Eur Food Res Technol (2004) 219:605613
DOI 10.1007/s00217-004-1002-6
ORIGINAL PAPER
Daniel R
sch Angelika Krumbein Lothar W. Kroh
Antioxidant gallocatechins, dimeric and trimeric proanthocyanidins
from sea buckthorn (Hippopha
rhamnoides) pomace
Received: 14 May 2004 / Published online: 12 October 2004

Springer-Verlag 2004
Abstract Residues such as peels and seeds that result
from fruit juice production may contain substantial
amounts of valuable natural antioxidants. In order to
isolate the potential antioxidants gallocatechins and
proanthocyanidins, pomace from sea buckthorn (Hip-
popha
rhamnoides) berries was extracted by acetone-
water (75:25, v/v) and fractionated by Sephadex LH-20
gel chromatography and semipreparative HPLC. The
structures of the monomeric flavan-3-ols isolated, (+)-
gallocatechin and ()-epigallocatechin, were elucidated
by electrospray mass spectrometry (ESI-MS) and 1H and
13
C NMR spectroscopy. Five dimeric proanthocyanidins
were identified by HPLC-ESI-MS(-MS) and by acid-
catalyzed cleavage in the presence of phloroglucinol.
Moreover, nine trimeric proanthocyanidins were tenta-
tively identified by HPLC-ESI-MS(-MS) in the Sephadex
fractions of sea buckthorn pomace. The isolated flavan-3-
ols and proanthocyanidins were potent in scavenging
Fremys salt, a synthetic free radical. They possessed
antioxidant capacities that were higher or comparable to
that of ascorbic acid or Trolox. On comparing the anti-
oxidant capacities of monomeric flavan-3-ols and di-
meric proanthocyanidins, no significant influence from
the degree of polymerization (DP) was observed.
Keywords Sea buckthorn Proanthocyanidins ESI-MS
NMR Antioxidant capacity
D. Rsch L. W. Kroh ())
Institut fr Lebensmitteltechnologie und Lebensmittelchemie,
Technische Universitt Berlin,
Gustav-Meyer-Allee 25, 13355 Berlin, Germany
e-mail: lothar.kroh@tu-berlin.de
Tel.: +49-30-31472584
Fax: +49-30-31472585
A. Krumbein
Institut fr Gemse- und Zierpflanzenbau Grobeeren/Erfurt e.V.,
Theodor-Echtermeyer-Weg 1, 14879 Grobeeren, Germany
Introduction
Increasing use has been made of food byproducts over
the last few years. Residues from plant food processing
(primarily from fruit and vegetables) represent a major
disposal problem for the industry, but they are also
promising sources of compounds that may be used be-
cause of their favorable technological and nutritional
properties [1]. Juice extraction from sea buckthorn (Hip-
popha
rhamnoides) berries leads to a residual press cake,
which can be used for seed separation following extrac-
tion of seed oil. The remaining pulp is reported to contain
mainly flavones [2]. Compared to other fruit byproducts,
like grape seeds and grape pomace, which are reported to
consist mainly of flavan-3-ols and proanthocyanidins [3,
4] little is known about the composition of these phenolic
compounds in sea buckthorn pomace.
Flavan-3-ols and proanthocyanidins (better known as
condensed tannins) are ubiquitous in the plant kingdom,
and are present in many foods, especially in fruit [5]. The
structures of the most abundant flavan-3-ols are shown in
Fig. 1. Flavan-3-ols are also present as esters of gallic
acid, and some glycosidated structures have been reported
[6]. Proanthocyanidins are oligomers and polymers con-
sisting of flavan-3-ol subunits linked through C4!C8 or
C4!C6 bonds (B-type). An additional ether bond can
also be formed between C2!O7 (A-type). Proantho-
cyanidins consisting only of (epi)catechin subunits are
called procyanidins, whereas prodelphinidins are oligo-
mers and polymers of (epi)gallocatechin. Most foods
contain procyanidins or heterogeneous proanthocyanidins
(procyanidins and prodelphinidins) [5, 6]. Flavan-3-ols
and proanthocyanidins show high antiradical activities
against highly reactive oxygen species like the superoxide
(O2), the hydroxyl radical (OH) [7, 8, 9], the synthetic
radical DPPH [10], and the radical cation ABTS+ [11].
They have also been shown to inhibit the oxidative
damage of low-density lipoprotein (LDL) in vitro [12],
and it has been suggested that they contribute to the
phenomenon called French Paradox [13].
606
Fig. 1 Structures of selected flavan-3-ols
In a previous work [14], we identified (+)-catechin and
()-epicatechin in the juice of sea buckthorn fruit. Be-
cause of the lack of reference substances, two compounds
suggesting an (epi)gallocatechin structure remained
unidentified. The aim of this work was the isolation and
structure elucidation of these monomeric gallocatechins,
and of small oligomeric proanthocyanidins. Furthermore,
in order to determine a possible influence from the de-
gree of polymerization (DP), our investigations included
measurement of the antioxidant capacities of the isolat-
