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Comprehensive Separation and Structural Analyses of Polyphenols and Related Compounds from Bracts of Hops (Humulus lupulus L.) Yoshihisa Tanaka,*,† Akio Yanagida,‡ Satoshi Komeya,‡ Miho Kawana,‡ Daiki Honma,† Motoyuki Tagashira,† Tomomasa Kanda,† and Yoichi Shibusawa‡ †

Research Laboratories for Fundamental Technology of Food, Asahi Group Holdings, Limited, 1-21, Midori 1-chome, Moriya-shi, Ibaraki 302-0106, Japan ‡ Division of Pharmaceutical and Biomedical Analysis, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan ABSTRACT: A novel sequential chromatographic technique was applied to the comprehensive separation of polyphenols and related compounds from a hop bract extract. Over 100 types of constituents were effectively isolated from only 25 g of extract in high yields by high-speed countercurrent chromatography followed by hydrophilic interaction chromatography and reversedphase high performance liquid chromatography. Among the materials isolated, the structures of 39 compounds were elucidated on the basis of their spectroscopic data including electrospray ionization time-of-flight mass spectrometry and one-dimensional/ two-dimensional nuclear magnetic resonance. Three new compounds, 1 known compound identified for the first time in plants, and 20 known compounds that have not been reported in hops, were found. The hop bract extract also contained an abundance of highly oligomeric proanthocyanidins, which consisted of B-type procyanidin structures. KEYWORDS: hop, Humulus lupulus L., polyphenol, proanthocyanidin, countercurrent chromatography

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INTRODUCTION Hop (Humulus lupulus L.) is a perennial herb, and its female cone is essential for beer brewing. The hop cone consists of two parts: lupulin glands and bract leaves. The lupulin glands are used as flavoring ingredients, which produce compounds responsible for bitterness and aroma in beer, whereas the remaining bract regions are unutilized waste products. The important compounds for the sensory/taste evaluation of beer are α-acids (humulones), which are precursors to iso-α-acids (isohumulones) and β-acids (lupulones); α-acids are largely found in the lupulin glands, not in the bracts.1 On the other hand, we reported that the unutilized hop bracts contained an abundance of polyphenolic compounds2−4 and that the crude extract of hop bracts exhibited several beneficial effects for anticaries2,5,6 and antiperiodontitis7−9 and the inhibition of some bacterial toxins.10−12 In the investigation of the effect for anticaries, it was suggested that the high-molecular-weight polyphenols (about 36 000−40 000 Da) of hop bracts inhibited the cellular adherence of Streptococcus mutans and S. sobrinus, the cariogenic bacteria.2 Furthermore, in the investigation of the effect for antiperiodontitis, it was reported that lowmolecular-weight polyphenols of hop bracts inhibited the cellular PGE2 production induced by Porphyromonas gingivalis, a major periodontal pathogen.8 These results suggest a possibility for the beneficial and industrial use of the hop bracts that are unutilized from beer brewing. However, unlike lupulin glands, the complete composition of the polyphenolic compounds in hop bracts has yet to be fully elucidated.4 Therefore, we started to perform research on the comprehensive separation and structural analyses of polyphenolic compounds from a hop bract extract. In this study, different separation modes of liquid chromatography were effectively applied to comprehensive © 2014 American Chemical Society

