Semisynthetic Preparation and Isolation of Dimeric Procyanidins B1

Article pubs.acs.org/JAFC

Semisynthetic Preparation and Isolation of Dimeric Procyanidins B1− B8 from Roasted Hazelnut Skins (Corylus avellana L.) on a Large Scale Using Countercurrent Chromatography Tuba Esatbeyoglu,† Andreas Juadjur,† Victor Wray,§ and Peter Winterhalter*,† †

Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany

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S Supporting Information *

ABSTRACT: Dimeric procyanidins B1−B8 were produced via semisynthesis from a polymeric proanthocyanidin fraction of hazelnut skins (Corylus avellana L.). This polymeric fraction was found to consist mostly of (+)-catechin and (−)-epicatechin as upper units. Therefore, according to the choice of nucleophile agent, it is possible to semisynthesize dimeric procyanidins B1, B3, B6, and B7 with (+)-catechin and B2, B4, B5, and B8 with (−)-epicatechin. The semisynthetic mixtures were separated on a preparative scale using high-speed countercurrent chromatography (HSCCC) and low-speed rotary countercurrent chromatography (LSRCCC). C4 → C8 linked dimeric procyanidins B1−B4 were isolated in amounts of 350−740 mg. To the best of the authors’ knowledge this is the first study isolating dimeric procyanidins B1−B8 in large amounts with countercurrent chromatography. Moreover, the dimeric prodelphinidins B1, B2, and B3 and their structural elucidation by 1H NMR spectroscopy without derivatization are described for hazelnuts as natural compounds for the first time. KEYWORDS: hazelnut (Corylus avellana L.), procyanidins, prodelphinidins, propelargonidins, semisynthesis, countercurrent chromatography, NMR spectroscopy

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hazelnut skins. In hazelnut skins flavan-3-ols ((+)-catechin, (−)-epicatechin, (−)-epicatechin 3-O-gallate), B-type dimeric procyanidins (B1, B2, B3), B-type dimeric galloylated procyanidins, B-type trimeric procyanidins (C2), and B-type dimeric and trimeric prodelphinidins were determined.8 It has been shown that roasting leads to a significant loss of phenolic compounds, that is, condensed tannins (proanthocyanidins) due to skin removal, indicating they contain high amounts in their skins compared to the kernel. To obtain the healthpromoting effects of polyphenols, consumption of whole unroasted hazelnuts instead of unroasted or roasted hazelnuts without skin is recommended.6,10 Because of the high price and commercially available low amounts of proanthocyanidins, including dimeric procyanidins as reference compounds, information about the bioavailability and bioactivity of these compounds is limited. To study the bioavailability and bioactivity of proanthocyanidins higher amounts are necessary. The amount of proanthocyanidins that is isolated from natural plant sources or foods is mostly very low. Recently, a successful, inexpensive formation of dimeric procyanidins by semisynthesis and their isolation by high-speed countercurrent chromatography on a large scale were described.11−13 Countercurrent chromatography is an inexpensive isolation technique because of low solvent costs and the absence of expensive adsorbents. During semisynthesis oligomeric and polymeric proanthocyanidins of proanthocya-

INTRODUCTION Polyphenols are secondary plant metabolites with a broad spectrum of chemical structures. Flavonoids with their C6− C3−C6 skeleton (2-phenylbenzopyran) are well-known phenolic compounds. One of the most important subclasses of flavonoids is the proanthocyanidins.1 Proanthocyanidins consisting exclusively of the flavan-3-ol units (epi)catechin are called procyanidins. With (epi)gallocatechin units they are named prodelphinidins and with (epi)afzelechin units, propelargonidins. In B-type proanthocyanidins these flavan-3ol units are linked through a C4−C8 and a C4−C6 bond. Atype proanthocyanidins have an ether bond between C2 and O7 or between C2 and O5 in addition to the C−C bond.2,3 Proanthocyanidins occur in many plant-derived foods. Nuts, especially hazelnuts (Corylus avellana L.), which are included in the Mediterranean diet, are a good source of proanthocyanidins.4 Proanthocyanidins are known, for example, to reduce the risk of cancer, cardiovascular diseases, and inflammation.5 Hazelnut, one of the most popular tree nuts in the world, belongs to the Betulaceae family. Turkey, especially the Black Sea region, is the major hazelnut producer worldwide.6,7 Hazelnut skins are byproducts of the roasting process in the food industry,8 which are mainly composed of B-type proanthocyanidins with an average degree of polymerization of 9.9 Recently, Del Rio et al.8 quantified >95% monomeric and polymeric proanthocyanidins (about 640 mg/100 g) of total polyphenols in hazelnut skins. (Epi)catechin and (epi)gallocatechin- and (epi)catechin-3-O-gallate units are bound in polymeric structures.2,9 To date, little is known about the qualitative and quantitative proanthocyanidin composition of © XXXX American Chemical Society