ed compounds by electron spin resonance spectroscopy
(ESR).
Experimental
Chemicals
(+)-Catechin, ()-epicatechin, vanillin, and phloroglucinol dihy-
drate were obtained from Fluka (Taufkirchen, Germany). Potassi-
um nitrosodisulfonate (Fremys salt) was purchased from Sigma-
Aldrich (Steinheim, Germany). (+)-Gallocatechin-(4a!2)-phloro-
glucinol, (+)-catechin-(4a!2)-phloroglucinol, and ()-epicatechin-
(4b!2)-phloroglucinol were isolated after acid-catalyzed cleavage
of oligomeric proanthocyanidins from sea buckthorn pomace in the
presence of phloroglucinol [15]. Reagents and solvents were pur-
chased from Roth (Karlsruhe, Germany) or Merck (Darmstadt,
Germany), and were of HPLC or analytical grade quality. HPLC
grade water was purified with a deionized water treatment system.
Extraction, isolation, and purification of gallocatechins
and dimeric proanthocyanidins
Sea buckthorn pomace (Hippopha
rhamnoides subsp. rhamnoides
cv. Hergo) was extracted and fractionated on a 604 cm i.d.
Sephadex LH-20 column (Pharmacia, Uppsala, Sweden), as pre-
viously described [16]. After the majority of flavonol glycosides
had been separated by increasing the methanol content of the eluent
from 0% to 60% (v/v) in increments of 10% to yield fractions
(400 mL each) named AG [16], fractions (20 mL each) named
H120, I120, J120 and K120 were collected by increasing the ated by semipreparative HPLC, as described above. From fractions
methanol content of the eluent from 70% to 100% (v/v) in incre-
ments of 10%. Finally, fractions named L and M were eluted with
Table 1 13C NMR spectroscopic data for (+)-gallocatechin 1 and
()-epigallocatechin 2 in CD3OD (300 MHz)a
Carbon 1 2
2 82.9 79.9
3 68.8 67.5
4 28.1 29.2
4a 100.7 100.1
5 156.8 157.3
6 96.2 96.3
7 157.6 157.6
8 95.5 95.5
8a 157.8 158.0
10 131.5 131.5
20 107.2 107.0
30 146.9 146.7
40 134.0 133.6
50 146.9 146.7
60 107.2 107.0
a
Chemical shifts (d) are in ppm. Assignments were confirmed by
HMQC and HMBC correlations, but carbons with almost the same
chemical shifts may be reversed. For structures, see Fig. 1
70% aqueous acetone (v/v) (400 mL each). The compositions of the
fractions obtained were monitored by HPLC-DAD, as previously
described [14], and by TLC [17]. Fractions exhibiting similar
composition patterns were pooled for further purification or for
HPLC-ESI-MS investigations.
To isolate compounds 1 or 2, pooled Sephadex LH-20 fractions
I4I12 or H14H20/I1I3 were evaporated, dissolved in ethanol
(1 mL) and fractionated on a 451.5 cm i.d. Sephadex LH-20
column using ethanol as eluent. The elution profile was monitored
by HPLC-DAD [14] and TLC [17]. Fractions containing com-
pounds 1 or 2 were evaporated and cleaned up by semipreparative
HPLC on a 25010 mm i.d., 5 mm Fluofix 120E column (NEOS
Company Ltd., Kobe, Japan) connected to a 1010 mm i.d., 5 mm
Hypersil BDS C8 guard column. The samples were fractionated
using isocratic elution with water-acetic acid (99.5:0.5, v/v). After
chromatographic separation has been performed at a flow rate of
5 mL/min using a Gynkothek Model 480 HPLC pump (Dionex,
Germering, Germany), the flow was split into 0.1 mL/min for de-
tection at 280 nm using a variable wavelength UV/Vis detector
(Linear Instruments, Reno, Nevada, USA), and 4.9 mL/min for
fraction collection.
(+)-Gallocatechin (1) was obtained as a white amorphous solid
(2.9 mg from Sephadex fractions I4I12 and 5.7 mg from phlo-
roglucinol degradation of Sephadex fraction M [15]. Rf 0.56 (A),
0.46 (B), 0.35 (C). ESI-MS, m/z (relative intensity): 611 [M+M-H]
(9), 341 [M+Cl] (23), 305 [M-H] (100); MS-MS of 305: 287 [M-
H-H2O] (4), 179 [M-H-C6H6O3] (100), 137 [M-H-C8H8O4] (31).
1
H NMR (CD3OD, 300 MHz) d (ppm): 2.55 (H-4a, dd, J=16.2,
7.8 Hz), 2.85 (H-4b, dd, J=16.1, 5.3 Hz), 4.02 (H-3, m), 4.58 (H-2,
d, J=7.2 Hz), 5.91 (H-8, d, J=2.2 Hz), 5.97 (H-6, d, J=2.2 Hz), 6.45
(H-20 , H-60 , s). 13C NMR data are shown in Table 1.
()-Epigallocatechin (2) was obtained as a white amorphous
solid (1.9 mg from Sephadex fractions H14H20/I1I3, and 2.4 mg
from the phloroglucinol degradation of Sephadex fraction M [15].
Rf 0.56 (A), 0.29 (B), 0.22 (C). ESI-MS, m/z (relative intensity):
611 [M+M-H] (11), 341 [M+Cl] (24), 305 [M-H] (100); MS-MS
of 305: 287 [M-H-H2O] (4), 179 [M-H-C6H6O3] (100), 137 [M-
H-C8H8O4] (33). 1H NMR (CD3OD, 300 MHz) d (ppm): 2.71 (H-
4a, dd, J=16.7, 3.0 Hz), 2.84 (H-4b, dd, J=16.7, 4.5 Hz), 4.16 (H-3,
m), 4.74 (H-2, s), 5.90 (H-8, d, J=2.3 Hz), 5.92 (H-6, d, J=2.1 Hz),
6.50 (H-20 , H-60 , s). 13C NMR data are shown in Table 1.
To isolate dimeric proanthocyanidins, Sephadex LH-20 frac-
tions J7J16 or J17J20/K1K10 were evaporated and fraction-
J7J16 dimers 3 (2.7 mg), 4 (1.2 mg), 5 (1.1 mg), and 7 (1.9 mg)
were isolated. Besides dimeric proanthocyanidins 3 (2.7 mg) and 7