separation. Separation was performed by a sequential liquid chromatography (LC) method in three or four steps: (1) highspeed countercurrent chromatography (HSCCC); (2) hydrophilic interaction chromatography (HILIC) using an amide column; and reversed-phase (RP) LC using a (3) octadecylsilyl (ODS) or (4) phenyl column. Step 4 was only performed when separation by step 3 was unsuccessful. Among the LC methods, HSCCC is a support-free liquid−liquid partition chromatography method that uses an immiscible two-phase solvent system.13 Recently, it has been used for the separation and purification of a wide variety of natural products14 because the permanent adsorption of analytes onto the column is avoided by eliminating solid supports in HSCCC, and nearly 100% recovery of the analytes can be achieved. HILIC was proposed by Alpert to describe a variant of normal-phase (NP) LC in 1990.15 Polar/hydrophilic analytes (including polyphenols) are strongly retained on the HILIC column (amide-, aminopropyl-, or diol-silica, etc.) by hydrogen bonding, but the nonaqueous organic mobile phase in traditional NP-LC is replaced with a more polar mobile phase containing water, which acts as a stronger solvent, in the HILIC mode. We expected that the combination of HSCCC, HILIC, and conventional RP-LC would contribute to the comprehensive separation of polyphenolic constituents from a hop bract extract. We describe the results of the above separation, as well as the structural analyses of isolated compounds, and we discuss the compositional characterization of hop bract polyphenols. Received: Revised: Accepted: Published: 2198

December 10, 2013 February 12, 2014 February 18, 2014 February 18, 2014 dx.doi.org/10.1021/jf405544n | J. Agric. Food Chem. 2014, 62, 2198−2206

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by a DAD. The eluate (12 mL) was collected every 3 min by a fraction collector, and each collected fraction was evaporated and lyophilized. For treatment of 25 g of HBP, HSCCC separation was repeated five times. HILIC Conditions. The lyophilized HSCCC fractions were further separated by HILIC using an amide column under the following conditions. HILIC separation was carried on a TSKgel Amide-80 column (21.5 mm i.d. × 300 mm, 10 μm, Tosoh, Tokyo, Japan) protected with a guard column (21.5 mm i.d. × 75 mm). The mobile phase consisted of eluent A (90% aqueous acetonitrile) and eluent B (50% aqueous acetonitrile). A multilinear gradient was applied from 100:0 (A:B) to 80:20 over 20 min, and then it was changed to 50:50 over 60 min. The flow rate was maintained at 13 mL/min, and the sample solution (a maximum of 200 mg of a lyophilized HSCCC fraction dissolved in a maximum of 10 mL of eluent A) was injected through a sample loop. During HILIC separation, the UV absorbance of the eluate was monitored by a DAD. The eluate (13 mL) was collected every 1 min by a fraction collector, and each collected fraction was evaporated and lyophilized. RP-HPLC Conditions Using ODS Column. The lyophilized HILIC fractions were further separated by RP-HPLC using an ODS column under the following conditions. The RP-HPLC separation was carried out on a Mightysil RP-18 GP column (10 mm i.d. × 250 mm, 5 μm, Kanto Chemical, Tokyo, Japan) protected with a guard column (10 mm i.d. × 10 mm). The mobile phase consisted of eluent A (10% aqueous acetonitrile) and eluent B (50% aqueous acetonitrile). An isocratic elution of 100:0 (A:B) was started for 20 min, and then a linear gradient was applied from 100:0 to 50:50 over 40 min. The flow rate was maintained at 13 mL/min, and the sample solution (a maximum of 100 mg of a lyophilized HILIC fraction dissolved in a maximum of 5 mL of eluent A) was injected through a sample loop. During RP-HPLC separation, the UV absorbance of the eluate was monitored by a DAD. The eluate (13 mL) was collected every 1 min by a fraction collector, and each collected peak fraction was evaporated and lyophilized. RP-HPLC Conditions Using Phenyl Column. When the isolation of a pure compound by the above RP-HPLC method was unsuccessful, a further RP-HPLC separation step using a phenyl column was performed under the following conditions. The RP-HPLC separation was carried out on an Inertsil Ph-3 column (10 mm i.d. × 250 mm, 5 μm, GL Science, Tokyo, Japan) protected with a guard column (10 mm i.d. × 10 mm). The isocratic elution of a mobile phase with aqueous acetonitrile (an appropriate volume ratio was applied on a case-by-case basis) was performed at a flow rate of 13 mL/min. The sample solution (a maximum of 100 mg of a lyophilized RP-HPLC fraction obtained from step 3 dissolved in a maximum of 5 mL of the mobile phase) was injected through a sample loop. During RP-HPLC separation, the UV absorbance of the eluate was monitored by a DAD. The eluate (13 mL) was collected every 1 min by a fraction collector, and each collected peak fraction was evaporated and lyophilized. Ultrafiltration. For the purification of high-molecular-weight compounds (proanthocyanidins) in HSCCC fraction J, ultrafiltration was performed under the following conditions. A portion of the lyophilized powder of fraction J dissolved in 50% methanol was applied to an Amicon ultra filter device (exclusion molecular weight limit: 3000 Da, Millipore) and centrifuged (14000g, 30 min). The concentrated solution containing highly oligomeric proanthocyanidins (over 20-mers) was washed with the dissolving solvent twice and was evaporated and lyophilized for further NMR analysis.