Received: March 17, 2014 Revised: June 23, 2014 Accepted: June 28, 2014

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dx.doi.org/10.1021/jf501312a | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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HPLC pump BT 3020 pumped the two-phase solvent systems. The aqueous lower phase was used as mobile phase (elution mode, head to tail). All samples were dissolved in 10 mL upper and 10 mL lower phases and loaded into the system by loop injection. The amount of loaded sample, revolution speed of the apparatus, flow rate, and solvent systems are given in the discussions for the respective runs. The HSCCC separation was monitored at λ = 280 nm with a Knauer UV−vis detector (Berlin, Germany) and recorded on a plotter (BBC Goerz SE 120, Vienna, Austria; 3 cm/h). Fractions of 12 mL were collected using a Super Frac fraction collector (Pharmacia LKB, Bromma, Sweden). LSRCCC. LSRCCC separation of dimeric procyanidins B1, B3, B6, and B7 was carried out according to the method of Esatbeyoglu et al.15 For the LSRCCC separation (single coil, total volume, 5500 mL; 8.2 mm i.d.) of dimeric procyanidins B2, B4, B5, and B8, 12.4 g of the lyophilized filtrate of the semisynthetic process with (−)-epicatechin was dissolved in 100 mL upper and 100 mL lower phases and was loaded into the system via a sample loop. tert-Butyl methyl ether/nbutanol/water (4.5:0.5:5, v/v/v) was used as the two-phase solvent system. The flow rate was set at 4 mL/min, and the revolution speed at 50 rpm. The upper organic phase represented the mobile phase. LSRCCC separation was carried out in the “head-to-tail” elution mode. After an elution time of 39 h, the elution mode was switched to extrusion. In this case, the aqueous lower phase was pumped into the LSRCCC column. UV absorbance of the effluent was monitored at λ = 280 nm with a Knauer UV−vis detector. The elution was recorded on a Servogor 120 plotter (BBC Goerz Metrawatt SE 120, Vienna, Austria). About 50 mL fractions were collected by a fraction collector (Pharmacia LKB Super Frac). Semipreparative HPLC. CCC fractions were purified on a preparative HPLC system from Knauer (Smartline 1000 HPLC pump, Smartline Manager 5000 solvent organizer and degasser, Wellchrom K-2600 UV detector). A Hypersil ODS C-18, 5 μm, 250 × 16 mm i.d. (M&W Chromatographietechnik GmbH, Berlin, Germany) semipreparative HPLC column was used for the isolation of proanthocyanidins. Water (solvent A) and acetonitrile (solvent B) were used as solvent systems. The flow rate was 6 mL/min. The following gradients were used for the isolation of proanthocyanidins: Gradient 1 for dimeric procyanidins B1 and B3 and dimeric prodelphinidin B2, 0 min, 8% B; 40 min, 25% B; 42 min, 100% B; 47 min, 100% B; 49 min, 8% B; 54 min, 8% B. Gradient 2 for dimeric procyanidins B2, B4, B5, and B7, 0 min, 10% B; 30 min, 30% B; 32 min, 100% B; 37 min, 100% B; 39 min, 10% B; 44 min, 10% B. Gradient 3 for dimeric procyanidin B6 and trimeric procyanidin C1, 0 min, 10% B; 40 min, 30% B; 42 min, 100% B; 47 min, 100% B; 49 min, 10%; 54 min, 10% B. Gradient 4 for dimeric procyanidin B8, 0 min, 10% B; 50 min, 35% B; 52 min, 100% B; 57 min, 100% B; 59 min, 10%; 64 min, 10% B. Gradient 5 for dimeric prodelphinidin B1, 0 min, 6% B; 50 min, 27% B; 52 min, 100% B; 57 min, 100% B; 59 min, 6% B; 65 min, 6% B. Gradient 6 for dimeric prodelphinidin B3, 0 min, 6% B; 40 min, 15% B; 42 min, 100% B; 47 min, 100% B; 49 min, 6% B; 54 min, 6% B. The fractions were monitored at λ = 280 nm. The purity of all compounds was confirmed by HPLC-PDA (>95%) at λ = 280 nm. Phloroglucinolysis. Analysis was carried out according to the methods of Esatbeyoglu et al. 13 and Kennedy and Jones.16 Phloroglucinol adducts (+)-catechin-(4α → 2)-phloroglucinol and (−)-epicatechin-(4β → 2)-phloroglucinol as reference compounds were obtained by HSCCC according to the method of Köhler and Winterhalter.17 Circular Dichroism. CD spectra were measured according to the method of Esatbeyoglu et al.15 Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H and 13 C NMR, 1H−1H correlated spectroscopy (COSY), 1H−1H phasesensitive nuclear Overhauser enhancement spectroscopy (NOESY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC) experiments were performed at 300 or 240 K on a Bruker Avance ARX 400 NMR spectrometer equipped with a variable-temperature unit B VT-2000 (Rheinstetten, Germany). Samples were dissolved in acetone-d6. Chemical shifts were

nidin-rich extracts were degraded under acidic conditions. These formed carbocations (upper units) react in the presence of flavan-3-ols as nucleophile agents to dimeric proanthocyanidins. In a previous study, 57 samples with regard to the occurrence of dimeric procyanidins B1−B8 were analyzed.14 In this context, dimeric procyanidins B1−B4, B6, and B7 in hazelnut skins were detected in low amounts.14 According to Gu et al.,2 hazelnut polymers are composed of 41.9% (−)-epicatechin, 39.5% (+)-catechin, 10.8% (epi)gallocatechin, and 0.5% epicatechin-3-O-gallate in their extension units. The conditions are appropriate for the use of semisynthesis to produce all eight dimeric procyanidins B1−B8 in higher amounts. Therefore, the aim of the present study is the isolation of B-type dimeric procyanidins in high amounts (i.e., 500 mg) after semisynthesis of hazelnut skins for the first time. Furthermore, the proanthocyanidin composition of hazelnut skins is not wellcharacterized in the literature until now. Here, we characterize for the first time various proanthocyanidins, that is, flavan-3-ol (epi)afzelechin, B-type dimeric propelargonidins, and different A- and B-type dimeric prodelphinidins, which occur naturally in hazelnut skins by LC-MS/MS and/or NMR spectroscopy.