(2.0 mg), the semipreparative HPLC of fractions J17J20/K1K10
also yielded compound 6 (2.2 mg).
TLC
Analytical TLC was performed, in order to monitor the Sephadex
LH-20 fractionation, on 2020 cm silica gel 60 plates (Merck,
Darmstadt, Germany) using acetone-toluene-formic acid (30:30:10,
v/v/v, solvent A) [17]. Isolated monomeric gallocatechins were also
characterized using 2020 cm cellulose plates (Merck, Darmstadt,
Germany), which were developed with t-butanol-acetic acid-water
(30:10:10, v/v/v, solvent B) or acetic acid-water (3:47, v/v/v, sol-
vent C) [18]. The spots were visualized with vanillin reagent: 1 g
vanillin was dissolved in 25 mL ethanol, 25 mL water, and 25 mL
o-phosphoric acid (85%).
NMR spectroscopy
1
H, 13C, 1H-1H-COSY, HMQC, and HMBC spectra of gallocate-
chins 1 and 2 dissolved in CD3OD were recorded using a DPX
300 MHz spectrometer (Bruker, Rheinstetten, Germany).
607
HPLC-DAD-ESI-MS
The Agilent 1100 series HPLC system (Agilent Technologies,
Waldbronn, Germany) consisted of a binary HPLC pump, an au-
tosampler, a degasser, a column thermostat and a photodiode array
detector, and was controlled by ChemStation software. Chromato-
graphic separation of membrane-filtered (0.45 mm PTFE) (Roth,
Karlsruhe, Germany) Sephadex LH-20 fractions J7J16, J17J20/
K1K10, K11K20 and L of pomace extract was carried out on a
2504.6 mm i.d., 5 mm, Fluofix 120E column (NEOS Company
Ltd., Kobe, Japan) connected to a 104.6 mm i.d. guard column of
the same material using two solvents [A: water-acetic acid
(99.5:0.5, v/v), B: acetonitrile]. Gradient elution was performed as
follows: 0% B (5 min); 05% B in 13.5 min; 550% B in 1.5 min;
50% B (5 min); 500% B in 1 min; 0% B (15 min) at a flow rate of
1.0 mL/min and a column temperature of 30 C. After detection by
DAD at 280 nm (spectroscopic contour plots from 200400 nm),
the flow was split and 0.35 mL/min were subjected to ESI-MS
analysis. ESI-MS experiments were performed as described above
with the exception of the conditions of dry and nebulizer gas, which
were set at 10 L/min (350 C) and 40 psi, respectively. MS-MS
experiments were performed by isolation and fragmentation of the
most abundant pseudomolecular ion.