MATERIALS AND METHODS 1

13

Apparatus. H nuclear magnetic resonance (NMR, 600 MHz), C NMR (150 MHz), 1H−1H correlation spectroscopy (1H−1H COSY), heteronuclear single quantum coherence, heteronuclear multiple bond correlation (HMBC), and rotating-frame overhauser enhancement (ROE) spectroscopy spectra were recorded on a Bruker AV600 instrument (Bruker Biospin GmbH, Rheinstetten, Germany); chemical shifts are given in δ (ppm) relative to the solvent signal (CD3OD, δH 3.31; δC 49.01, 300, or 243 K). Electrospray ionization (ESI) time-offlight mass spectrometry (TOFMS) was carried out on a QSTAR Elite system (AB Sciex, Tokyo, Japan). All chromatographic experiments were carried out on an high performance liquid chromatography (HPLC) system (Hitachi, Tokyo, Japan), which consisted of an L7150 pump, a 7161 injector (Rheodyne, loop capacity: 10 mL), an L7455 diode-array detector (DAD, optical path length of a flow cell: 0.2 mm), and an SF-2120 fraction collector (Advantec, Tokyo, Japan). Chromatograms were recorded on a personal computer using D-7000 chromatography data station software (Hitachi). HSCCC experiments were carried out using an HPLC system (Hitachi) equipped with a type-J coil-planet centrifuge (J-CPC) (Easy-PREP 320, Kutsuwa Sangyo, Hiroshima, Japan) between the 7161 injector and L-7455 DAD. The J-CPC apparatus contains three multilayer-coil separation columns placed symmetrically at a distance of 7 cm from the central axis of the centrifuge. Each separation column was fabricated by directly winding a single piece of polytetrafluoroethylene (PTFE) tubing (1.6 mm i.d. × 50 m length) onto each column holder. The total capacity of the three columns is about 320 mL. Optical rotations were measured with a P-2200 polarimeter (JASCO, Tokyo, Japan). UV data were measured with a U-2900 spectrophotometer (Hitachi). All solvents (high or HPLC grade) for extraction and chromatographic separation were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Plant Material and Sample Preparation. In this study, the spray-dried powder of the polyphenol-rich extract from hop bracts (HBP; food additive grade in Japan) was used as a raw material. The factory-scale preparation method for HBP is described in previous papers.2,4 To describe it briefly, HBP was prepared from the crude hop bract extract supplied by Simon H. Steiner Hopfen GmbH (Germany). The extract was produced by extracting 101 t of the female inflorescence of hop bracts with 50% aqueous ethanol (100 kL, 50 °C, 10 h), centrifuging the material to remove solids, and evaporating ethanol. The bracts were obtained to crush hop cones (cropped in Hallertau, Germany, in 2003) and to remove the lupulin glands by sifting. Next, the crude extract from the 101 t of hop bracts was dissolved in distilled water and passed through a SEPABEADS SP850 column (150 cm i.d. × 230 cm, Mitsubishi Chemical Corporation, Tokyo, Japan). The column was washed with distilled water (12 kL) and eluted with 50% aqueous ethanol (12 kL). After concentrating the eluting fraction, the concentrated fraction was spray-dried to obtain 2 t of HBP powder (yield: 2.0%). In this study, a small portion of HBP powder (25 g) was used for the following HSCCC separation. HSCCC Conditions. The HSCCC separation of HBP was carried out using a two-phase solvent system composed of methyl t-butyl ether (MTBE)−acetonitrile−0.1% aqueous trifluoroacetic acid (TFA) (2:2:3) in the NP partition mode (initial mobile phase: organic upperphase (UP) solvent; stationary phase: aqueous lower-phase (LP) solvent). First, the three-coil PTFE columns (capacity: 320 mL) of JCPC were entirely filled with the LP solvent as a stationary phase. Then, the columns were rotated at 1000 rpm (112g), while the UP mobile phase was pumped into the columns in the tail-to-head direction at a flow rate of 4.0 mL/min. After hydrodynamic mixing between the two phases in the columns reached equilibrium, 10 mL of sample solution (5 g of HBP dissolved with 5 mL each of the UP and LP solvents) was injected, and the hydrophobic constituents of the HBP sample were separated using the UP mobile phase. Following elution using UP for 120 min, the mobile phase was switched to LP, which completely eluted the polar constituents retained on the column, and elution was performed using LP for 100 min. During HSCCC separation, the UV absorbance of the eluate was monitored

