catechin (7, R ¼ H; 7a, R ¼ Ac)
232
Pharmazie 62 (2007) 3

ORIGINAL ARTICLES
Fig.:
13C NMR spectrum of an oligomeric flavan-3-
ol fraction of Salicis purpureae cortex (100 MHz,
MeOH-d4). (A) C-5/C-7/C-8a of A-rings; (B)
C-30/C-40of procyanidin units; (C) C-10of B-
rings ; (D––E) C-20/C-50/C-60of PC units; (F)
substituted A-ring carbons; (G) C-4a of PC
units; (H) C-6/C-8 of A-rings; (I) C-2 of ex-
tender 2,3-trans units; (J) C-2 of terminal 2,3-
trans units; (K) C-2 of 2,3-cis units; (L) C-3
of all extender units; (M) C-3 of terminal
units, (N) C-4 of 2,3-trans units, (O) C-4 of
2,3-cis units
3.3. Extraction and isolation
of flavan-3-ols with dihydroxylated B-rings and the predo-
minance of 2,3-cis configurated units. Thus, concerning its
flavanol derivates, the bark of S. purpurea resembles
much the bark of the East-asian S. sieboldiana Blume that
has been shown to contain compounds 2–8 (Hsu et al.
1985). From the bark of S. pet-susu Kimura 2 and 6 were
isolated (Ohara and Hujimori 1996); Salicis cortex crude
drug material obtained from the pharmaceutical market
and not further defined taxonomically contained 2, 3, 5, 6
and 8 (Kolodziej 1990).
The dry pulverized extract was dissolved in water and extracted with ethy-
lacetate exhaustively to yield an ethylacetate phase (46%) an a water phase
(54%).
The EtOAc fraction (148,4 g) was chromatographed on Sephadex 1 in four
portions to give 15 fractions each run. Fraction 7 (2320 to 2780 ml,
2868 mg) was further separated by MCI I (2380 to 2580 ml, 498 mg) and
MPLC I (1630 to 1970 ml) to give 382 mg of compound 4. MCI1-separa-
tion (MCI I) of the Sephadex1 fraction 9 (3140 to 3540 ml, 1347 mg) lead
directly to compounds 1 (1880 to 2280 ml, 90 mg) and 2 (2530 to
3030 ml, 42 mg). Compound 3 (3130 to 3280 ml, 27 mg) was further pur-
ified by HPLC I (Rt ¼ 10,5 min, 6 mg). Compounds 5 and 6 were sepa-
rated from Sephadex1 fraction 11 (4100 to 4835 ml, 1749 mg) by chroma-
tography with MCI I (1300 to 1690 ml, 287 mg) and again with MCI II
(5: 1160 to 1285 ml, 129 mg; 6: 1410 to 1600 ml, 64 mg).
The water fraction (171.8 g) was chromatographed with Sephadex II to
yield a sugar enriched fraction (the first 3540 ml, 94,9%) and a flavan-3-ol
enriched fraction (3540 to 8840 ml, 5,1%). The flavan-3-ol-fraction again
was separated with Sephadex III to give 11 fractions. Compounds 7 and 8
were purified from fraction 4 (4690 to 5490 ml, 201 mg) with MCI III (7:
865 to 960 ml, 23 mg; 8: 1095 to 1775 ml, 44 mg). Fraction 11 (9000 to
10730 ml, 3342 mg) was used as the higher oligomeric fraction (Newman
et al. 1987; Eberhardt and Young 1994).
3. Experimental
3.1. Plant material
Dry 70% ethanolic extract of Salicis pupureae cortex with a DER 8–14 : 1.
The extract was obtained from Bionorica (Neumarkt/Obpf, Germany);
batch number AE 0665/2 (voucher at Bionoria, D-Neumarkt; another vou-
cher is deposited at the Institute of Pharmaceutical Biology and Phyto-
chemistry, WWU Mu¨nster under PBMS 212). Its content of salicin deriva-
tives was determined to 16,9% according to the European Pharmacopoeia
(2005), and tannins following (Glasl 1983) to 8.5%.
3.4 Structural data
The physico-chemical data of 4, 7a and 8 will be presented here, because
the quality of published data are worse (4) or not existing (7a) or differ (8)
from those presented in the literature.