ESI-MS ESR analysis

ESI mass spectra (negative ionization) of isolated gallocatechins 1
and 2 were recorded using an Agilent 1100 Series LC/MSD trap
controlled by LCMSD software (version 4.1). Methanolic solutions
of 1 and 2 (10 mg/L) were injected using a syringe pump (5 mL/
min). Nitrogen was used as dry gas (5 L/min, 300 C) and nebulizer
gas (15 psi). The capillary, end plate, and capillary exit voltages
were set at 3500 V, 500 V, and 120 V, respectively. The full scan
mass spectra were measured from m/z 100 up to m/z 1000. MS-MS
and MSn spectra were recorded by isolation and fragmentation of
the pseudomolecular ions of interest.
Acid-catalyzed cleavage of dimeric proanthocyanidins with phlo-
roglucinol
Methanolic solutions (200 mL) of purified dimeric proanthocyani-
dins (700 mg/L) were added to 200 mL of a freshly prepared so-
lution of phloroglucinol (800 mg/L) in acetic acid. The mixture was
reacted for 30 min in a pressure stable vial at 90 C under a nitrogen
atmosphere. After cooling, the solution was evaporated to dryness
on a nitrogen stream, dissolved in water, and analyzed by HPLC-
DAD-ECD as previously described [14], with the exception of the
applied gradient between eluent A (water-phosphoric acid,
99.5:0.5, v/v) and eluent B (acetonitrile-water-phosphoric acid,
50:45.5:0.5, v/v/v), which was as follows: 0% B (5 min); 015% B
in 24 min; 15100% B in 5 min; 100% B (7 min); 1000% B in
1 min; 0% B (15 min).
To determine their antioxidant capacities, the isolated gallocate-
chins 1 and 2 and the dimeric proanthocyanidins 37 were dis-
solved in methanol in order to prepare 0.1 mM solutions. Aliquots
(500 mL) were allowed to react with an equal volume of a solution
of Fremys salt (1 mM in phosphate buffer pH 7.4) under the
conditions previously described [14].
Results and discussion
The defatted 75% aqueous acetone extract of sea buck-
thorn pomace was fractionated on a Sephadex LH-20
column. While most of the flavonol glycosides eluted by
increasing the methanol content from 0 to 60% [16],
fractions containing flavan-3-ols were collected using a
water-methanol-gradient (70100% methanol) and sub-
sequent elution with 70% aqueous acetone. The flavan-3-
ol compositions of the fractions were monitored by TLC.
Rf values (Table 2) were compared to the results of Lea et
al [17]. Proanthocyanidins of higher molecular weight are
capable of forming strong hydrogen bonds with the
Sephadex material. Therefore, the elution order of sea