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RESULTS AND DISCUSSION HSCCC Separation and Fractionation of Hop Bract Extract. In this study, the spray-dried powder of HBP was used as raw material. HBP contains various types of polyphenolic compounds,4 but the phenolic composition of HBP has yet to be fully elucidated. First, the constituents of HBP were fractionated by HSCCC using a two-phase solvent system composed of MTBE−acetonitrile−0.1% aqueous TFA (2:2:3) in the NP partition mode (initial mobile-phase: organic UP 2199

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Sequential Chromatographic Isolation of Polyphenolic Constituents of the HSCCC Fractions of HBP. The phenolic constituents of the HSCCC fractions C−I were further separated by HILIC using an amide column followed by RP-HPLC using ODS and phenyl columns. The schemes for sequential HPLC separation of HSCCC fractions C−I are shown in Figure 2. As a result of comprehensive HPLC separation, 102 types of single-peak compounds were isolated and recovered with amounts ranging from 0.1 mg to 40 mg. Furthermore, the structures of the compounds were elucidated on the basis of their spectroscopic data including ESI-TOFMS and 1D/2D NMR. Structural Analyses of the Isolated Compounds. Among the isolated compounds described above, 35 known compounds, 1 known compound identified for the first time in plants, and 3 new compounds were found. As shown in Table 2, the structures of the 35 known compounds (5−39) were identified by comparing their LC retention times, MS, NMR, and other spectroscopic data with those of experimental and literature values of standard compounds. They included 2-(2methylbutyryl)phloroglucinol-1-O-β-glucopyranoside (5);16 2isobutyrylphloroglucinol-1-O-β-glucopyranoside (6);16 3-O-caffeoylquinic acid (7);17 4-O-caffeoylquinic acid (8);17 5-Ocaffeoylquinic acid (9);17 4-O-β-glucopyranosyl-p-coumaric acid (10);18 trans-3-O-p-coumaroylquinic acid (11);19 cis-3-Op-coumaroylquinic acid (12);19 4-O-p-coumaroylquinic acid (13);20 3-O-feruloylquinic acid (14);21 4-O-feruloylquinic acid (15);21 kaempferol-3-O-β-glucopyranoside (16);22 quercetin-3O-β-glucopyranoside (17);22 kaempferol-3-O-β-rutinoside (18);22 kaempferol-3-O-β-neohesperidoside (19);22 quercetin3-O-β-neohesperidoside (20);22 quercetin-3-O-(2″-O-α-rhamnosyl-6″-O-malonyl)-β-glucopyranoside (21);22 myricetin-3-Oβ-glucopyranoside (22);22 kaempferol-3-O-β-(6-O-malonyl)glucopyranoside (23);23 quercetin-3-O-β-(6-O-malonyl)glucopyranoside (24);23 procyanidin B1 (25);24 epicatechin(4β-8)-epicatechin-(4β-8)-catechin (26);25 epicatechin-(4β-6)epicatechin-(4β-8)-catechin (27);25 tryptophan (28); 2-(3hydroxy-2-oxoindolin-3-yl)acetic acid (29);26 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (30);27 1-methyl-1,2,3,4tetrahydro-β-carboline-3-carboxylic acid (31);28 pinoresinol4″-O-β-glucopyranoside (32);29 isolariciresinol-3α-O-β-glucopyranoside (33);30 lariciresinol-4-O-β-glucopyranoside (34);31 (3S)-O-β-glucopyranosyl-6-[3-oxo-(2R)-butenylidenyl]-1,1,5trimethylcyclohexane-(5R)-ol (35);32 dihydrovomifoliol-3′-Oβ-glucopyranoside (36);33 5-O-β-xylopyranosylgentisic acid (37);34 4-O-β-glucopyranosyl-p-hydroxybenzoic acid (38);35 and (2E,4E)-8-β-glucopyranosyloxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid (39).36 The 20 compounds (10−15, 21, 22, 27, 29−39) have not been reported from hops. The spectroscopic data of compounds (1−4) are also shown in Tables 2−4. Compound 1 was obtained as a yellow amorphous powder. Its TOFMS signal (m/z 431.2273 [M + H]+) confirmed that its molecular formula was C21H34O9. The 1 H NMR spectrum of 1 showed two trans-coupled olefinic signals (3JH‑4,H‑5 = 15.6), a singlet olefinic signal, an oxygen-