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MATERIALS AND METHODS

Chemicals. Water (deionized, Nanopure, Werner, Leverkusen, Germany), acetonitrile of HPLC quality (Fisher Scientific, Loughborough, UK), tert-butyl methyl ether (distilled, industrial quality), methanol (distilled, industrial quality), n-hexane (distilled, industrial quality), n-butanol, p.a. (Fisher Scientific), ethyl acetate, p.a. (Fisher Scientific), 2-propanol, p.a. (Sigma, Steinheim, Germany), (+)-catechin hydrate, ≥98% (Sigma), (−)-epicatechin, p.a. (Sigma), hydrochloric acid, 37% (Riedel-de-Haën, Seelze, Germany), ethanol, p.a. (Riedel-de-Haën), sodium hydrogen carbonate, p.a. (Merck, Darmstadt, Germany), and acetone-d6 (Deutero GmbH, Kastellaun, Germany) were used. High-Performance Liquid Chromatography Photodiode Array (HPLC-PDA) and HPLC−Electrospray Ionization Multiple Mass Spectrometry (HPLC-ESI-MSn) Analysis. HPLC-PDA and HPLC-ESI-MSn conditions were the same as described earlier.12 Optimization of the Reaction Conditions for Semisynthesis of Dimeric Procyanidins. The reaction conditions, such as ratio of substrates, reaction time, and reaction temperature, were described in Esatbeyoglu and Winterhalter.12 Sample Preparation for High-Speed (HSCCC) and LowSpeed Rotary Countercurrent Chromatography (LSRCCC) Separation. Hazelnut skin precipitate was obtained from 70% aqueous acetone extract of hazelnut skins after ethanol/n-hexane (5:13, v/v) precipitation as described earlier.15 The precipitate, received after filtration, was used for semisynthesis. For HSCCC. Amounts of 1 g of (+)-catechin or (−)-epicatechin and 500 mg of hazelnut skin precipitate were dissolved in 50 mL of 0.1 N methanolic HCl and kept at 40 °C for 30 min in a water bath. The reaction mixture was neutralized with about 10 mL of 0.5 N sodium hydrogen carbonate solution and evaporated, and the residue was lyophilized. About 1 g of the reaction mixture was applied for HSCCC separation. For LSRCCC. For semisynthesis of dimeric procyanidins B1, B3, B6, and B7, 10 g of (+)-catechin and 10 g of hazelnut skin precipitate were used, and 10 g of (−)-epicatechin and 10 g of hazelnut skin precipitate were applied to semisynthesize dimeric procyanidins B2, B4, B5, and B8. Further conditions for sample preparation are given in Esatbeyoglu et al.15 About 10 g of the reaction mixture was used for LSRCCC separation. HSCCC. A high-speed countercurrent chromatograph model CCC1000 (Pharma-Tech Research Corp., Baltimore, MD, USA) was equipped with three preparative coils of PTFE tubing, connected in series (total volume, 800 mL; 2.6 mm i.d.; 160 m length). A Biotronik B



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Figure 1. HPLC-PDA chromatograms at λ = 280 nm of the 70% acetone extract of hazelnut skins before (A) and after ethanol/n-hexane precipitation (B, filtrate; C, precipitate). Modified from Esatbeyoglu.38 Hz, H6′t), 6.98 (1OH, OH7t), 7.14 (d, 1H, J = 1.8 Hz, H2′t). (a shifts are interchangeable.) (+)-Gallocatechin-4α → 8-(+)-catechin (dimeric prodelphinidin B3): amorphous white powder; λmax = 228 and 276 nm; ESIMS/MS m/z 593 [M − H]−; MS/MS fragments m/z 575, 467, 425, 407, 303, 289, 245; CD (0.17 mmol/L in methanol) [θ]204 106643, [θ]215 −328118, [θ]240 −73079, [θ]271 3371, [θ]284 −6290. 1 H NMR (400 MHz, aceton-d6, 300 K), two rotamers A and B in a ratio of 55:45, δ 2.53* (dd, 1H, J = 7.9 Hz, J = 16.2 Hz, H4Bt), 2.61 (dd, 1H, J = 8.1 Hz, J = 16.2 Hz, H4Bt), 2.76* (dd, 1H, J = 5.4 Hz, J = 16.3 Hz, H4At), 2.89 (dd, 1H, J = 5.3 Hz, J = 15.9 Hz, H4At), 3.81* (m, 1H, H3t), 4.06 (m, 1H, H3t), 4.23* (d, 1H, J = 9.6 Hz, H2u), 4.28 (d, 1H, J = 9.1 Hz, H2u), 4.35* (ddd, 1H, J = 1.3 Hz, J = 8.1 Hz, J = 8.1 Hz, H3u), 4.45* (d, 1H, J = 7.9 Hz, H4u), 4.50 (t, 1H, J = 8.0 Hz, J = 9.0 Hz, H3u), 4.54 (d, 1H, J = 7.9 Hz, H4u), 4.62* (d, 1H, J = 7.1 Hz, H2t), 4.73 (d, 1H, J = 7.5 Hz, H2t), 5.78*a (d, 1H, J = 2.4 Hz, H8u), 5.84*a (d, 1H, J = 2.4 Hz, H6u), 5.85a (d, 1H, J = 2.4 Hz, H6u), 5.93a (d, 1H, J = 2.4 Hz, H8u), 6.04b (s, 1H, H6t), 6.17b (s, 1H, H6t), 6.19* (dd, 1H, J = 2.0 Hz, J = 8.2 Hz, H6′t), 6.37* (s, 2H, H2′u, H6′u), 6.55 (s, 2H, H2′u, H6′u), 6.59* (d, 1H, J = 2.0 Hz, H2′t), 6.65* (d, 1H, J = 8.2 Hz, H5′t), 6.77 (d, 1H, J = 8.1 Hz, H5′t), 6.88 (dd, 1H, J = 2.0 Hz, J = 8.1 Hz, H6′t), 7.02 (d, 1H, J = 2.0 Hz, H2′t). (* rotamer B; a, b shifts are interchangeable.) 13 C NMR (100 MHz, acetone-d6, 300 K) δ 28.4* (C4t), 28.7 (C4t), 37.9* (C4u), 38.2 (C4u), 68.1* (C3t), 68.1 (C3t), 73.1* (C3u), 73.1 (C3u), 82.0* (C2t), 83.0 (C2t), 83.9* (C2u), 84.0 (C2u), 96.0 (C6t), 96.1a (C6u), 96.8a (C6u), 97.0a (C8u), 97.2a (C8u), 97.4* (C6t), 100.6 (C4at), 101.9* (C4at), 106.1* (C4au), 106.4 (C4au), 107.7* (C8t), 107.5 (C8t), 108.0* (C2′u, C6′u), 108.0 (C2′u, C6′u), 115.0 (C2′t), 115.1* (C2′t), 115.6 (C5′t), 115.8* (C5′t), 119.5* (C6′t), 120.0 (C6′t), 131.6b (C1′u), 131.7b (C1′u), 131.7b (C1′t), 131.9b (C1′t), 133.2 (C4′u), 133.3* (C4′u), 144.7*c (C3′t), 145.1*c (C4′t),