Table 2 TLC Rf values (sol- Rf value DPa Structurea Sephadex fraction Rf value (Lea et al [17])
vent A) for flavan-3-ols (DP 1)
and proanthocyanidins (DP 2 0.68 1 (E)C H14-H20, I1-I20, J1-J6 0.68
4) in fractions obtained from the 0.57 1 (E)GC H14-H20, I1-I12
fractionation of sea buckthorn 0.42 2 (E)C-(E)C J7-J16 0.44
pomace extract by Sephadex 0.34 2 (E)C-(E)GC J7-J20, K1-K10
LH-20 gel chromatography, and 0.29 2 (E)GC-(E)GC J7-J20, K1-K10
comparison with literature data 0.29 3 (E)C-(E)C-(E)C K11-K20, L 0.28
from Lea et al [17] 0.24 3 (E)C-(E)C-(E)GC K11-K20, L
0.20 3 (E)C-(E)GC-(E)GC K11-K20, L
0.17 3 (E)GC-(E)GC-(E)GC K11-K20, L
0.00.2c 4 M 0.19
a b
DP=degree of polymerization; C=catechin, GC=gallocatechin, EC=epicatechin, EGC=epigallo-
catechin, alternative sequences are possible for heterogeneous proanthocyanidins; c Rf values are
indicative of the presence of further proanthocyanidins (>DP 4)
608
buckthorn flavan-3-ols followed the DP by Sephadex LH-
20 fractionation (Table 2).
An Rf value of 0.68 indicated the presence of mono-
meric catechins [17], which have been already identified
in sea buckthorn juice [14]. The Rf value 0.57 was as-
signed to monomeric gallocatechins, that were more re-
tarded on silica gel than catechins because of their higher
hydrophilicity. Spots at Rf 0.42 were consistent with the
presence of dimeric procyanidins [17]. Additional spots at
Rf 0.34 and Rf 0.29 suggested the presence of dimeric
heterogeneous proanthocyanidins and dimeric prodel-
phinidins, respectively. The presence of trimeric proan-
thocyanidins was suggested by the spots at Rf 0.29 (pro-
cyanidins), Rf 0.24 and 0.20 (heterogeneous proantho-
cyanidins), and Rf 0.17 (prodelphinidins). After TLC de-
velopment of Sephadex fraction M, a band was detected
ranging from Rf 0.0 to 0.2, which was believed to result
from high molecular weight proanthocyanidin oligomers
(>DP 3). The structural elucidation of Sephadex fraction
M is described elsewhere [15].
Structural elucidation of monomeric gallocatechins
Monomeric gallocatechins 1 and 2 from Sephadex LH-20
fractions I4I12 and H14H20/I1I3 were isolated by
further gel chromatography on Sephadex LH-20 followed
by semipreparative HPLC. The ESI mass spectra (nega-
tive ionization) of compounds 1 and 2 were quite similar
and showed their most abundant signals at m/z 305 [M-
H]. Further ions were detected at m/z 341 [M+Cl] and
m/z 611 [M+M-H]. MS-MS fragmentation of m/z 305
produced the characteristic ion m/z 137 [M-H-C8H8O4]
resulting from retro Diels-Alder (RDA) fission. The 13C
NMR spectra of 1 and 2 showed similar carbon signals for
the phloroglucinol A-ring and the pyrogallol B-ring, but
differences in the signals for the pyran C-ring (Table 1).
In contrast to 2 (d 79.9 and d 67.5 ppm), the C-2 and C-3
signals of 1 appeared downfield at d 82.9 and d 68.8 ppm.
This observation, and the large coupling observed for the
H-2 to H-3 (d 4.58 and d 4.02 ppm, d, J=7.2 Hz) in the 1H
NMR spectrum indicated a 2,3-trans-orientation in 1 [4].
A 2,3-cis-orientation between C-2 and C-3 in 2 was de-
duced from the singlet at d 4.74 ppm corresponding to
H-2. According to spectroscopic data and Rf values [19,
20], compounds 1 and 2 were identified as (+)-gallocat-
echin and ()-epigallocatechin (Fig. 1), respectively. To
our knowledge the occurrence of gallocatechins 1 and 2 in
the fruit of sea buckthorn has never been reported before.
It has, however, been described for the leaves of this plant
[21].
Structural elucidation of dimeric proanthocyanidins
First of all, the dimeric proanthocyanidins of Sephadex
fractions J7J16 and J17J20/K1K10 were investigat-
ed by HPLC-DAD-ESI-MS (Fig. 2). The mass-to-charge
ratio (m/z) of the pseudomolecular ion-peaks allowed the
identification of three different kinds of dimers. The value
m/z 609 [M-H] observed for 3 indicated the presence of a
dimeric prodelphinidin (Table 3). Compounds 4 and 5
showed an ion-peak at m/z 577 [M-H] which was char-
acteristic of dimeric procyanidins (Table 3). Their pseu-
domolecular ion peak [M-H] at m/z 593 suggested
that compounds 6 and 7 were heterogeneous proantho-
cyanidins consisting of one (epi)gallocatechin and one
(epi)catechin subunit (Table 3). In order to determine the
sequence of the monomeric subunits in the heterogeneous
dimers, data on their MS-MS fragmentation pattern were
compared to the observations of Friedrich et al [22].
Unfortunately, mass spectroscopic techniques will not
provide information regarding the stereochemistry of the
flavan-3-ol subunits. After isolation by semipreparative
HPLC, this information could be drawn from the results
of acid-catalyzed cleavage of the dimeric flavan-3-ols in
the presence of phloroglucinol [23]. However, the posi-
tions of the interflavanoid linkages of the proantho-
cyanidins could not be assigned by this method.
MS-MS fragmentation of compound 3 ([M-H] at m/
z 609) produced characteristic fragments at m/z 591 [M-
H-H2O], m/z 483 [M-H-C6H6O3] (loss of phlorogluci-
nol), m/z 441 [M-H-C8H8O4] (RDA fission), and m/z 423
[M-H-C8H8O4-H2O] (Table 3). Cleavage of the inter-
flavanoid bond led to m/z 305 [Mter-H] (lower terminal
subunit) and m/z 303 [Mext-3H] (upper extension sub-
unit) [22]. After isolation, dimer 3 was reacted with
phloroglucinol, which led to the release of the terminal
subunit that was identified as (+)-gallocatechin by HPLC
comparison with the reference compound (Fig. 3). The
extension subunit was captured by phloroglucinol, which
yielded (+)-gallocatechin-(4a!2)-phloroglucinol. There-
fore, the structure of 3 was concluded to be gallocatechin-
gallocatechin.
MS-MS fragments of 4 and 5 ([M-H] at m/z 577)
appeared at m/z 559 [M-H-H2O], m/z 451 [M-H-
C6H6O3] (loss of phloroglucinol), m/z 425 [M-H-
C8H8O3] (retro Diels-Alder fission), m/z 407 [M-H-
C8H8O3-H2O], m/z 289 [Mter-H], and m/z 287 [Mext-
3H] (Table 3). After reaction with phloroglucinol, dimer
4 yielded (+)-catechin and (+)-catechin-(4a!2)-phloro-
glucinol. While the extension subunit of 5 was also cap-
tured as (+)-catechin-(4a!2)-phloroglucinol, ()-epicat-
echin was released as the terminal subunit. Therefore, the
structures of 4 and 5 were catechin-catechin and catechin-
epicatechin, respectively.
Information about the sequence in heterogeneous
proanthocyanidins could be drawn from the intensity of
RDA fission products after MS-MS fragmentation. From
investigations with authentic reference material, it is
known that RDA fission mainly occurs in the upper ex-
tension subunit [22] which gives rise to a fragment that is
more energetically favorable [5]. In the case of the di-
meric proanthocyanidin 6 ([M-H] at m/z 593), the most
abundant RDA fragments were detected at m/z 441 [M-H-
C8H8O3] (loss of m/z 152) and m/z 423 [M-H-C8H8O3-
H2O] (loss of m/z 170) (Fig. 4). Fragments at m/z 425
[M-H-C8H8O4] (loss of m/z 168) and m/z 407 [M-H-
 609
Fig. 2 HPCL-DAD (280 nm)
plots of Sephadex LH-20 frac-
tions J7-J16, J17-J20/K1-K10,
K11-K20 and L of sea buck-
thorn extract. For peak identifi-
cation see Tables 3 and 4