3.2. Chromatographic systems
Sephadex Chromatography: Sephadex1LH20 [Pharmacia, Uppsala, Swe-
den]; column: 5.5 ꢂ 90 cm, flow: 0.5 mL/min; eluents: Sephadex I: 4 L
EtOH/H2O (3 þ 1), followed by EtOH 2 L, EtOH/MeOH (1 þ 1) 3 L and
Me2CO/H2O (7 þ 3) 3 L; Sephadex II: 4 L EtOH/H2O (3 þ 1), EtOH/MeOH
(1 þ 1) 1 L and Me2CO/H2O (7 þ 3) 4 L; Sephadex III: EtOH/MeOH
(1 þ 1) 5 L, MeOH 7 L and Me2CO/H2O (7 þ 3) 4 L.
Catechin-3-O-(1-hydroxy-6-oxo-2-cyclohexene-1-carboxylic acid)-ester (4):
[a]D20 ꢀ31 (c ¼ 0,1; acetone); 1H NMR (400 MHz, MeOH-d4,
*
d ¼ 3.30 ppm, data confirmed by simulation; d ¼ [ppm], [J ¼ [Hz]]):
00
00
*
*
2.28 [dd, 2.0/5.0, H-5 a ], 2.32 [ddd, 1.8/3.8/ꢀ16.0, H-4 a ], 2.39 [dd,
00
00
*
*
1.8/3.9, H-4 b ], 2.49 [ddd, 2.0/9.0/ꢀ16.0, H-5 b ], 2.64 [dd, 6.7/ꢀ16.5,
LPLC on MCI: MCI1-gel [Mitsubishi Corp., Tokyo]; column:
2.5 ꢂ 100 cm, flow: 10 mL/min; linear gradient with 2 Waters Millipore1
HPLC-pumps (model 510) with a Waters Millipore1 Automated Gradient
Controller; step gradient with one pump and 500 ml per step; MCI I: line-
ar gradient of 20% MeOH to 70% MeOH in 400 min, then 100% MeOH;
MCI II: step gradient of 30% MeOH to 50% MeOH, 5% steps, then 100%
MeOH; MCI III: 10% step gradient from 20% to 70% MeOH, 500 ml per
step, then 100% MeOH.
H-4a], 2.77 [dd, 5.1/ꢀ16.5, H-4b], 4.9 [d, 6.5, H-2], 5.24 [ddd, 5.1/6.1/
6.7, H-3], 5.54 [ddd, 1.8/1.8/9.8, H-200 ], 5.89 [d, 2.3, H-8], 5.94 [d, 2.3,
*
H-6], 5.97 [ddd, 3.9/3.9/9.8, H-300 ], 6.65 [dd, 2.1/8.1, H-6 ], 6.73 [d, 8.1,
H-50], 6.75 [d, 2.1, H-20]; 13C-NMR (100 MHz, MeOH-d4; *interchange-
able; d ¼ [ppm]): 24.35 [C-4], 26.74 [C-400], 36.17 [C-500], 72.87 [C-3],
79.05 [C-2], 79.16 [C-100], 95.43 [C-8], 96.49 [C-6], 99.29 [C-4a], 114.7
[C-2000], 116.3 [C-50], 0 119.35 [C-60], 128.7 [C-200], 130.7 [C-10], 133.22
0
*
0
*
*
[C-3 ], 146.39 [C-4 ], 146.52 [C-3 ], 156.37 [C-8a], 157.62 [C-5],
158.26 [C-7], 170.57 [C-700], 206.95 [C-6 00]; ESI-MS: [M þ Na]þ m/z
¼ 451,2; [M þ K]þ m/z ¼ 467,2; [M ꢀ H]ꢀ m/z ¼ 427,4; [M – acid moi-
ety]ꢀ m/z ¼ 289,3; ESI-MS-MS: daughters of 451,2: [M – acid moiety –
H2O þ Na]þ m/z ¼ 295; [acid-moiety þ Na]+ m/z ¼ 178,9; [acid moiety –
CO2 þ Na]þ m/z ¼ 134,9.
MPLC on RP-18: Orpegen HD-Sil-RP-18, 18–60 mm, column:
26 ꢂ 460 mm, pump as above; MPLC I: 10% step gradient from 20% to
80% MeOH, 500 ml per step, then 100% MeOH, flow: 10 mL/min,
Preparative HPLC: eluents: MeCN (A) and in aqua millipore1 (B); flow:
6 mL; HPLC I: system: Knauer RP-18, Eurospher1100, 7 mm,
250 ꢂ 20 mm with 30% A to 60% A in 15 min; detection at 278 nm.
