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Eur Food Res Technol (2004) 219:605–613 DOI 10.1007/s00217-004-1002-6

ORIGINAL PAPER

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 1 H and

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 Fremy’s 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.

13

Keywords Sea buckthorn · Proanthocyanidins · ESI-MS · NMR · Antioxidant capacity

D. R sch · L. W. Kroh ( ) )

Institut f r Lebensmitteltechnologie und Lebensmittelchemie,

Technische Universit t 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 f r Gem se- und Zierpflanzenbau Großbeeren/Erfurt e.V.,

Theodor-Echtermeyer-Weg 1, 14879 Großbeeren, 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 (O 2 · ), 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

606 Fig. 1 Structures of selected flavan-3-ols In a previous work [14], we identified (+)-catechin and

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 (Fremy’s salt) was purchased from Sigma- Aldrich (Steinheim, Germany). (+)-Gallocatechin-(4 a!2)-phloro- glucinol, (+)-catechin-(4 a !2)-phloroglucinol, and ( )-epicatechin- (4 b!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 60 4 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 A G [16], fractions (20 mL each) named H1 20, I1 20, J120 and K1 20 were collected by increasing the 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 13 C NMR spectroscopic data for (+)-gallocatechin 1 and

( )-epigallocatechin 2 in CD 3 OD (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

1

0

131.5

131.5

2

0

107.2

107.0

3

0

146.9

146.7

4

0

134.0

133.6

5

0

146.9

146.7

6 0

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 I4 I12 or H14H20 / I1 I3 were evaporated, dissolved in ethanol (1 mL) and fractionated on a 45 1.5 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 250 10 mm i.d., 5 mm Fluofix 120E column (NEOS Company Ltd., Kobe, Japan) connected to a 10 10 mm i.d., 5 m m 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 I4 I12 and 5.7 mg from phlo- roglucinol degradation of Sephadex fraction M [15]. R f 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-H O] (4), 179 [M-H-C H O ] (100), 137 [M-H-C H O ] (31). 1 H NMR (CD 3 OD, 300 MHz) d (ppm): 2.55 (H-4 a , dd, J =16.2, 7.8 Hz), 2.85 (H-4 b , 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-2 0 , H-6 0 , s). 13 C NMR data are shown in Table 1.

( )-Epigallocatechin ( 2) was obtained as a white amorphous

solid (1.9 mg from Sephadex fractions H14–H20/I1–I3 , and 2.4 mg from the phloroglucinol degradation of Sephadex fraction M [15]. R f 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-H 2 O] (4), 179 [M-H-C 6 H 6 O 3 ] (100), 137 [M- H-C 8 H 8 O 4 ] (33). H NMR (CD 3 OD, 300 MHz) d (ppm): 2.71 (H- 4a , dd, J =16.7, 3.0 Hz), 2.84 (H-4 b, 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-2 0 , H-6 0 , s). 13 C NMR data are shown in Table 1. To isolate dimeric proanthocyanidins, Sephadex LH-20 frac- tions J7 J16 or J17–J20/K1–K10 were evaporated and fraction- ated by semipreparative HPLC, as described above. From fractions 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

6

6

3

8

8

4

1

(2.0 mg), the semipreparative HPLC of fractions J17–J20/K1–K10 also yielded compound 6 (2.2 mg).

TLC

Analytical TLC was performed, in order to monitor the Sephadex LH-20 fractionation, on 20 20 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 20 20 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, 13 C, 1 H- 1 H-COSY, HMQC, and HMBC spectra of gallocate- chins 1 and 2 dissolved in CD 3 OD were recorded using a DPX 300 MHz spectrometer (Bruker, Rheinstetten, Germany).

ESI-MS

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 MS n 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 m L 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); 0–15% B in 24 min; 15–100% B in 5 min; 100% B (7 min); 100–0% B in 1 min; 0% B (15 min).

HPLC-DAD-ESI-MS

607

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, J17–J20/ K1–K10, K11 K20 and L of pomace extract was carried out on a

250 4.6 mm i.d., 5 mm, Fluofix 120E column (NEOS Company

Ltd., Kobe, Japan) connected to a 10 4.6 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); 0–5% B in 13.5 min; 5–50% B in 1.5 min;

50% B (5 min); 50–0% 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 200–400 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.