referenced to the residual solvent signals and are reported in parts per million and coupling constants in hertz. (−)-Epigallocatechin-4β → 8-(+)-catechin (dimeric prodelphinidin B1): amorphous white powder; λmax = 228 and 276 nm; ESIMS/MS m/z 593 [M − H]−; MS/MS fragments m/z 575, 467, 425, 407, 303, 289, 245; CD (0.17 mmol/L in methanol) [θ]207 −84791, [θ]218 84795, [θ]235 35278, [θ]281 −5207. 1 H NMR (400 MHz, acetone-d6, 240 K) δ 2.57 (dd, 1H, J = 6.8 Hz, J = 16.9 Hz, H4Bt), 2.63 (dd, 1H, J = 5.5 Hz, J = 17.0 Hz, H4At), 3.89 (m, 1H, H3u), 4.02 (1OH, OH3u), 4.06 (m, 1H, H3t), 4.52 (1OH, OH3t), 4.69 (sbr, 1H, H4u), 4.87 (d, 1H, J = 6.0 Hz, H2t), 5.03 (sbr, 1H, H2u), 5.89 (s, 1H, H6t), 5.92 (d, 1H, J = 1.9 Hz, H8u), 5.99 (d, 1H, J = 1.9 Hz, H6u), 6.45 (s, 2H, H2′u, H6′u), 6.68 (d, 1H, J = 8.1 Hz, H5′t), 6.86 (dd, 1H, J = 2.1 Hz, J = 8.1 Hz, H6′t), 6.88 (d, 1H, J = 2.1 Hz, H2′t). Assignment of aromatic hydroxyl groups: 7.00 (OH7t), 7.70a (OH4′u), 8.27 (OH3′u, OH5′u), 8.41a (OH3′t), 8.62b (OH7u), 8.63b (OH5t), 8.72a,c (OH5u, OH4′t), 8.75a, c (OH5u). (a, b, c shifts are interchangeable.) (−)-Epigallocatechin-4β → 8-(−)-epicatechin (dimeric prodelphinidin B2): amorphous white powder; λmax = 228 and 276 nm; ESI-MS/MS m/z 593 [M − H]−; MS/MS fragments m/z 575, 467, 425, 407, 303, 289, 245; CD (0.15 mmol/L in methanol) [θ]205 −84507, [θ]221 63392, [θ]240 38165, [θ]273 −10694, [θ]287 −452. 1 H NMR (400 MHz, acetone-d6, 240 K), 90% major rotamer, δ 2.67 (dd, 1H, J = 2, which remained on the CCC coil, the more apolar two-phase solvent system was used for fractionation. The chromatogram of the CCC separation acquired at 280 nm is shown in Figure 3B. Fraction I did not

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RESULTS AND DISCUSSION Initially, the defatted 70% acetone extract of hazelnut skins was precipitated with ethanol and n-hexane in a ratio of 5:13 (v/v). This allows an enrichment of higher oligomeric and polymeric proanthocyanidins in the precipitate and an enrichment of lower oligomeric proanthocyanidins in the filtrate. Figure 1 shows the HPLC-PDA chromatograms of 70% acetone extract of hazelnut skins before and after precipitation. The proportion of flavan-3-ol constituents in the upper and terminal units of the hazelnut skin precipitate was determined by phloroglucinolysis, as published earlier.4,14 The analysis showed that 30.7 ± 0.55% (+)-catechin-(4α → 2)-phloroglucinol and 62.9 ± 1.45% (−)-epicatechin-(4β → 2)-phloroglucinol are present in the upper units of the polymeric proanthocyanidin fraction of hazelnut skins. Also, 3.67 ± 0.15% (−)-epigallocatechin and 2.97 ± 0.11% (+)-gallocatechin as part of the upper unit were detected. To find the optimal reaction conditions for semisynthesis of dimeric procyanidins, the influence of the reaction time, temperature, and ratio of reagents such as nucleophile agent, that is, (+)-catechin and (−)-epicatechin, as well as the polymeric procyanidin fraction (hazelnut precipitate) was determined (data not shown). The optimal reaction conditions for semisynthetic studies were 30 min at 40 °C with a ratio of substrates of 1:2 (hazelnut precipitate and nucleophile agent). D



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Figure 3. (A) HSCCC chromatogram of the reaction mixture of hazelnut skin precipitate and (+)-catechin. Sample load, 928 mg; solvent system, ethyl acetate/n-butanol/water (14:1:15, v/v/v); flow rate, 2.7 mL/min; revolution speed, 900 rpm. (B) HSCCC chromatogram of the coil (A). Sample load, 550 mg; solvent system, n-hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v); flow rate, 3 mL/min; revolution speed, 1000 rpm. (C) HSCCC chromatogram of the reaction mixture of hazelnut skin precipitate and (−)-epicatechin. Sample load, 1000 mg; solvent system, ethyl acetate/isopropanol/water (20:1:20, v/v/v); flow rate, 3 mL/min; revolution speed, 1000 rpm. (D) HSCCC chromatogram of the coil (C). Sample load, 395 mg; solvent system, n-hexane/ethyl acetate/methanol/water (1.2:10:1.2:10, v/v/v/v); flow rate, 2.7 mL/min; revolution speed, 1000 rpm. Modified from Esatbeyoglu.38

contain any relevant compounds. Fraction II contained mainly trimeric procyanidin C1. In fraction III unknown compounds, that is, galloylated dimeric procyanidins (m/z 729 [M − H]−),8 A-type trimeric procyanidin (m/z 863 [M − H]−), B-type dimeric propelargonidin (m/z 561 [M − H]−), and an A-type dimeric prodelphinidin (m/z 591 [M − H]−) were enriched. The byproduct gambiriin A1 was found in fraction IV. Fraction V consisted of dimeric procyanidin B7 (18.8 mg, purity = 82.6%). B6 was enriched in fraction VI (10.8 mg, purity = 26.1%). Unreacted (+)-catechin was obtained from fraction VII (purity = 99.0%). The last fraction contained the second byproduct gambiriin A3. To isolate dimeric procyanidins B2, B4, B5, and B8 the reaction mixture of the semisynthesis carried out with (−)-epicatechin was separated with the solvent system ethyl acetate/isopropanol/water (20:1:20, v/v/v) (Figure 3C).