TLC: all fractions were tested by TLC on silica gel plats [Merck, Darm-
stadt, Germany]; mobile phase: EtOAc/HCO2H/H2O 90 þ 5 þ 5; detection
by Naturstoffreagent (diphenylboryloxy-ethylamine), vanillin/HCl and ani-
saldehyde/sulphuric acid.
NMR and MS: all NMR-spectra were measured on a Varian AS 400 or
Varian Unity 600 in CD3OD; CDCl3 was used for acetylated flavan-3-ols
(peracetylation with Ac2O/pyridine 1 þ 1 for 24 h at room temperature);
ESI-MS-data were measured on a Finnigan LCQ or a Micromass Quattro
LC.
Epicatechin-(4ß!8)-catechin-(4a!8)-catechin (7): [a]D20 ꢀ193,3 (c ¼ 0,1;
MeOH); ESI-MS: [M þ Na]þ m/z ¼ 1519. 1H NMR of peracetate (7a;
*
400 MHz, CDCl3, d ¼ 7.26 ppm; interchangeable; d ¼ [ppm], [J ¼ [Hz]]):
2.42 [dd, 3.9/ꢀ17.1, H-4a (I)], 2.73 [dd, 3.9/ꢀ17.1, H-4b (I)], 4.32 [d,
10.4, H-2 (F)], 4.41 [d, 2.3, H-4 (C)], 4.42 [d, 8.4, H-4 (F)], 4.91 [dd, 1.4/
2.3, H-3 (C)], 5.19 [br s, H-2 (C)], 5.24 [ddd, 3.1/3.9/3.9, H-3 (I)], 5.31
[d, 3.1, H-2 (I)], 5.66 [dd, 8.4/10,4, H-3 (F)], 5.91 [d, 2.2, H-8 (A)], 6.25
[dd, 2.0/8.4, H-60 (B)], 6.28 [d, 2.2, H-6 (A)], 6.3 [dd, 2.0/8.4, H-60 (H)],
6.66 [s, H-6 (G)], 6.69 [s, H-6 (D)], 6.70 [d, 2.0, H-20 (H)], 6.84 [d, 2.0,
H-20 (E)], 6.92 [dd, 2.0/8.4, H-60 (E)], 7.05 [d, 8.4, H-50 (B) ], 7.06 [d,
*
Pharmazie 62 (2007) 3
233

ORIGINAL ARTICLES
0
0
8.4, H-5 (E) ], 7.16 [d, 8.4, H-5 (H)], 7.22 [d, 2.0, H-20 (B)]; 13C NMR
of peracetate (7a; 100 MHz, CDCl3, d ¼ 77.0 ppm; a––dassignments with
the same letter are interchangeable; d ¼ [ppm]): 22.24 [C-4 (I)], 34.46 [C-
4 (C)], 37.31 [C-4 (F)], 67.78 [C-3 (I)], 70.68 [C-3 (C)], 71.40 [C-3 (F)],
73.77 [C-2 (C)], 77.9 [C-2 (I)], 79.98 [C-2 (F)], 107.68 [C-8 (A)], 108.9
[C-6 (A)], 109.52 [C-6 (G)], 110.69 [C-4a (G)], 112.16 [C-4a (A)], 112.3
[C-6 (D)], 117.51 [C-8 (G)], 117.97 [C-4a (D)a], 118.56 [C-8 (D)a],
120.47 [C-20 (H)], 122.89 [C-20 (B)], 123.11 [C-20 (E)], 123.56 [C-50
(E)b], 123.48 [C-60 (H)], 123.9 [C-50 (B)b], 124.48 [C-60 (B)], 125.33 [C-50
(H)], 125.78 [C-60 (E)], 135.19 [C-10 (E)], 135.90 [C-10 (H)], 136.64 [C-10
(B)], 142.17 [C-30 (B)], 142.21 [C-40 (B)c], 142.58 [C-30 (H)c], 142.6 [C-30
(E)c], 142.66 [C-40 (H)c], 142.91 [C-40 (E)], 147.96 [C-7 (G)], 148.34 [C-5
(A)], 148.51 [C-7 (D)d], 148.66 [C-5 (G)d], 149.19 [C-5 (D)d], 149.88 [C-
7 (A)], 151.69 [C-8a (G)], 155.75 [C-8a (D)], 156.12 [C-8a (A)].