solvent; stationary phase: aqueous LP solvent). Ten milliliters of sample solution (5 g of HBP dissolved with 5 mL each of the UP and LP solvents) was injected into the HSCCC column (capacity: 330 mL). To treat 25 g of HBP, HSCCC separation was repeated 5 times. Figure 1 shows the NP-HSCCC

Figure 1. HSCCC separation and fractionation profile of HBP (5 g). Separation was carried out using a two-phase solvent system composed of MTBE−acetonitrile−0.1% aqueous TFA (2:2:3) and NP partition mode (initial mobile phase: organic UP; stationary phase: aqueous LP). Flow rate: 4 mL/min. Retention % of stationary phase: 65%. Other chromatographic conditions: see Materials and Methods. All eluates were collected and divided into 10 fractions (fractions A−J).

chromatogram of HBP (5 g) separation. In the separation profile, hydrophobic constituents of HBP were quickly eluted with the organic UP mobile-phase solvent within 145 min, whereas the polar/hydrophilic constituents were strongly retained on the aqueous LP stationary phase in the HSCCC column. Then, the mobile-phase solvent was switched from UP to LP at 120 min after HBP injection, and then the remaining polar/hydrophilic constituents eluted through the column by 215 min. As shown in Figure 1, all eluates were collected and divided into 10 fractions (fractions A−J). The weights and yields of the fractions are listed in Table 1. The total yield of HBP from the HSCCC method (5 g × 5 times) was calculated to be 90.4%. The yield reveals that almost all of the constituents of HBP are recovered with no loss due to HSCCC treatment because the moisture content of the spray-dried HBP powder is about 5−10%. Among all the HSCCC fractions, the weight and yield of fraction J were the highest13.5 g, 54.0%. Fraction J was the most hydrophilic fraction, and it contained a wide variety of oligomeric proanthocyanidins (details of the characterization of the fraction J constituents are described below). Furthermore, the moderate hydrophobic polyphenols of the above HSCCC fractions (except for hydrophilic fraction J and hydrophobic fractions A and B) were sequentially separated by conventional HPLC. Unfortunately, the sequential separation for the fractions A and B were incomplete in the present study because the constituents in the both fractions were too hydrophobic for next HILIC separation.

