Preparation of dimeric procyanidins B1, B2, B5, and B7 from a polymeric procyanidin fraction of black chokeberry (Aronia melanocarpa)

Article abstract of DOI:10.1021/jf904354n

A semisynthetic approach has been used for the preparative formation of dimeric procyanidins B1, B2, B5, and B7. As starting material for the semisynthesis, polymeric procyanidins from black chokeberry were applied. These polymers were found to consist almost exclusively of (-)-epicatechin units. Under acidic conditions the interflavanoid linkages of the polymeric procyanidins are cleaved and the liberated (-)-epicatechin can react with nucleophiles, such as (+)-catechin or (-)-epicatechin. In this way, the polymeric procyanidins are degraded while dimeric procyanidins are formed. During this reaction only dimeric procyanidins are formed that contain (-)-epicatechin in the upper unit, that is, B1 [(-)-EC-4β→8-(+)-C)], B2 [(-)-EC-4β→8-(-)-EC], B5 [(-)-EC-4β→6-(-)-EC], and B7 [(-)-EC-4β→6-(+)-C]. The reaction mixtures of the semisynthesis can be successfully fractionated with high-speed countercurrent chromatography (HSCCC), and it is possible to isolate pure procyanidins B1, B2, B5, and B7 on a preparative scale.

Full text of DOI:10.1021/jf904354n

J. Agric. Food Chem. 2010, 58, 5147–5153 5147
DOI:10.1021/jf904354n
Preparation of Dimeric Procyanidins B1, B2, B5, and B7 from
a Polymeric Procyanidin Fraction of Black Chokeberry
(Aronia melanocarpa)
T
UBA
E
SATBEYOGLU AND
P
ETER
W
INTERHALTER
*
€
Institute of Food Chemistry, Technische Universitat Braunschweig, Schleinitzstrasse 20,
38106 Braunschweig, Germany
A semisynthetic approach has been used for the preparative formation of dimeric procyanidins B1,
B2, B5, and B7. As starting material for the semisynthesis, polymeric procyanidins from black
chokeberry were applied. These polymers were found to consist almost exclusively of
(-)-epicatechin units. Under acidic conditions the interflavanoid linkages of the polymeric procya-
nidins are cleaved and the liberated (-)-epicatechin can react with nucleophiles, such as (þ)-
catechin or (-)-epicatechin. In this way, the polymeric procyanidins are degraded while dimeric
procyanidins are formed. During this reaction only dimeric procyanidins are formed that contain
(-)-epicatechin in the upper unit, that is, B1 [(-)-EC-4βf8-(þ)-C)], B2 [(-)-EC-4βf8-(-)-EC], B5
[(-)-EC-4βf6-(-)-EC], and B7 [(-)-EC-4βf6-(þ)-C]. The reaction mixtures of the semisynthesis
can be successfully fractionated with high-speed countercurrent chromatography (HSCCC), and it is
possible to isolate pure procyanidins B1, B2, B5, and B7 on a preparative scale.
KEYWORDS: Dimeric procyanidins B1, B2, B5, and B7; black chokeberry (Aronia melanocarpa);
preparation; high-speed countercurrent chromatography
INTRODUCTION
MATERIALS AND METHODS
Reagents. Black chokeberry pomace was supplied by Walther GmbH
(Arnsdorf, Germany). The freeze-dried pomace was separated into seeds
and skins using a sieve tower with eight different mesh sizes (2.8 mm-
200 μm). The seeds were collected in sieve fraction 0.5-1 mm. Chemicals
and solvents were as follows: (-)-epicatechin, p.a. (Sigma, Steinheim,
Germany); (þ)-catechin-hydrate, g98% (Sigma); sodium acetate (anhy-
drous), p.a. (Merck, Darmstadt, Germany); phloroglucinol, p.a. (Merck);
Aronia melanocarpa or black chokeberry is a member of the
Rosaceae family. Black chokeberry originates from the eastern
parts of North America and was introduced in Europe at the
beginning of the 20th century. It is used for the production of,
for example, juices, syrups, and soft spreads. Aronia fruits are
known to exhibit a wide range of biological and pharmacological
properties, such as antioxidative, anti-inflammatory, antiathero-
genic, and antidiabetic activities (1, 2). Important phenolic
constituents of black chokeberry are polymeric procyani-
dins (3, 4). Procyanidins consist of the flavan-3-ols (þ)-catechin
and (-)-epicatechin. In most cases, the linkage is between C4 of
the upper unit and C8 of the lower unit (5). Most important,
A. melanocarpa was found to contain homogeneous B-type pro-
cyanidins (4). The concentration of procyanidins in A. melanocarpa
is among the highest determined in berries (664 mg/100 g of fresh
weight) (6).