Balde´ AM, Pieters LA, Gergely A, Kolodziej H, Claeys M, Vlietinck AJ
(1991) A-Type proanthocyanidins from stem bark of Pavetta owariensis.
Phytochemistry 30: 337–342.
Cos P, De Bruyne T, Hermans N, Apers S, Vanden Berghe D, Vlietinck
AJ (2003) Proanthocyanidins in health care: current and new trends.
Curr Med Chem 10: 1345–1359.
Eberhardt TL, Young RA (1994). Conifer seed cone proanthocyanidin
polymers: Characterization by 13C NMR spectroscopy and determination
of antifungal activities. J Agric Food Chem 42: 1704–1708.
European Pharmacopoeia: Weidenrinde, Salicis cortex. 5. Ed., Vol. 2,.
Dtsch. Apoth. Verl. 2005. p. 3666–3668.
Ferreira D, van Rensburg H, Malan E, Coetzee J, Nel RJJ (1999) Recent
advances in the chemistry of proanthocyanidins. In: Recent Advances in
Phytochemistry 33 (Phytochemicals in Human Health Protection, Nutri-
tion and Plant Defense), p. 255–288.
*
Epicatechin-(4ß!8)-epicatechin-(4ß!8)-catechin (8): [a]D20 þ132 (c
¼ 0,1; acetone); ESI-MS: [M ꢀ H]ꢀ m/z ¼ 865. 1H NMR (600 MHz,
MeOH-d4, d ¼ 3.30 ppm, 253 K, a, bassignments with the same letter are
interchangeable; d ¼ [ppm], [J ¼ [Hz]]): 2.55 [dd, 4.5/ꢀ17.1, H-4a (I)],
2.62 [dd, 5.2/ꢀ16.5, H-4b (I)], 3.99 [d, 2.0, H-3 (C)], 4.07 [brs, H-3 (F)],
4.19 [m, H-3 (I)], 4.48 [brs, H-4 (C)], 4.76 [brs, H-4 (F)], 5.02 [brs, H-2
(F)a], 5.03 [brs, H-2 (I)a], 5.07 [brs, H-2 (C)], 5.88 [s, H-6 (D)b], 5.89 [s,
H-6 (G)b], 5.97 [d, 2.4, H-6 (A)], 6.01 [d, 2.4, H-8 (A)], 6.68 [d, 8.0, H-
50 (E)], 6.69 [dd, 2.3/8.0, H-60 (B)], 6.72 [d, 8.0, H-50 (H)], 6,73 [d, 8.0,
H-50 (B)], 6.78 [dd, 2.3/8.0 H-60 (E)], 6.86 [d, 2.3, H-20 (H)], 6.89 [d,
2.3, H-20 (B)], 6.90 [dd, 2.3/8.0, H-60 (H)], 7.04 [d, 2.3, H-20 (E)];
13C NMR (150 MHz, MeOH-d4, d ¼ 49.0 ppm; 253 K, a––cassignments
with the same letter are interchangeable; d ¼ [ppm]): 26.2 [C-4 (I)], 37.0
[C-4 (C)], 37.1 [C-4(F)], 68.1 [C-3 (I)], 72.3 [C-3 (F)], 73.5 [C-3(C)],
76.8 [C-2 (C)], 76.9 [C-2 (F)], 81.6 [C-2 (I)], 95.9 [C-6 (A)a], 96.0 [C-8
(A)a], 96.8 [C-6 (D)], 97.0 [C-6 (G)], 100.5 [C-4a (G)], 101.7 [C-4a (A)],
102.5 [C-4a (D)], 106.4 [C-8 (D)], 108.1 [C-8 (G)], 113.9 [C-20 (H)],
114.8 [C-20 (B)b], 114.9 [C-20 (E)b], 115.78 [C-50 (B)c], 115.8 [C-50 (E)c],
115.9 [C-50 (H)c], 118.6 [C-60 (E)], 119.0 [C-60 (H)], 119.1 [C-60 (B)],
132.4 [C-10 (B)], 132.6 [C-10 (H)], 132.7 [C-10 (E)], 145.2 [C-40 (E)],
145.3 [C-40 (H)], 145.6 [C-40 (B)], 145.7 [C-30 (H)], 145.8 [C-30 (E)],
146.0 [C-30 (B)], 153.7 [C-8a (G)], 154.8 [C-8a (C)], 155.7 [C-5 (G)],
156.4 [C-7 (G)], 156.9 [C-7 (D)], 157.1 [C-5 (D)], 157.8 [C-5 (A)], 158.0
[C-8a (A)], 158.2 [C-7 (A)].