Table 3 HPLC-DAD-ESI-MS data for dimeric proanthocyanidins obtained from sea buckthorn pomace
Peak Rt (min) Fractions UV (nm) [MH] (m/z) MS-MS (m/z)a Structure assignmentb
3 4.5 J7-J20/K1-K10 272 609 591 (2), 483 (12), 441 (100), 423 (90), GC-GC
305 (25), 303 (2)
4 10.7 J7-J20/K1-K10 280 577 559 (6), 451 (39), 425 (100), 407 (93), C-C
289 (32), 287 (15)
5 16.6 J7-J16 280 577 559 (6), 451 (39), 425 (100), 407 (56), C-EC
289 (44), 287 (5)
6 9.7 J7-J20/K1-K10 277 593 575 (27), 467 (57), 441 (47), 425 (56), C-EGC
423 (100), 407 (10), 305 (41), 303 (10),
289 (16), 287 (10)
7 6.4 J7-J16 277 593 575 (66), 467 (48), 441 (100), 425 (86), C-GC and GC-C
423 (95), 407 (32), 305 (78), 303 (10)
a b
Relative intensities are given in parentheses; C=catechin, GC=gallocatechin, EC=epicatechin, EGC=epigallocatechin

C8H8O4-H2O] (loss of m/z 186) produced a lower in-
tensity (Table 3). Therefore, the structure (epi)catechin-
(epi)gallocatechin was concluded for compound 6. This
assignment was confirmed by the higher intensity of the
daughter ion m/z 305 compared to m/z 289 (Table 3)
which corresponded to the fragment [Mter-H] resulting
from the release of the terminal subunit (Fig. 4). This
cleavage also released the extension subunit which was
detected as the daughter ion m/z 287 [Mext-3H] (Fig. 4).
The stereochemistry of the terminal subunit was deduced
after reaction with phloroglucinol/HCl, which yielded
()-epigallocatechin. The extension subunit was captured
as (+)-catechin-(4a!2)-phloroglucinol, and the structure
catechin-epigallocatechin was assigned to 6.
Compound 7 exhibited a MS-MS fragmentation pat-
tern quite similar to that of compound 6 (Table 3). After
reaction with phloroglucinol/HCl, (+)-catechin and (+)-
catechin-(4a!2)-phloroglucinol, as well as (+)-gallocat-
echin and (+)-gallocatechin-(4a!2)-phloroglucinol were
formed. Although compound 7 gave only one signal on
HPLC analysis, we concluded that it consisted of gallo-
catechin-catechin and catechin-gallocatechin.
610
Fig. 3 Possible structure of
gallocatechin-gallocatechin 3
and its acid-catalyzed cleavage
in the presence of phlorogluci-
nol