€
hydrochloric acid, 37% (Riedel-de-Haen, Seelze, Germany); and ascorbic
acid, pure (Merck). Solvents for high-performance liquid chromatography
(HPLC) analysis were as follows: acetonitrile, HPLC quality (Sigma);
acetic acid (Mallinckrodt Baker B.V., Deventer, The Netherlands); and
water (deionized, Nanopure). Solvents used for high-speed counter-
current chromatography (HSCCC) and solvent precipitation were as
follows: ethyl acetate (Acros Organics, Geel, Belgium); methanol
(distilled, industrial quality); n-hexane (distilled, industrial quality);
water (deionized, Nanopure); 1-butanol, p.a. (Fisher Scientific, Lough-
borough, U.K.); ethanol (distilled, industrial quality); 2-propanol, p.a.
(Sigma); dichloromethane, p.a. (Fisher Scientific); water (deionized,
Nanopure); and acetone (distilled, industrial quality).
Solvent Precipitation of a Polymeric Procyanidin Fraction.
Approximately 21 g of milled aronia seeds or approximately 23 g of milled
aronia pomace was defatted with n-hexane (3 ꢀ 200 mL) and dichlo-
romethane (3 ꢀ 200 mL). Subsequently, the defatted aronia seeds were
extracted with 70% aqueous acetone solution (3 ꢀ 200 mL). Defatted
aronia pomace was extracted first with methanol (3 ꢀ 200 mL) and then
with 70% aqueous acetone solution (3 ꢀ 200 mL). Three grams of the
freeze-dried 70% aqueous acetone extracts were stirred for 1 h in 150 mL
of ethanol and the insoluble residues were removed by filtration. Then
150 mL of n-hexane was dropped into the solution (10 mL/min). After
filtration, the obtained precipitate was evaporated with a rotary evapora-
tor and freeze-dried.
The aim of the present study was the development of a strategy
for the preparation of dimeric procyanidins B1, B2, B5, and B7 on
a large scale (Figure 1). Dimeric procyanidins can be formed
directly by the reaction of procyanidin-rich extracts with flavan-
3-ols. This approach was first applied to the synthesis of procya-
nidins with C2 epimers (7) using harsh reaction conditions
€
(e.g., 95 °C for 22 h) and more recently modified by Kohler
et al. (8).
*Author to whom correspondence should be addressed (telephone
þ49-531-391-7200; fax þ49-531-391-7230; e-mail p.winterhalter@
tu-bs.de).
© 2010 American Chemical Society
Published on Web 03/02/2010
pubs.acs.org/JAFC

5148 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Esatbeyoglu and Winterhalter
Figure 2. HPLC-PDA chromatograms of Aronia melanocarpa before (A)
and after (B, C) solvent precipitation.
aqueous acetic acid (v/v) (A) and acetonitrile (B). The separation was
carried out at 25 °C, under the following conditions: 3-10% acetonitrile
(0-25 min), 10-35% acetonitrile (25-45 min), 35-75% acetonitrile
(45-50 min), 75% acetonitrile (50-55 min), 75-3% acetonitrile (55-
60 min), followed by a reequilibration of the column for 10 min. Detection
was at 280 nm; flow rate, 0.8 mL/ min. Procyanidins were identified with
the help of isolated reference compounds and were quantified as dimeric
procyanidin B1 equivalents.
HPLC-ESI/MS/MS Analysis. Chromatographic analyses were
performed on an Agilent 1100 HPLC system (Waldbronn, Germany)
equipped with a 1200 autosampler and an 1100 HPLC pump. The HPLC
was also interfaced to an Esquire HPLC-MS/MS system from Bruker
GmbH (Bremen, Germany). HP ChemStation was used for data collec-
tion. MS parameters: negative mode; capillary, 3000 V; end plate, -500 V;
capillary exit, -105 V; dry gas, 325 °C; gas flow, 10 L/min; and nebulizer,
40 psi. During the chromatographic run mass spectra of the eluate were
recorded from m/z 50 to 2200. HPLC conditions as well as mobile phases
were as described above.