ESR analysis

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 Fremy’s 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 (70–100% methanol) and sub- sequent elution with 70% aqueous acetone. The flavan-3- ol compositions of the fractions were monitored by TLC. R f 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 R f values (sol- vent A) for flavan-3-ols (DP 1) and proanthocyanidins (DP 2– 4) in fractions obtained from the fractionation of sea buckthorn pomace extract by Sephadex LH-20 gel chromatography, and comparison with literature data from Lea et al [17]

R f value

DP a

Structure a

Sephadex fraction

R f value (Lea et al [17])

0.68

1

(E)C

H14-H20, I1-I20, J1-J6 H14-H20, I1-I12

0.68

0.57

1

(E)GC

0.42

2

(E)C-(E)C

J7-

J16

0.44

0.34

2

(E)C-(E)GC

J7- J20, K1-K10 J7- J20, K1-K10

0.29

2

(E)GC-(E)GC

0.29

3

(E)C-(E)C-(E)C

K11-K20, L

0.28

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

0.0 0.2 c

4

M

0.19

a DP=degree of polymerization; b C=catechin, GC=gallocatechin, EC=epicatechin, EGC=epigallo- catechin, alternative sequences are possible for heterogeneous proanthocyanidins; c R f 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 R f value of 0.68 indicated the presence of mono-

meric catechins [17], which have been already identified

in sea buckthorn juice [14]. The R f 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 R f 0.42 were consistent with the presence of dimeric procyanidins [17]. Additional spots at R f 0.34 and R f 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 R f 0.29 (pro- cyanidins), R f 0.24 and 0.20 (heterogeneous proantho- cyanidins), and R f 0.17 (prodelphinidins). After TLC de- velopment of Sephadex fraction M , a band was detected ranging from R f 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 H14–H20/I1–I3 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-C 8 H 8 O 4 ] resulting from retro Diels-Alder (RDA) fission. The 13 C 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 1 H

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 R f 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 J17–J20/K1–K10 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-H 2 O] , m/z 483 [M-H-C 6 H 6 O 3 ] (loss of phlorogluci- nol), m/z 441 [M-H-C 8 H 8 O 4 ] (RDA fission), and m/z 423 [M-H-C 8 H 8 O 4 -H 2 O] (Table 3). Cleavage of the inter- flavanoid bond led to m/z 305 [M ter -H] (lower terminal subunit) and m/z 303 [M ext -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-(4 a !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-H 2 O] , m/z 451 [M-H- C 6 H 6 O 3 ] (loss of phloroglucinol), m/z 425 [M-H- C 8 H 8 O 3 ] (retro Diels-Alder fission), m/z 407 [M-H- C 8 H 8 O 3 -H 2 O] , m/z 289 [M ter -H] , and m/z 287 [M ext - 3H] (Table 3). After reaction with phloroglucinol, dimer 4 yielded (+)-catechin and (+)-catechin-(4 a! 2)-phloro- glucinol. While the extension subunit of 5 was also cap- tured as (+)-catechin-(4 a! 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- C 8 H 8 O 3 ] (loss of m/z 152) and m/z 423 [M-H-C 8 H 8 O 3 - H 2 O] (loss of m/z 170) (Fig. 4). Fragments at m/z 425 [M-H-C 8 H 8 O 4 ] (loss of m/z 168) and m/z 407 [M-H-

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

609

extract. For peak identifi- cation see Tables 3 and 4 609 Table 3 HPLC-DAD-ESI-MS data for

Table 3 HPLC-DAD-ESI-MS data for dimeric proanthocyanidins obtained from sea buckthorn pomace

Peak

R t (min) Fractions

UV (nm) [M H] ( m/z )

MS-MS ( m/z ) a

Structure assignment b

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 Relative intensities are given in parentheses; b C=catechin, GC=gallocatechin, EC=epicatechin, EGC=epigallocatechin

C 8 H 8 O 4 -H 2 O] (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 [M ter -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 [M ext -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-(4 a !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

acid-catalyzed cleavage in the presence of phlorogluci- nol Fig. 4 Structure of selected daughter ions resulting

Fig. 4 Structure of selected daughter ions resulting from MS-MS fragmentation of the dimeric proanthocyanidin cate- chin-epigallocatechin 6

the dimeric proanthocyanidin cate- chin-epigallocatechin 6 Identification of trimeric proanthocyanidins Trimeric

Identification of trimeric proanthocyanidins

Trimeric proanthocyanidins of Sephadex fractions K11K20 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-

Structure assignment b

(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

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

711 711 575 607 (100), 609 609 575 305 695 (100), (66), (11), (51), (23), (21), (4), (23), 607 607 303 289 305 607 607 289 593 (6), (30), (12), (11), (14) (58), (30), (6), (42), 592 593 287 289 287 (44), 305 429 591 (37), (47) (8), (44) (48), (97) 303 (8), 287 592 303 305 (25), 303 (8) (22), (2), (46), (44) 289 289 303 303 (4) (20) (6), (8) 289 (7) (E)C-(E)GC-(E)C