Fraction I contained several compounds, that is, B-type trimeric procyanidin, two B-type dimeric prodelphinidins, and two Atype dimeric prodelphinidins. One dimeric prodelphinidin was identified as EGC-4β → 8-EC (see below) and one A-type dimeric prodelphinidin as EGC-(2β → O5, 4β → 6)-C.15 Fraction II contained compounds similar to those of fraction I. Two B-type trimeric procyanidins were identified as EC-4β → 6-EC-4β → 8-EC and trimer C1 (EC-4β → 8-EC-4β → 8EC).13 B2 was enriched (69 mg; purity = 88.7%) in fraction III and B4 in fraction IV (45.6 mg, purity = 70.5%). Fraction V consisted of A- and B-type dimeric prodelphinidins, an A-type dimeric procyanidin, and a trimeric procyanidin. A galloylated dimeric procyanidin (m/z 729 [M − H]−)8 was enriched in fraction VI. This CCC separation was repeated twice. Coil fractions of two CCC separations were divided into five fractions, and fraction II and III were combined to isolate B5 E



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Figure 4. LSRCCC chromatogram of the filtrate of the reaction mixture of hazelnut skin precipitate and (+)-catechin after ethanol/n-hexane precipitation (5:13, v/v) (LSRCCC-1). Sample load, 9.58 g; solvent system, tert-butyl methyl ether/n-butanol/water (4.3:0.7:5, v/v/v); flow rate, 5 mL/min; revolution speed, 50 rpm; elution mode, U−H (upper organic phase as mobile phase; head to tail). Modified from Esatbeyoglu.38

Figure 5. LSRCCC chromatogram of the reaction mixture of hazelnut skin precipitate and (−)-epicatechin (LSRCCC-2). Sample load, 12.4 g; solvent system, tert-butyl methyl ether/n-butanol/water (4.5:0.5:5, v/v/v); flow rate, 4 mL/min; revolution speed, 50 rpm; elution mode, U−H (upper organic phase as mobile phase; head to tail). Modified from Esatbeyoglu.38

and B8 (Figure 3D). The solvent system n-hexane/ethyl acetate/methanol/water (1.2:10:1.2:10, v/v/v/v) was selected for this separation. Fraction I was composed of polymeric procyanidins. Dimeric procyanidins B5 and B8 were found in fraction II (56 mg). High-purity (−)-epicatechin (98.4%) was recovered from fraction III, whereas fraction IV contained (+)-catechin (purity = 93.9%). In summary, hazelnut skins are suitable substrates for semisynthesis of all eight B-type dimeric procyanidins B1− B8. To achieve a higher yield of dimeric procyanidins and enrichment of minor compounds, the separation of semisynthetic reaction mixture was carried out on a large scale by LSRCCC. Fractionation of the Semisynthetic Reaction Mixture by LSRCCC. The reaction mixture, after semisynthesis with (+)-catechin, was precipitated with ethanol/n-hexane in a ratio of 5:13 (v/v). After filtration of this solution, the resulting filtrate (9.58 g) after evaporation and lyophilization was fractionated with the solvent system tert-butyl methyl ether/ n-butanol/water (4.3:0.7:5, v/v/v) by LSRCCC. The first LSRCCC separation of procyanidins, from a semisynthetic

reaction mixture of a commercial grape seed extract with (+)-catechin, has been described by Köhler.18 In the current study, the isolation of proanthocyanidins from hazelnut skins after semisynthetic preparation was described for the first time. Figure 4 shows the LSRCCC separation of the filtrate of the reaction mixture of hazelnut skin precipitate and (+)-catechin after ethanol/n-hexane precipitation. The collected fractions were analyzed by HPLC-PDA at λ = 280 nm and combined to eight fractions. The coil fraction, which was obtained by extrusion, was divided into three fractions. Figure S1 in the Supporting Information shows the composition of some selected LSRCCC fractions. Due to the similar K values of dimeric procyanidins B6 and B7, (+)-catechin, and (−)-epicatechin, all compounds eluted in fractions I−III. Fraction I was composed of various proanthocyanidins, that is, 65.7% (+)-catechin, 7.6% (−)-epicatechingallate, 7.1% dimeric procyanidin B7 (coelution with gambiriin A3), and 2.0% dimeric procyanidin B6. Smaller amounts of A-type dimeric prodelphinidins, A-type trimeric proanthocyanidins (m/z 879 [M − H]− (Epi)C-(Epi)C-(Epi)GC, tentatively), B-type dimeric propelargonidins, and prodelphinidins [(Epi)GCF



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(Epi)GC (m/z 609 [M − H]−)] as well as galloylated dimeric procyanidins (m/z 729 [M − H]−) were found. From this fraction EGC-(2β → O5, 4β → 6)-C15 and dimeric procyanidin B6 were isolated with the solvent system n-hexane/ethyl acetate/methanol/water (1.4:10:1.4:10, v/v/v/v) by HSCCC (data not shown). Fraction II was composed of 88.8% (+)-catechin, 4.2% dimeric procyanidin B7, 1.4% (−)-epicatechin, and 1.1% dimeric procyanidin B6. About 90% (+)-catechin, 3.9% (−)-epicatechin, and 2.0% dimeric procyanidin B7 were enriched in fraction III. Fraction IV contained dimeric procyanidin B3 (560 mg; purity = 76.9%). Fraction V consisted of 67.0% dimeric procyanidin B1 (168 mg) and the trimeric procyanidins EC-4β → 8-EC-4β → 8-C and EC-4β → 6-EC-4β → 8-C.13 About 600 mg of dimeric procyanidin B1 was obtained from fractions VI and VII (purity = 81.1 and 90.1%, respectively). In fraction VIII dimeric procyanidin B1, A- and B-type dimeric prodelphinidins, B-type trimeric procyanidin, and a further A-type trimeric proanthocyanidin (m/z 879 [M − H]− (Epi)C-(Epi)C-(Epi)GC) were enriched. GC-4α → 8-C was isolated from this fraction by preparative HPLC. EGC-4β → 8-C and EGC-(2β → O7, 4β → 8)-C were obtained from coil fractions I and II. The structures of these B-type dimeric prodelphinidins are elucidated by NMR spectroscopy (see below). Trimeric procyanidin EC-4β → 8-C4α → 8-C was enriched (276 mg; purity = 62.0%) in coil fraction III.13 Dimeric procyanidins B2, B4, B5, and B8 were isolated from the reaction mixture of the semisynthesis carried out with (−)-epicatechin and hazelnut skin precipitate by LSRCCC. To eliminate the polymeric compounds, the reaction mixture was treated with ethanol/n-hexane (5:13, v/v). This solution was filtered, and the obtained filtrate after evaporation and lyophilization was fractionated with the solvent system tertbutyl methyl ether/n-butanol/water (4.5:0.5:5, v/v/v). The LSRCCC separation is shown in Figure 5. After separation by elution as well as extrusion and analysis of selected samples by HPLC-PDA, 11 fractions from the elution and five coil fractions from the extrusion were obtained. Selected fractions are depicted in Figure S2 in the Supporting Information. Fraction I contained the more apolar compounds gambiriin A5, (−)-epicatechingallate, galloylated dimeric procyanidin, and (epi)afzelechin. (Epi)afzelechin is until now unknown for hazelnut skins.2,8,9,19 Besides the compounds found in fraction I, fraction II contained dimeric procyanidins B5 and B8, (+)-catechin, and (−)-epicatechin. Fraction III was composed mainly of (−)-epicatechin (37.5%), (+)-catechin (28.1%), dimeric procyanidins B5 (13.0%), and B8 (3.7%). Besides these dimeric procyanidins, 83.4% (−)-epicatechin was obtained from fraction IV. Compounds in fractions I− IV show similar K values. For further isolation of compounds in fractions I−IV, separation was performed with HSCCC (data not shown). The main compound in fraction V was (−)-epicatechin (82.5%), and one B-type galloylated dimeric procyanidin was detected in minor amounts, as well. Fraction VI consisted of target compounds B2 (23.5%) and B4 (33.6%) as well as EC-4β → 8-EC-4β → 6-EC (5.1%).13 Fraction VII contained dimeric procyanidins B2 and B4 in large amounts of 740 and 350 mg, respectively (calculated at λ = 280 nm by HPLC-PDA). Fraction VIII was composed of 44.3% dimeric procyanidin B2 and 10.9% B-type trimeric procyanidin C1, whereas fraction IX contained about 70% trimeric procyanidin C1. Up to 110 mg of procyanidin trimer C1 was isolated from this fraction. One B-type galloylated dimeric prodelphinidin