HSCCC. A high-speed countercurrent chromatograph model CCC-
1000 manufactured by Pharma-Tech Research Corp. (Baltimore, MD)
was equipped with three preparative coils, connected in series (total
volume = 800 mL). The revolution speed of the apparatus was 1000
rpm. Flow rates between 2.7 and 3.0 mL/min were used. A Biotronik
HPLC pump BT 3020 was used to pump the liquids. The aqueous lower
phase was applied as the mobile phase in the head to tail elution mode. All
samples were dissolved in a 1:1 mixture of upper and lower phase and
injected into the system by loop injection (20 mL). The amount of sample
injected was 1 g. During each HSCCC separation, UV absorbance of the
effluent was monitored by a Knauer UV-vis detector (280 nm) and
recorded using a BBC Goerz SE 120 plotter (3 cm/h). Twelve milliliter
fractions were collected by using a Super Frac fraction collector
(Pharmacia, LKB Super Frac fraction collector). The two-phase solvent
system ethyl acetate/n-butanol/water (14:1:15, v/v/v) was applied to the
separation of dimeric procyanidin B1 (see CCC-1, CCC-3) and ethyl
acetate/2-propanol/water (20:1:20, v/v/v) to the separation of dimeric
procyanidin B2 (CCC-5, CCC-7). The dimeric procyanidins B5 and B7,
which remained on the coil after the first fractionation, were rechromato-
graphed with the more lipophilic solvent system: n-hexane/ethyl acetate/
methanol/water (1:10:1:10, v/v/v/v) (CCC-2, CCC-4, CCC-6, CCC-8).
Figure 1. Structures of dimeric procyanidins of the B-type: (a) 4βf8
linked dimeric procyanidins B1 and B2; (b) 4βf6 linked dimeric procya-
nidins B5 and B7.
Phloroglucinolysis. Analysis was carried out according to the method
of Kennedy et al. (9).
Optimization of Reaction Conditions. Optimization of the Ratio
of Substrates. Aronia seed or aronia pomace precipitate (7.05 mg/mL)
was reacted with various ratios of flavan-3-ol [(þ)-catechin or (-)-
epicatechin)], that is, 7.05, 14.10, and 21.15 mg/mL, corresponding to
ratios of 1:0.75 (5.29 mg/mL), 1:1, 2:1, and 3:1 of flavan-3-ol to aronia seed
or aronia pomace precipitate, in 1 N methanolic hydrochloric acid (HCl)
solution for 20 min at 40 °C.
Optimization of Temperature. Aronia seed or aronia pomace precipi-
tate (7.05 mg/mL) was reacted with flavan-3-ol (14.10 mg/mL) in 1 N
methanolic HCl solution for 20 min at 30, 35, 40, and 50 °C, respectively.
Optimization of Reaction Time. Aronia seed or aronia pomace pre-
cipitate (7.05 mg/mL) was reacted with flavan-3-ol (14.10 mg/mL) in 1 N
methanolic HCl solution at 40 °C for 10, 15, 20, and 30 min. In all cases the
reaction was stopped by adding 5 times the volume of 80 mM aqueous
sodium acetate solution.
Preparative Formation of Dimeric Procyanidins. Seven hundred
milligrams of (þ)-catechin or (-)-epicatechin and 350 mg of aronia seed or
aronia pomace precipitate were used for semisynthesis. The reactants were
dissolved in 50 mL of 0.1 N methanolic HCl and kept at 40 °C for 20 min.
The reaction mixture was neutralized with 0.5 N sodium hydrogen
carbonate solution. After evaporation with a rotary evaporator, the
residual aqueous solution was freeze-dried. Yields of all reaction mixtures
were about 1.35 g, but of these only 1 g was applied to HSCCC separation.
HPLC Photodiode Array (PDA) Analysis. A HPLC system from
Jasco (Gross-Umstadt, Germany), with a PU-2080 plus pump combined
with a DG-2080053 three-line degasser and an LG 2080-02 ternary
gradient unit, and MD-2010 plus DAD were used. HPLC separation
was achieved on a 250 mm ꢀ 4.6 mm i.d., 5 μm, Aqua column
(Phenomenex, Aschaffenburg, Germany) protected with a guard column
of the same material (4 mm ꢀ 4 mm). The mobile phases consisted of 2%
RESULTS AND DISCUSSION
Extraction of defatted aronia seeds or pomace with 70%
aqueous acetone (v/v) and subsequent freeze-drying yielded a

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5149
Figure 3. Mechanism of semisynthesis.