Fig. 4 Structure of selected

daughter ions resulting from
MS-MS fragmentation of the
dimeric proanthocyanidin cate-
chin-epigallocatechin 6

Identification of trimeric proanthocyanidins
Trimeric proanthocyanidins of Sephadex fractions K11
K20 and L were also investigated by HPLC-DAD-ESI-
MS (Fig. 2). Compounds 8, 9 and 10 showed pseudo-
molecular ion peaks [M-H] at m/z 913.5, which was
consistent with trimeric prodelphinidins (Table 4). The
occurrence of trimeric procyanidins 11, 12 was deduced
from their pseudomolecular ion peak [M-H] at m/z 865.6
(Table 4). The daughter ions resulting from MS-MS frag-
 611

mentation of compounds 812 were in accordance with

Structure assignmentb

(E)GC-(E)GC-(E)GC
(E)GC-(E)GC-(E)GC
(E)GC-(E)GC-(E)GC

(E)GC-(E)GC-(E)C
(E)GC-(E)GC-(E)C
the loss of phloroglucinol, RDA fission, and cleavage of

(E)C-(E)GC-(E)C
(E)GC-(E)C-(E)C
the interflavanoid bond (Table 4).

(E)C-(E)C-(E)C
(E)C-(E)C-(E)C
The pseudomolecular ion [M-H] of 13 and 14 at
m/z 897.5 indicated that these compounds were trimeric
proanthocyanidins consisting of two (epi)gallocatechin
and one (epi)catechin subunits (Table 4). As for the di-
meric proanthocyanidins, RDA fission of trimers pre-
dom-inately affects the upper extension subunit [22].

(32), 745 (13), 729 (32), 727 (4) 711 (100), 607 (30), 593 (37), 592 (22), 303 (6), 289 (7)
In the case of 13 and 14, this fission resulted in the for-
mation of m/z 711 [M-H-C8H8O4-H2O] as the most
(12), 745 (78), 743 (100) 727 (78), 609 (23), 607 (30), 429 (97) 305 (46), 303 (8)

(54), 729 (43), 713 (17), 711 (100), 695 (23), 593 (42), 591 (8), 303 (2), 289 (20)
abundant daughter ion (Table 4), which suggests the
presence of (epi)gallocatechin as the upper extension
(8), 745 (4), 729 (41), 727 (9) 711 (100), 607 (6), 592 (44), 303 (25), 289 (4) subunit. The occurrence of the daughter ion m/z 289
(100),784 (53), 745 (19), 727 (95), 609 (51), 607 (58), 305 (48), 303 (44)

(12), 695 (100), 591 (25), 577 (24), 305 (4), 303 (12), 289 (8), 287 (8)
[Mter-H] indicates that (epi)catechin is the terminal
subunit (Table 4). Therefore, the sequences of 13
and 14 were tentatively concluded as (epi)gallocatechin-
(epi)gallocatechin-(epi)catechin.
(54), 713 (70), 695 (100), 577 (51), 575 (21), 289 (6), 287 (47)
(50), 713 (9), 695 (82), 577 (100), 575 (66), 289 (11), 287 (44)

Compound 15 exhibited a pseudomolecular ion peak

[M-H] at m/z 881.5 (Table 4). The most abundant
C=catechin, GC=gallocatechin, EC=epicatechin, EGC=epigallocatechin

daughter ion in MS-MS was m/z 711 [M-H-C8H8O3-

(46), 745 (6), 727 (100), 609 (51), 607 (11), 305 (14)

H2O]. Furthermore, the ion m/z 289 [Mter-H] was

detected with a high intensity, suggesting that the tenta-
tive sequence of 15 is (epi)catechin-(epi)gallocatechin-
(epi)catechin. In contrast to 15, RDA fission of 16 ([M-
H] at m/z 881.1) led to m/z 695 [M-H-C8H8O4-H2O] as
the most abundant daughter ion (Table 4). Therefore, it
was suggested that 16 ((epi)gallocatechin-(epi)catechin-
Table 4 HPLC-DAD-ESI-MS data for trimeric proanthocyanidins from sea buckthorn pomace

(epi)catechin) consists of (epi)gallocatechin as the upper

extension.
In principle, further structural elucidation of the tri-
meric proanthocyanidins could be performed by the
phloroglucinol reaction. Due to the limited concentration
of each trimer, and the lack of reference substances, they
were not purified from the Sephadex fractions.
MS-MS (m/z)a

Antioxidant capacities of isolated gallocatechins

787
787
896
739
739
771
771
755
713

and dimeric proanthocyanidins

Each of the isolated compounds were tested for their ef-

[MH]

ficiency to scavenge the free radical species Fremys salt.