(m/z 745 [M − H]−) was also detected in smaller amounts. The main compound in fraction X was EC-4β → 6-EC-4β → 8EC,13 together with dimeric procyanidin B1 and A-type trimeric proanthocyanidin (m/z 879 [M − H]− (Epi)C-(Epi)C(Epi)GC). The last fraction was composed of A- and B-type dimeric prodelphinidins as well as two B-type trimeric proanthocyanidins (m/z 881 [M − H]−, according to ref 2 tentatively (Epi)GC-(Epi)C-(Epi)C). B-type dimeric prodelphinidin (−)-EGC-4β → 8-(−)-EC was isolated from coil fraction I, and its structure was elucidated by NMR spectroscopy (see below). This minor compound occurred naturally in hazelnut skins, confirmed by LC-MS/MS, and is described for the first time in this study. Smaller amounts of Aand B-type dimeric prodelphinidins (with (epi)gallocatechin as upper unit), trimeric proanthocyanidins, and trimeric proanthocyanidins (Epi)GC-(Epi)C-(Epi)C and (Epi)C-(Epi)GC(Epi)C were identified by LC-MS/MS. Similar compounds were found in coil fractions II−IV. The last coil fraction contained EC-4β → 8-C-4α → 8-EC13 and small amounts of a B-type trimeric proanthocyanidin (m/z 897 [M − H]−, according to ref 20 tentatively (Epi)GC-(Epi)GC-(Epi)C). C4 → C8 linked dimeric procyanidins B1 and B3 (about 600 mg; purity = 76.9−90.1%) as well as dimeric procyanidins B2 and B4 (740 and 350 mg) were isolated in large amounts for the first time. Dimeric procyanidins B5−B8 are formed in lower amounts because of steric hindrance.21−23 Moreover, the semisynthesis of different B-type trimeric procyanidins (EC4β → 8-EC-4β → 8-C, EC-4β → 6-EC-4β → 8-C, EC-4β → 6EC-4β → 8-EC, trimer C1 (EC-4β → 8-EC-4β → 8-EC), EC4β → 8-C-4α → 8-C, EC-4β → 8-EC-4β → 6-EC and EC-4β → 8-C-4α → 8-EC) was shown. The structure of the minor compounds such as A- and B-type dimeric prodelphinidins EGC-4β → 8-EC, EGC-4β → 8-C, GC-4α → 8-C, EGC-(2β → O7, 4β → 8)-C, and EGC-(2β → O5, 4β → 6)-C are elucidated by NMR spectroscopy13,15 and were described in hazelnut skins for the first time as well as dimeric prodelphinidins composed of (Epi)GC-(Epi)GC (m/z 609 [M − H]−). Further unknown A- and B-type dimeric prodelphinidins (m/z 591 and 593 [M − H]−) and B-type galloylated dimeric prodelphinidins (m/z 745 [M − H]−) were detected. To date, the occurrence of prodelphinidins in hazelnuts was known, but not their exact structures and their galloylated forms.2,4,8 The detection of the flavan-3-ol (epi)afzelechin, B-type dimeric propelargonidins (m/z 561 [M − H]−) and A-type dimeric procyanidin (m/z 575 [M − H]−) were detected for the first time in hazelnut skins. Recently, the occurrence of A- and B-type trimeric proanthocyanidins consisting of (epi)catechin, (epi)gallocatechin, and (epi)catechingallate units was described in roasted hazelnut skins.8,9 In the present study, different types of proanthocyanidins such as an A-type trimeric procyanidin (m/z 863 [M − H]−), A-type trimeric proanthocyanidins (m/z 879 [M − H]− tentatively (Epi)C-(Epi)C-(Epi)GC), and B-type trimeric proanthocyanidins (m/z 881 [M − H]− tentatively (Epi)GC(Epi)C-(Epi)C and (Epi)C-(Epi)GC-(Epi)C; m/z 897 [M − H]− tentatively (Epi)GC-(Epi)GC-(Epi)C) were identified in hazelnut skins. The natural occurrence of these compounds in hazelnut skins was confirmed by LC-MS/MS by analyzing the hazelnut skin filtrate, which was obtained after precipitation of the 70% acetone extract. Structure Elucidation of B-Type Dimeric Prodelphinidins. B-type dimeric prodelphinidins (−)-EGC-4β → 8-(+)-C, (−)-EGC-4β → 8-(−)-EC, and (+)-GC-4α → 8-(+)-C were G



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Figure 6. Chemical structures of the dimeric prodelphinidins B1 (a), B2 (b), and B3 (c). u, upper; t, terminal.