(E)C-(E)C-(E)C

(E)C-(E)C-(E)C

a Relative intensities are given in parentheses; b C=catechin, GC=gallocatechin, EC=epicatechin, EGC=epigallocatechin

695 729 745 713 713 745 745 (4), (100), (43), (13), (9), (70), (6), (78), (53), 729 695 727 713 729 695 743 745 591 (41), (82), (100), (17), (32), (100), (100) (19), (25), 727 577 711 727 609 727 727 577 577 (9) (100), (100), (4) (51),

(24),

(51),

(78),

(95),

Table 4 HPLC-DAD-ESI-MS data for trimeric proanthocyanidins from sea buckthorn pomace

MS-MS ( m/z ) a

(8), (32), (50), (54), (100),784 (46), (12), 745

(54),

713 755 771 771 739 739 896 787 787 (12),

( [M m/z ) H]

865.6

865.6

913.5

913.5

913.5

897.5

897.5

881.5

881.1

UV (nm)

278 278 274 276 276 280 276 273 274

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

K20

K20

K20

Fractions

R t (min)

17.0 6.3 7.0 5.6 5.2 9.8 4.9 4.0 3.8

Peak

15 14 13 12 11 10 9 8

16

611

mentation of compounds 812 were in accordance with the loss of phloroglucinol, RDA fission, and cleavage of the interflavanoid bond (Table 4). 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]. In the case of 13 and 14, this fission resulted in the for- mation of m/z 711 [M-H-C 8 H 8 O 4 -H 2 O] as the most abundant daughter ion (Table 4), which suggests the presence of (epi)gallocatechin as the upper extension subunit. The occurrence of the daughter ion m/z 289 [M ter -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. Compound 15 exhibited a pseudomolecular ion peak [M-H] at m/z 881.5 (Table 4). The most abundant daughter ion in MS-MS was m/z 711 [M-H-C 8 H 8 O 3 - H 2 O] . Furthermore, the ion m/z 289 [M ter -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-C 8 H 8 O 4 -H 2 O] as the most abundant daughter ion (Table 4). Therefore, it was suggested that 16 ((epi)gallocatechin-(epi)catechin- (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.

Antioxidant capacities of isolated gallocatechins and dimeric proanthocyanidins

Each of the isolated compounds were tested for their ef-

ficiency to scavenge the free radical species Fremy’s salt. All tested flavan-3-ol monomers and proanthocyanidins were very potent scavengers of Fremy’s radical, as demonstrated by their antioxidant capacities, which were higher than or comparable to ascorbic acid or the Vita- min E derivative Trolox (Table 5). Because of their ad- ditional hydroxyl group (pyrogallol structure), gallocate- chins are reported to be more efficient radical scavengers than catechins [8, 24]. However, our investigations with Fremy’s salt suggested lower antioxidant capacities for (+)-gallocatechin 1 and ( )-epigallocatechin 2 (4.7 and 4.1 mol Fremy’s salt/mol) than for (+)-catechin and

( )-epicatechin (6.1 and 6.7 mol Fremy’s salt/mol) (Ta-

ble 5). This observation could be attributed to a higher concentration of impurities within the isolated gallocate- chins than in the catechins that were used as commercial available standards. Another possible explanation could

be the higher sensitivity of the gallocatechins toward

612

Table 5 Antioxidant capacity (mol Fremy’s salt/mol mono- meric subunit; mean€SD of duplicate assays) of monomeric flavan-3-ols and proantho- cyanidins

Compound

Antioxidant capacity [mol Fremy’s salt/mol monomeric subunit] a

Monomeric flavan-3-ols

Proanthocyanidins

Others

( )-epicatechin b (+)-catechin (+)-gallocatechin 1 )-epigallocatechin 2

gallocatechin-gallocatechin 3 catechin-catechin 4

catechin-epicatechin 5 catechin-epigallocatechin 6 catechin-gallocatechin / gallocatechin-catechin 7 ascorbic acid Trolox b,c

b

(

b

6.7€0.2

6.1€0.3

4.7€0.1

4.1€0.1

4.8€0.2

6.1€0.1

3.3€0.3

5.4€0.2

5.0€0.3

4.0€0.3

4.2€0.0

a For all proanthocyanidins, the value was calculated on the basis of the molecular weight of (+)-

catechin (290 Da);

data from R sch et al [14]; c commonly used as a reference substance when

comparing antioxidant activities

b

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- my’s 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 Fremy’s 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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