polyphenol-rich extract that mainly consisted of anthocyanins
and polymeric procyanidins. Enrichment of polymeric procyani-
dins from this extract is possible by a simple cleanup step using
solvent precipitation. For solvent precipitation the extract is
redissolved in ethanol, and n-hexane is added as a nonpolar
solvent. Of the different ratios of ethanol/n-hexane tested (0.5:1,
1:1, 1:2, 1:3, and 1:4), the 1:1 mixture was found to give the best
yield of polymeric procyanidins in the precipitate (Figure 2). Our
analysis of the so-obtained polymeric procyanidin fraction by
phloroglucinolysis (9) revealed that besides a small amount of
(þ)-catechin (<1.5%), only (-)-epicatechin units are present in
the polymer chain. Galloylated subunits are not present. As mean
degree of polymerization (mDP) a value of 25-30 has been
determined.
polymeric procyanidins are cleaved and simultaneously dimeric
procyanidins are formed. Crucial conditions for this reaction, for
example, the ratio of nucleophile to aronia seed or aronia pomace
precipitate, temperature, and reaction time, must be optimized
(Figure 4) to reduce the formation of so-called gambiriins
(chalcane flavan-3-ol dimers, Figure 5), which are known by-
products of this semisynthesis (8). The pathway of chalcane
flavan-3-ol adduct formation was described in a previous
paper (8). Formation of dimeric procyanidins as well as gambir-
iins was found to increase with higher amounts of added flavan-
3-ol. The maximum formation of procyanidin dimers was ob-
served at temperatures between 30 and 40 °C, whereas the yield of
gambiriins continued to increase at higher temperature. Optimi-
zation of reaction time between 10 and 30 min showed the
slightest influence on the formation of procyanidin dimers and
gambiriins. Hence, the selected conditions for semisynthesis were
a reaction temperature between 35 and 40 °C, a reaction time
between 15 and 20 min, and a ratio of reactions partners,
nucleophiles (i.e., catechin or epicatechin) and polymeric procya-
nidins, of 2:1.
€
It has recently been shown by Kohler et al. (8) that even under
gentle reaction conditions, the interflavanoid linkage of poly-
meric procyanidins can be cleaved with 1 N methanolic HCl by
releasing (þ)-catechin or (-)-epicatechin as carbocation together
with an uncharged terminal unit. The liberated carbocation can
immediately react with an excess of added nucleophiles, such as
(þ)-catechin or (-)-epicatechin, giving rise to the formation
of dimeric procyanidins (Figure 3). During this process the
Polymeric procyanidins of aronia seeds as well as aronia
pomace elute as an unresolved broad peak (polymer hump)

5150 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Esatbeyoglu and Winterhalter
Figure 4. Optimization of reaction conditions: (A-C) with (þ)-catechin; (D-F) with (-)-epicatechin (pro, procyanidin dimer; gam, gambiriin).
Figure 6. HPLC-PDA chromatograms of the Aronia melanocarpa preci-
pitate before semisynthesis (A) and after semisynthesis with (þ)-catechin
(B) or (-)-epicatechin (C) as nucleophile under optimized conditions
Figure 5. Structures of chalcane flavan-3-ol dimers (gambiriins): (a) 1f8
(given in the text) (Gam, gambiriin). See also the Supporting Information.
linked gambiriins; (b) 1f6 linked gambiriins.
and B2 are formed in a higher yield compared to procyanidins
between 30 and 50 min in the HPLC-PDA chromatogram.
Figure 6A shows the HPLC-PDA analysis of the aronia precipi-
tate. After reaction with (þ)-catechin or (-)-epicatechin, the
concentration of dimeric procyanidins increased and the poly-
meric hump nearly disappeared. Consequently, only dimeric
procyanidins with (-)-epicatechin in the upper unit, such as
B1 (EC-4f8-C), B2 (EC-4f8-EC), B5 (EC-4f6-EC), and B7
(EC-4f6-C), are formed during semisynthesis. This is due to the
composition of the polymeric procyanidins of A. melanocarpa.
Our analyses (phloroglucinolysis) have shown that the polymeric
fraction consists almost exclusively of (-)-epicatechin in the
upper unit. Hence, with (þ)-catechin as nucleophile dimeric
procyanidins B1 and B7 are formed (Figure 6B), whereas addition
of epicatechin produces dimeric procyanidins B2 and B5
(Figure 6C). The C4-C8 connected dimeric procyanidins B1
B5 and B7.