Table 1. Weights and Yields of Fractions A−J Obtained from HBP (25 g) by NP-HSCCC Separation HSCCC fractions weights (g) yields (%)

A

B

C

D

E

F

G

H

I

J

total

0.2 0.8

0.5 2.0

0.7 2.8

1.5 6.0

0.8 3.2

0.9 3.6

0.8 3.2

1.7 6.8

2.0 8.0

13.5 54.0

22.6 90.4

2200

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Figure 2. Schemes of sequential HPLC separations of HSCCC fractions C−I. Numbers in red indicate new compounds and a known one, which has been identified for the first time in plants. Numbers in blue indicate known compounds that have not been reported from hops. Asterisk (*) indicates isolated compounds whose structures could not be determined in this study.

H-6 indicated the cis orientation of H-2 with respect to H-6 (Table 3). Coupling constants in 1H NMR and chemical shifts in 13C NMR supported the presence of the O-β-glucopyranosyl moiety (Table 3). The HMBC correlation between H-Glc1 and C-1 confirmed that the sugar moiety was attached at C-1. Therefore, the structure of 1 was confirmed to be 1-O(2′,3′,4′,4′-tetrahydroabscisoyl)-β-glucopyranose (Figure 4). Compound 2 was obtained as a yellow amorphous powder. Its TOFMS signal (m/z 142.0497 [M + H]+) confirmed that the molecular formula was C6H7NO3. The 1H and 13C NMR spectra of 2 were consistent with those of a known chemical synthetic intermediate, (R)-4-methylene-5-oxopyrrolidine-2carboxylic acid.38 The absolute configuration of the structure was expected to have the 2S-form because of positive optical rotation, the sign of which was reversed compared to the literature value of the above compound, and this was consistent with L-pyroglutamic acid in methanol. On the basis of these

bearing methine signal, a sugar moiety, four methylene signals, a methine signal, a secondary methyl group, two tertiary methyl groups, and an olefinic methyl group (Table 3). The 1H−1H COSY correlation from H-4 to H-5 and the HMBC correlations from H-2 to C-1 and from H-6 to C-2, C-3, and C-4 indicated the presence of the 3-methyl-2,4-pentadienoyl group (Figure 3). The proton connectivities of H-7′, H-2′, H3′, H-4′, and H-5′ assigned by 1H−1H COSY and the HMBC correlations from H-2′ax, H-3′eq, H-5′eq, H-8′, and H-9′ to C1′ and from H-5′ax, H-5′eq, H-8′, and H-9′ to C-6′ supported the presence of the 1,4-dihydroxy-2,6,6-trimethylcyclohexane moiety (Figure 3). The HMBC correlation between H-5 and C-1′ indicated that the acyl group was attached at C-1′, suggesting that 1 has 2′,3′,4′,4′-tetrahydroabscisic acid as the aglycon portion (Figure 3).37 The ROE correlations from H2′ax to H-4′ax and H-8′ and from H-5 to H-3′ax and H-5′ax supported the chair conformation. The ROE between H-2 and 2201

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Table 2. Yields, Maximum Absorption Wavelengths, and Mass Spectral Data of Purified Compounds

a

no.

yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

0.01 0.02 0.02 0.05 0.04 0.07 0.39 0.04 0.09 0.04 0.18 0.01 0.32 0.13 0.07 0.42 0.40 0.12 0.05 0.08 0.03 0.02 0.12 0.51 0.02 0.01 0.01 0.10 0.05 0.07 0.02 0.01 0.01 0.01 0.04 0.01 0.04 0.02 0.03

λmax (nm) 270 206 312 324 222, 222, 320 322 322 223, 308 304 226, 322 324 264, 254, 265, 265, 255, 257, 254, 262, 254, 278 280 280 219, 253 219, 278 228, 283 226, 234 248 314 248 267

285 284

293

310

345 352 346 344 352 355 378 345 351

278 278 278 278

[M − H]−

[M + H]+

attribution

429.2 140.1 323.1 353.1 371.1 357.1 353.1 353.1 353.1 325.1 337.1 337.1 337.1 367.1 367.1 447.1 463.1 593.2 593.2 609.2 695.2 479.1 533.1 549.1 577.1 865.3 865.3 203.1 206.0 215.1 229.1 519.2 521.2 521.2 n.d.a 387.2 285.1 299.1 403.2