913.5
913.5
913.5
865.6
865.6
897.5
897.5
881.5
881.1
(m/z)

All tested flavan-3-ol monomers and proanthocyanidins

were very potent scavengers of Fremys radical, as
b
Relative intensities are given in parentheses;

demonstrated by their antioxidant capacities, which were

(nm)

higher than or comparable to ascorbic acid or the Vita-

274
273
276
280
276
276
274
278
278
UV

min E derivative Trolox (Table 5). Because of their ad-

ditional hydroxyl group (pyrogallol structure), gallocate-
K11-K20/L

K11-K20/L
K11-K20/L
K11-K20/L
K11-K20/L

chins are reported to be more efficient radical scavengers

K11-K20

K11-K20
K11-K20
Fractions

than catechins [8, 24]. However, our investigations with

Fremys salt suggested lower antioxidant capacities for
(+)-gallocatechin 1 and ()-epigallocatechin 2 (4.7 and
L

4.1 mol Fremys salt/mol) than for (+)-catechin and

()-epicatechin (6.1 and 6.7 mol Fremys salt/mol) (Ta-
Rt (min)

ble 5). This observation could be attributed to a higher

3.8
4.0
4.9
9.8
17.0
5.2
5.6
7.0
6.3

concentration of impurities within the isolated gallocate-

chins than in the catechins that were used as commercial
available standards. Another possible explanation could
Peak

8
9
10
11
12
13
14
15
16

be the higher sensitivity of the gallocatechins toward

a
612
Table 5 Antioxidant capacity Compound Antioxidant capacity [mol Fremys
(mol Fremys salt/mol mono- salt/mol monomeric subunit]a
meric subunit; meanSD of
duplicate assays) of monomeric Monomeric flavan-3-ols ()-epicatechinb 6.70.2
flavan-3-ols and proantho- (+)-catechinb 6.10.3
cyanidins (+)-gallocatechin 1 4.70.1
()-epigallocatechin 2 4.10.1
Proanthocyanidins gallocatechin-gallocatechin 3 4.80.2
catechin-catechin 4 6.10.1
catechin-epicatechin 5 3.30.3
catechin-epigallocatechin 6 5.40.2
catechin-gallocatechin / 5.00.3
gallocatechin-catechin 7
Others ascorbic acidb 4.00.3
Troloxb,c 4.20.0
a
For all proanthocyanidins, the value was calculated on the basis of the molecular weight of (+)-
catechin (290 Da); b data from Rsch et al [14]; c commonly used as a reference substance when
comparing antioxidant activities

oxidation, which caused their rapid degradation during
storage or measurement. For comparison with the mono-
mers, the antioxidant capacities of the isolated dimeric
proanthocyanidins were not calculated on their molecu-
lar weight but on the basis of the monomeric subunit
(+)-catechin (Table 5). With the exception of dimer 5, the
antioxidant capacities of the dimeric proanthocyanidins
were comparable to those of the monomeric flavan-3-ols
(Table 5). Comparison of these results with those ob-
tained by other methods proved difficult, because differ-
ent substrates, system compositions and analytical meth-
ods may result in different orders of antioxidant effec-
tiveness for the same phenolic compounds [25]. In our
previous paper [14], we reported that the antioxidant ef-
fectivenesses of most phenolic compounds against Fre-
mys salt were quite similar to their abilities to scavenge
the radical cation ABTS+ [24]. We can now confirm this
observation by comparing our results for monomeric
flavan-3-ols and dimeric proanthocyanidins with the re-
sults of Plumb et al [11]. These authors also reported no
differences in the antioxidant activities of monomers
and dimers (expressed in monomer equivalents) against
ABTS+.
Conclusions
In conclusion, the present study demonstrates the isola-
tion and structural elucidation of monomeric gallocate-
chins and low molecular weight oligomeric proantho-
cyanidins from sea buckthorn pomace. The isolated com-
pounds were potent in scavenging the synthetic free rad-
ical Fremys salt. Therefore, utilization of sea buckthorn
pomace extract as food supplement would be conceivable.
The physiological significance of antioxidants from food
depends on their absorption and biotransformation. Sig-
nificant amounts of the intact (+)-catechin molecule are
absorbed in vivo [26]. Because of the complexities of
proanthocyanidins, very few studies on their bioavail-
abilities exist. In an in vitro study, it was demonstrated
that the monomer, dimer and trimer were all absorbed to a
similar extent through a cell layer derived from human
intestinal cell line Caco-2 [27]. Furthermore, dimeric
procyanidins were detected in human plasma after con-
sumption of a flavanol-rich food such as cocoa [28].
Further in vivo data are necessary to evaluate the real
health benefits of the proanthocyanidins.
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