HSCCC separations of the reaction mixtures of aronia seed
(CCC-1) or aronia pomace precipitate (CCC-3) with (þ)-catechin
are shown in Figure 7A,C. As solvent system ethyl acetate/
n-butanol/water (14:1:15, v/v/v) was selected, which was pre-
viously found to be suitable for the isolation of dimeric procya-
nidin B1 (10). From fraction 1 of CCC-1 26.8 mg of nonreacted
polymeric procyanidins was recovered. In the case of the aronia
pomace precipitate (CCC-3) this fraction was separated into three
fractions. Fraction 1 consisted of polymeric procyanidins and
fractions 2 and 3 of tetrameric procyanidins. From fraction 2 of
CCC-1 129.4 mg of dimeric procyanidin B1 (purity 93.5%) was
obtained and 122.5 mg of B1 (86.1%) from fraction 4 of CCC-3.
A mixture oftwo unknown trimericprocyanidins(54.7%; 28.9%)
was found in fraction 3 of CCC-1 as well as in fraction 5 of CCC-3

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J. Agric. Food Chem., Vol. 58, No. 8, 2010 5151
Figure 7. (A) CCC-1: HSCCC chromatogram of the reaction products of aronia seed precipitate and (þ)-catechin. Injection of sample, 1000 mg; flow rate, 3
mL/min; retention ofthe stationary phase(Rs), 48.1%. (B) CCC-2: HSCCC chromatogram of the coil fraction of CCC-1. 564mg sample; flow rate, 2.7 mL/min;
Rs, 63.5%. (C) CCC-3: HSCCC chromatogram of the reaction products of aronia pomace precipitate and (þ)-catechin. 1000 mg sample; flow rate, 2.7 mL/
min; Rs, 45.8%. (D) CCC-4: HSCCC chromatogram of the coil fraction of CCC-3. 507 mg sample; flow rate, 2.7 mL/min; Rs, 49.7%. See also the Supporting
Information.
(69.0%; 19.0%). The last fraction 4 of CCC-1 contained two
epimerized dimeric procyanidins. The coil fractions of CCC-1
and CCC-3 were afterward separated using the more lipophilic
solvent system n-hexane/ethyl acetate/methanol/water (1:10:1:10,
v/v/v/v), which allowed the isolation of dimeric procyanidin
B7 (10). Figure 7B,D displays the results of the HSCCC separa-
tions of the coil fractions that were labeled CCC-2 and CCC-4.
Oligomeric procyanidins, like tetrameric and pentameric speci-
mens, were found in fraction 1 (2.8 mg) from CCC-2, whereas in
fractions 2 (1.8 mg) and 3 (3.6 mg, 75.6%) trimeric procyanidins
were enriched. These compounds eluted in fraction 1 of CCC-4
together (15.5%, 52.2%). Fraction 4 of CCC-2 consisted of
9.5 mg of gambiriin A1 and an unknown trimeric procyanidin.
Fraction 2 of CCC-4 was composed of 7.3 mg of gambiriin A1 and
the unknown trimeric procyanidin in concentrations of 41.6 and
42.0%, respectively. Fraction 5 (CCC-2; 33.9 mg) contained
dimeric procyanidin B7 (purity = 92.2%), and fraction 3
(CCC-4) consisted of 31.4 mg of procyanidin B7 (purity =
93.6%). The nonreacted (-)-catechin was recovered in >98%
purity from fraction 7 of CCC-2 (368.9 mg) and from fraction 4 of
CCC-4 (316.9 mg).
HSCCC separations of the reaction mixtures of the semisyn-
theses with the precipitates from aronia seed (CCC-5) and aronia
pomace (CCC-7) with (-)-epicatechin are displayed in Figure 8,
panels A and C, respectively. As solvent system for HSCCC ethyl
acetate/2-propanol/water (20:1:20, v/v/v) was employed (10).

5152 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Esatbeyoglu and Winterhalter
Figure 8. (A) CCC-5: HSCCC chromatogram of thereaction products of aronia seed precipitate and(-)-epicatechin. Injection of sample, 1000mg; flow rate,
2.7 mL/min; Rs, 51.6%. (B) CCC-6: HSCCC chromatogram of the coil fraction of CCC-5. 402 mg sample; flow rate, 2.7 mL/min; Rs, 67.1%. (C) CCC-7:
HSCCCchromatogramofthereactionproducts of aroniapomaceprecipitateand(-)-epicatechin. 1000 mgsample; flow rate, 3 mL/min;Rs, 37%.(D) CCC-8:
HSCCC chromatogram of the coil fraction of CCC-7. 461 mg sample; flow rate, 2.7 mL/min; Rs, 65.8%.