431.2 142.0 325.1 355.1 373.1 359.1 355.1 355.1 355.1 327.1 339.1 339.1 339.1 369.1 369.1 449.1 465.1 595.2 595.2 611.2 697.2 481.1 535.1 551.1 579.1 867.2 867.2 205.1 208.1 217.1 231.1 521.2 523.2 n.d.a 431.2 389.2 287.1 301.1 405.2

1-O-(2′,3′,4′,4′-tetrahydroabscisoyl)-β-glucopyranose (S)-4-methylene-5-oxopyrrolidine-2-carboxylic acid 6-O-p-coumaroyl-L-galactono-1,4-lactone 6-O-feruloyl-L-galactono-1,4-lactone 2-(2-methylbutyryl)phloroglucinol-1-O-β-glucopyranside 2-isobutyrylphloroglucinol-1-O-β-glucopyranoside 3-O-caffeoylquinic acid 4-O-caffeoylquinic acid 5-O-caffeoylquinic acid 4-O-β-glucopyranosyl-p-coumaric acid trans-3-O-p-coumaroylquinic acid cis-3-O-p-coumaroylquinic acid 4-O-p-coumaroylquinic acid 3-O-feruloylquinic acid 4-O-feruloylquinic acid kaempferol-3-O-β-glucopyranoside quercetin-3-O-β-glucopyranoside kaempferol-3-O-β-rutinoside kaempferol-3-O-β-neohesperidoside quercetin-3-O-β-neohesperidoside quercetin-3-O-(2″-O-α-rhamnosyl-6″-O-malonyl)-β-glucopyranoside myricetin-3-O-β-glucopyranoside kaempferol-3-O-β-(6-O-malonyl)- -glucopyranoside quercetin-3-O-β-(6-O-malonyl)-glucopyranoside procyanidin B1 epicatechin-(4β-8)-epicatechin-(4β-8)-catechin epicatechin-(4β-6)-epicatechin-(4β-8)-catechin tryptophan 2-(3-hydroxy-2-oxoindolin-3-yl)-acetic acid 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid pinoresinol-4″-O-β-glucopyranoside isolariciresinol-3α-O-β-glucopyranoside lariciresinol-4-O-β-glucopyranoside (3S)-O-β-glucopyranosyl-6-[3-oxo-(2R)-butenylidenyl]-1,1,5-trimethylcyclohexane-(5R)-ol dihydrovomifoliol-3′-O-β-glucopyranoside 5-O-β-xylopyranosylgentisic acid 4-O-β-glucopyranosyl-p-hydroxybenzoic acid (2E,4E)-8-β-glucopyranosyloxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid

Not detected.

11.1 in 4), the HMBC correlation between H-2 and C-1, and the isotopic shifts of C-2, C-3, and C-5 in the H−D exchange experiments (CD3OH and CD3OD); the moiety was confirmed by a comparison of experimental values with a standard compound (Table 4). The HMBC correlation between H-6 and C-9′ confirmed that the sugar moiety was attached at C-9′. The absolute configuration of the sugar moiety in 4 was expected to have the L-form because of positive optical rotation, which was consistent with L-galactono-1,4-lactone in methanol. The absolute configuration of 3 was expected to have the same form, although the optical rotation of 3 could not be observed because of its small amount. Therefore, the structures of 3 and 4 were determined to be 6-O-p-coumaroyl-L-galactono-1,4lactone and 6-O-feruloyl-L-galactono-1,4-lactone, respectively (Figure 4). To our knowledge, compounds 1−4 have been identified for the first time in plants. Structural Characterization of Hydrophilic Proanthocyanidins in HBP. As shown in Figure 1 and Table 1, the constituents of HSCCC fraction J were a mixture of the most