Compound EGC-4β → 8-EC showed a major rotamer (90%) and a minor rotamer (10%), whereas no rotamers were detected for EGC-4β → 8-C. The presence of (+)-gallocatechin in the upper unit leads to hindered rotation around the interflavanoid linkage, producing two major rotational isomers in a ratio of about 1:1 (55 to 45%). This leads to signal duplication in 1H and 13C NMR spectra at 300 K. In addition to EGC-4β → 8-EC and EGC-4β → 8-C, one- and twodimensional 13C NMR spectra were recorded for GC-4α → 8C. H2′ and H6′ protons of the trihydroxylated B-ring C atoms ((epi)gallocatechin) showed a signal at about δC 108.0. Inspection of the region δC 115−120 show the signals of H2′, H5′, and H6′ of the dihydroxylated B-ring C atoms ((epi)catechins).33−35 Signals at δC 83.0 and 84.0 suggested the 2,3-trans configuration of both units.27 The observed data were in accordance with literature data.34 The presence of two rotamers (25 °C, 500 MHz, acetone-d6) was described later by Karonen et al.26 Structure Elucidation by Phloroglucinolysis and CD. The flavan-3-ol units and the interflavanoid bond of these compounds were confirmed by phloroglucinolysis. Degradation of >70% indicated a 4 → 8 interflavanoid linkage of these Btype dimeric prodelphinidins. Mild degradation yielded for compound EGC-4β → 8-EC 20.1% EGC-phloroglucinol for the upper unit and 54% EC for the terminal unit. EGC-4β → 8C gave 18.2% EGC-phloroglucinol and 53.9% C and GC-4α → 8-C, 26.0% GC-phloroglucinol and 64.9% C. Moreover, these results indicated that 4 → 8-linked B-type dimeric prodelphinidins with (+)-gallocatechin in the upper unit were cleaved more quickly than those with (−)-epigallocatechin in the upper unit. The absolute configuration at positions C2 and C4 was determined by CD spectra. All compounds ((−)-EGC-4β → 8(+)-C, (−)-EGC-4β → 8-(−)-EC, (+)-GC-4α → 8-(+)-C) with 2R absolute stereochemistry showed a negative Cotton effect at 280 nm. Compounds (−)-EGC-4β → 8-(+)-C and (−)-EGC-4β → 8-(−)-EC showed a positive Cotton effect at 220−240 nm, indicating the 2β-4β-orientation (4R configuration).36 However, the 2α−4α orientation (4S configuration) in (+)-GC-4α → 8-(+)-C was confirmed by the negative Cotton effect at 220−240 nm. To date, the structure elucidation of these three dimeric prodelphinidins was done by 13C NMR spectroscopy and acidcatalyzed degradation or 1 H NMR spectroscopy after derivatization (i.e., peracetylation), whereas Malik et al.37 elucidated only the structure of EGC-4β → 8-EC by 1H NMR spectroscopy without derivatization or degradation. Here, we describe to the best of our knowledge the NMR and assignment of the interflavanoid bond from ROESY spectra of B-type dimeric prodelphinidins for the first time in detail.

isolated as white amorphous solids from coil fraction I (LSRCCC-1 and LSRCCC-2) and fraction VIII (LSRCCC-1) of the LSRCCC separation after purification by preparative HPLC. Their chemical structures are shown in Figure 6. The structure of these compounds was determined on the basis of electrospray ionization mass spectrometry as well as by 1D and 2D 1H NMR spectroscopic analysis that included COSY and NOESY. 1D and 2D 13C NMR spectroscopy (HSQC, HMBC) were used for structure determination of (+)-GC-4α → 8(+)-C, as well. All 1H NMR spectra for (−)-EGC-4β → 8(+)-C and (−)-EGC-4β → 8-(−)-EC were recorded at 240 K (400 MHz, acetone-d6), and all 1H and 13C NMR spectra (400 and 100 MHz, respectively) of (+)-GC-4α → 8-(+)-C were recorded in acetone-d6 at 300 K. Chemical shifts and coupling constants are given under Materials and Methods. LC-MS/MS analysis yielded a molecular ion of [M − H]− m/z 593 with fragment ions m/z 575, 467, 425, 407, 303, 289, and 245, which indicated the presence of a B-type prodelphinidin (Supporting Information Figure S3). On the basis of the fragment ions m/z 425 and 289 derived from the quinone-methide cleavage and absence of m/z 305, it was concluded that (epi)gallocatechin was the upper unit and (epi)catechin the terminal unit.2,20,24 NMR spectra of dimeric prodelphinidins are similar to those of dimeric procyanidins.25,26 Structure elucidation was performed as in refs 13 and 15, and only relevant data are shown here. The relative configuration of both units was determined from the magnitude of the coupling constants J2, 3 and J3, 4 of the heterocyclic protons (C-ring).27−32 EGC-4β → 8-C showed a 2,3-cis-3,4-trans- and 2,3-trans-configuration, EGC-4β → 8-EC a 2,3-cis-3,4-trans- and 2,3-cis-configuration, and GC-4α → 8-C a 2,3-trans-3,4-trans- and 2,3-trans-configuration. Proton singlets at about δ 6.41, 6.45, and 6.55 (H2′ and H6′ are chemically equivalent) are evidence of trihydroxylated Brings (pyrogallol ring: 3′,4′,5′-substitution) of the upper unit. All C-ring protons were assigned from inspection of COSY spectra. Long-range correlations between H2′ and H6′ (B-ring) as well as H2 (C-ring) allowed the identification of B- and Crings of both units. Only one proton for H4 of the upper unit was determined. This observation confirmed (epi)gallocatechin as the upper unit. NOE correlations of H2′ and H6′ of the terminal unit with H4 of the upper unit were characteristic of a 4 → 8 interflavanoid linkage. All compounds are linked C4 → C8. Due to the absence of NOE correlations between H2 and H4, the terminal unit is bound quasi-axially in EGC-4β → 8-EC and EGC-4β → 8-C. Detection of NOE correlations between H2 and H4 indicated that the terminal unit of GC-4α → 8-C is bound quasi-equatorially. H