Fletcher AC, Porter LJ, Haslam E, Gupta RK (1977) Plant Proanthocyani-
dins. Part 3. Conformational and configurational studies of natural pro-
cyanidins. J Chem Soc Perkin Trans I: 1628–1637.
Foo LJ, Lu J, Molan AL, Woodfield D, McNabby W (2000) The phenols
and prodelphinidins of white clover flowers. Phytochemistry 54: 539–
548.
Foo LY, Karchesy JJ (1989) Procyanidin dimers and trimers from Douglas
Fir inner bark. Phytochemistry 28: 1743–1747.
Glasl H (1983) Zur Photometrie in der Drogenstandardisierung. 3. Gehalts-
bestimmung von Gerbstoffdrogen. Dtsch Apoth Ztg 123: 1979–1987.
Hsu FL, Nonaka GI, Nishioka I (1985) Acylated flavanols and procyani-
dins from Salix sieboldiana. Phytochemistry 24: 2089–2092.
Kolodziej H (1986) Synthesis and characterization of procyanidin dimers
as their peracetates and octamethyl ether diacetates. Phytochemistry 25:
1209–1215.
Kolodziej H (1990) Oligomeric flavan-3-ols from medicinal willow bark.
Phytochemistry 29: 955–960.
Meier B, Meier-Liebi M (1998), Salix. In: Hagers Handbuch der Pharma-
zeutischen Praxis. Blaschek, W. et al. (eds), Folgeband 3, Springer Ber-
lin, p. 469–496.
Newman RH, Porter LJ, Foo LY (1987) High-resolution 13C NMR studies
of proanthocyanidin polymers (condensed tannins). Magn Res Chem 25:
118–124.
Cleavage reaction according to Thompson et al. (1972); in brief, 25 mg of
8 were dissolved in 4 ml 0.1 N ethanolic HCl and heated at 60 ꢁC for
15 min. The solution was dried, again dissolved in 1.5 ml ethanol 96% and
chromatographed on Sephadex1LH20 (1.5 ꢂ 7.5 cm; 5 ml fractions) with
ethanol as eluent to yield 2 (2.5 mg) and 5 (3.0 mg).
Ohara S, Hujimori H (1996) Polyphenols in Salix species II. Polyphenols
from the bark of Salix pet-susu Kimura. Makuzai Gakkaishi 42: 618–
623.
Pearl IA, Darling SF (1970) Phenolic extractives of Salix purpurea bark.
Phytochemistry 9: 1277–1281.
Schmid B, Ko¨tter I, Heide L (2001) Pharmacokinetics of salicin after oral
administration of a standardized willow bark extract. Eur J Clin Pharma-
col 57: 387–391.
Shoji T, Motoh M, Nakamura T, Kanda T, Akiyama H, Goda Y (2003)
Isolation and structural elucidation of some procyanidins from apple by
low-temperature nuclear magnetic resonance. J Agric Food Chem 51:
3806–3813.
Acknowledgements: Thanks are due to Bionorica AG, Neumarkt, for finan-
cial support and the extract material; to Mrs. A. Klinke and Mr. B. Deiters
for technical assistance; to Dr. H. Luftmann and Mr. T. Meiners, Mu¨nster,
for ESI-MS experiments; to Dr. K. Bergander and Mrs. M. Heim, Mu¨nster,
for NMR-experiments.
Thompson R, Jacques D, Haslam E, Tanner R (1972) Plant proanthocyani-
dins. Part 1. Introduction; The isolation, structure, and distribution in
nature of plant procyanidins. J Chem Soc, Perkin Trans I: 1387–1399.
Weinges K, Go¨ritz K, Nader F (1968) Konfigurationsbestimmung von
References
Balas L, Vercauteren J, Laguerre M (1995) 2D-NMR structure elucidation
of proanthocyanidins: the special case of the catechin-(4a!8)-catechin-
(4a!8)-catechin trimer. Magn Res Chem 32: 85–94.
C
30H26O12-Procyanidinen und Strukturaufkla¨rung eines neuen Procyani-
dins. Liebigs Ann Chem 715: 164–171.
234
Pharmazie 62 (2007) 3