The target compound procyanidin B2 was isolated from
fraction 5 of CCC-5 in a purity of 96.4% (81.8 mg) as well as
from fraction 2 of CCC-7 (75.8 mg) in a purity of 80.0%. Addi-
tional compounds of CCC-5 were in fraction 1 polymeric pro-
cyanidins (3.8 mg), in fraction 2 tetrameric procyanidins (3.9 mg),
in fraction 3 an unknown trimeric procyanidin (6.4 mg, purity =
66.1%) and an (epi)catechin/(epi)gallocatechin derivative with a
molecular ion [M - H]- at m/z 879 and fragmentations at m/z
861, 757, 727, 709, 633, 591, 547, 467, 439, 377, 301, and 259, and
in fraction 4 trimeric procyanidin C1 (10.3 mg, purity = 87.7%).
The latter compounds (42.0 mg) were also found in fraction 1 of
CCC-7 in purities of 39.0 and 37.9%, respectively.
of the separation are shown in Figure 8, panels B (CCC-6) and D
(CCC-8). Fraction 5 of CCC-6 (20.0 mg) contained 88.2% pure
dimeric procyanidin B5, which was also found in fraction 2 of
CCC-8 (18.6 mg) in a purity of 86.3%. Further compounds of
CCC-6 were distributed as follows: polymeric procyanidins
(fraction 1, 1.8 mg), unknown trimeric procyanidin (fraction 2,
2.1 mg), an (epi)catechin/(epi)afezelechin derivative (fraction 3;
3.6 mg) with a molecular ion [M - H]- at m/z 561 and frag-
mentations at m/z 517, 435, 425, 409, 391, 299, 287, 273, 255, 243,
229, and 161, a complex mixture of the gambiriin A4, propelar-
gonidin, and unknown trimeric procyanidin (fraction 4, 4.3 mg).
The excess of nonreacted (-)-epicatechin was isolated from
fraction 6 of CCC-6 (284.4 mg) and from fraction 3 of CCC-8
(291.9 mg).
Compounds that remained on the coil were fractionated with
the same solvent as in the case of CCC-2 and CCC-4. The results

Article
In conclusion, aronia seeds have been applied for the semisyn-
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5153
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thetic formation of dimeric procyanidins. Equally suitable is aronia
pomace, the waste product in the production of chokeberry juices.
Both sources represent a good starting material for the formation
of dimeric procyanidins B1, B2, B5, and B7. Polymeric procya-
nidins of black chokeberry are composed almost exclusively of (-)-
epicatechin; therefore, during semisynthesis only dimeric procya-
nidins are formed that contain (-)-epicatechin in the upper unit,
such as dimeric procyanidins B1, B2, B5, and B7. HSCCC can be
successfully used for the isolation on a preparative scale. In this
way, dimeric procyanidins B1, B2, B5, and B7 were obtained in
high purity (80.0-98.4%). Dimeric procyanidins B1 and B2 were
obtained in highest yields. Otherwise, the C4βfC6 connected
procyanidins, such as dimeric procyanidins B5 and B7, are formed
in a significantly smaller amount.
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ACKNOWLEDGMENT
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(8) Kohler, N.; Wray, V.; Winterhalter, P. New approach for the
Aronia melanocarpa pomace was kindly provided by Kirstin
synthesis and isolation of dimeric procyanidins. J. Agric. Food Chem.
2008, 56, 5374–5385.
Walther (Walther GmbH, Arnsdorf, Germany).
(9) Kennedy, J. A.; Jones, G. P. Analysis of proanthocyanidin cleavage
products following acid-catalysis in the presence of excess phlor-
oglucinol. J. Agric. Food Chem. 2001, 49, 1740–1746.
Supporting Information Available: Tables 1-8. This infor-
mation is available free of charge via the Internet at http://
pubs.acs.org.
€
(10) Kohler, N.; Wray, V.; Winterhalter, P. Preparative isolation of
procyanidins from grape seed extracts by high-speed counter-current
chromatography. J. Chromatogr., A 2008, 1177, 114–125.
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Received for review December 11, 2009. Revised manuscript received
February 17, 2010. Accepted February 20, 2010. We thank the German
Federal Ministry of Education and Research (BMBF) for financial
support of the project “Dietary Procyanidins” (Grant 0313828C).