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(9) Monagas, M.; Garrido, I.; Lebrón-Aguilar, R.; Gómez-Cordovés, M. C.; Rybarczyk, A.; Amarowicz, R.; Bartolomé, B. Comparative flavan-3-ol profile and antioxidant capacity of roasted peanut, hazelnut, and almond skins. J. Agric. Food Chem. 2009, 57, 10590−10599. (10) Schmitzer, V.; Slatnar, A.; Veberic, R.; Stampar, F.; Solar, A. Roasting affects phenolic composition and antioxidative activity of hazelnuts (Corylus avellana L.). J. Food Sci. 2011, 76, S14−S19. (11) Köhler, N.; Wray, V.; Winterhalter, P. New approach for the synthesis and isolation of dimeric procyanidins. J. Agric. Food Chem. 2008, 56, 5374−5385. (12) Esatbeyoglu, T.; Winterhalter, P. Preparation of dimeric procyanidins B1, B2, B5, and B7 from a polymeric procyanidin fraction of black chokeberry (Aronia melanocarpa). J. Agric. Food Chem. 2010, 58, 5147−5153. (13) Esatbeyoglu, T.; Jaschok-Kentner, B.; Wray, V.; Winterhalter, P. Structure elucidation of procyanidin oligomers by low temperature 1H NMR spectroscopy. J. Agric. Food Chem. 2011, 59, 62−69. (14) Esatbeyoglu, T.; Wray, V.; Winterhalter, P. Dimeric procyanidins: screening for B1 to B8 and semisynthetic preparation of B3, B4, B6, and B8 from a polymeric procyanidin fraction of white willow bark (Salix alba). J. Agric. Food Chem. 2010, 58, 7820−7830. (15) Esatbeyoglu, T.; Wray, V.; Winterhalter, P. Identification of two novel prodelphinidin A-type dimers from roasted hazelnut skins (Corylus avellana L.). J. Agric. Food Chem. 2013, 61, 12640−12645. (16) Kennedy, J. A.; Jones, G. P. Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J. Agric. Food Chem. 2001, 49, 1740−1746. (17) Köhler, N.; Winterhalter, P. Large-scale isolation of flavan-3-ol phloroglucinol adducts by high-speed counter-current chromatography. J. Chromatogr., A 2005, 1072, 217−222. (18) Kö hler, N. Entwicklung und Anwendung leistungsfähiger präparativer gegenstromverteilungschromatographischer Trenntechniken; Cuvillier Verlag: Göttingen, Germany, 2006 (Ph.D. thesis, in German). (19) Phenol-Explorer 3.0: database on polyphenol contents in foods; http://www.phenol-explorer.eu/contents/food/716 (accessed Jan 24, 2014). (20) Friedrich, W.; Eberhardt, A.; Galensa, R. Investigation of proanthocyanidins by HPLC with electrospray ionization mass spectrometry. Eur. Food Res. Technol. 2000, 211, 56−64. (21) Hemingway, R. W.; Foo, L. Y.; Porter, L. J. Linkage isomerism in trimeric and polymeric 2,3-cis-procyanidins. J. Chem. Soc., Perkin Trans. 1 1982, 5, 1209−1216. (22) Viviers, P. M.; Kolodziej, H.; Young, D. A.; Ferreira, D.; Roux, D. G. Synthesis of condensed tannins. Part 11. Intramolecular enantiomerism of the constituent units of tannins from the Anacardiaceae: stoichiometric control in direct synthesis: derivation of 1H nuclear magnetic resonance parameters applicable to higher oligomers. J. Chem. Soc., Perkin Trans. 1 1983, 10, 2555−2562. (23) Baldé, A. M.; Pieters, L. A.; Wray, V.; Kolodziej, H.; Vanden Berghe, D. A.; Claeys, M.; Vlietinck, A. J. Dimeric and trimeric proanthocyanidins possessing a doubly linked structure from Pavetta owariensis. Phytochemistry 1991, 30, 4129−4135. (24) Monagas, M.; Garrido, I.; Lebrón-Aguilar, R.; Bartolomé, B.; Gómez-Cordovés, C. Almond (Prunus dulcis (Mill.) D.A. Webb) skins as a potential source of bioactive polyphenols. J. Agric. Food Chem. 2007, 55, 8498−8507. (25) Danne, A.; Petereit, F.; Nahrstedt, A. Proanthocyanidins from Cistus incanus. Phytochemistry 1993, 34, 1129−1133. (26) Karonen, M.; Leikas, A.; Loponen, J.; Sinkkonen, J.; Ossipov, V.; Pihlaja, K. Reversed-phase HPLC-ESI/MS analysis of birch leaf proanthocyanidins after their acidic degradation in the presence of nucleophiles. Phytochem. Anal. 2007, 18, 378−386. (27) Fletcher, A. C.; Porter, L. J.; Haslam, E.; Gupta, R. K. Plant proanthocyanidins. Part 3. Conformational and configurational studies of natural procyanidins. J. Chem. Soc., Perkin Trans. 1 1977, 14, 1628− 1637. (28) Botha, J. J.; Young, D. A.; Ferreira, D.; Roux, D. G. Synthesis of condensed tannins. Part 1. Stereoselective and stereospecific syntheses of optically pure 4-arylflavan-3-ols, and assessment of their absolute

Structure elucidation was performed without the necessity of derivatization and degradation (i.e., phloroglucinolysis), 1H NMR spectroscopy was sufficient.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(P.W.) Phone: +49-531-391-7200. Fax: +49-531-391-7230. Email: [email protected]. Funding

This project was supported by a grant of the German Federal Ministry of Education and Research (BMBF − Bundesministerium für Bildung und Forschung, Grant 0313828C). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to B. Jaschok-Kentner and C. Kakoschke (Helmholtz Centre for Infection Research, Braunschweig, Germany) for NMR measurements and Wild Flavors GmbH & Co. KG (Berlin, Germany) for providing roasted hazelnut skins (Corylus avellana L.).

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ABBREVIATIONS USED HSCCC, high-speed countercurrent chromatography; LSRCCC, low-speed rotary countercurrent chromatography; EC, (−)-epicatechin; C, (+)-catechin; EGC, (−)-epigallocatechin; GC, (+)-gallocatechin; (Epi)C, (+)-catechin or (−)-epicatechin; (Epi)GC, (+)-gallocatechin or (−)-epigallocatechin

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REFERENCES

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Semisynthetic Preparation and Isolation of Dimeric Procyanidins B1

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