data, 2 was determined to be (S)-4-methylene-5-oxopyrrolidine-2-carboxylic acid, which was identified for the first time in plants (Figure 4). Compounds 3 and 4 were obtained as yellow amorphous powders. Their molecular formulas were determined to be C15H16O8 and C16H18O9 (m/z 325.0912 [M + H]+ and 355.1007 [M + H]+), respectively, by TOFMS analyses. The 1 H NMR spectrum of 4 was similar to that of 3, except for the presence of a methoxy signal and the integral values of the olefinic signals. The 13C NMR spectra of 3 and 4 supported the presence of the p-coumaroyl group (δ 127.1, 131.2, 116.8, 161.4, 116.8, 131.2, 147.0, 114.8, and 168.9) in 3 and the feruloyl group (δ 127.7, 111.8, 149.4, 150.7, 116.5, 124.2, 147.3, 115.1, 168.8, and 56.4) in 4 (Table 4). The 1H and 13C NMR spectra of 3 and 4 indicated that they contain the same sugar moiety; it was identified to be galactono-1,4-lactone39 by coupling constants (3JH‑2,H‑3 = 8.7, 3JH‑3,H‑4 = 8.7, 3JH‑4,H‑5 = 2.4, 3 JH‑5,H‑6 = 6.0, 6.9, and 3JH‑6,H‑6 = 11.1 in 3 and 3JH‑2,H‑3 = 8.1, 3 JH‑3,H‑4 = 8.1, 3JH‑4,H‑5 = 2.4, 3JH‑5,H‑6 =5.7, 7.5, and 3JH‑6,H‑6 = 2202

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Table 3. 1H and 13C NMR Spectroscopic Data (δH, 600 MHz; δC, 150 MHz, CD3OD) for 1 δHa

position 1 2 3 4 5 6 1′ 2′ax 3′eq 3′ax 4′ax 5′eq 5′ax 6′ 7′ 8′ 9′ Glc1 Glc2 Glc3 Glc4 Glc5 Glc6 a

5.77 s 7.80 d (15.6) 6.52 d (15.6) 2.09 d (1.2) 2.06 1.83 1.20 3.92 1.68 1.51 0.81 1.08 0.83 5.51 3.36 3.43 3.35 3.39 3.84 3.68

m m dt (13.2, 11.7) m ddd (12.6, 4.8, 1.8) t (12.6) d (6.6) s s d (8.4) t (8.4) t (8.4) t (8.4) m dd (12.0, 2.4) dd (12.0, 4.8)

δC 166.1 116.6 154.3 129.1 140.1 21.5 80.1 37.0 42.5 67.1 47.9 40.6 27.0 23.5 16.6 95.4 74.0 78.1 71.1 78.8 62.4

Table 4. 1H and 13C NMR Spectroscopic Data (δH, 600 MHz; δC, 150 MHz, CD3OD) for 3 and 4

ROE correlations

3 δ Ha

position 6

1 2 3 4 5 6

3′ax, 5′ax, 9′ 2 4′ax, 7′, 8′ 3′ax, 4′ax, 7′ 5, 3′eq 2′ax, 3′eq, 5′eq, 8′ 4′ax, 8′, 9′ 5, 9′

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3′-OMe

2′ax, 3′eq 2′ax, 4′ax, 5′eq 5, 5′eq, 5′ax

4.38 4.33 4.19 4.07 4.29 4.33

d (8.7) t (8.7) dd (8.7, 2.4) m dd (11.1, 6.0) dd (11.1, 6.9)

7.47 d (9.0) 6.81 d (9.0) 6.81 7.47 7.67 6.37

d d d d

(9.0)b (9.0)c (15.9) (15.9)

4 δC 176.0 75.6 74.4 81.4 67.8 65.9 127.1 131.2 116.8 161.4 116.8b 131.2c 147.0 114.8 168.9

δ Ha 4.39 4.33 4.20 4.07 4.29 4.33

d (8.1) t (8.1) dd (8.1, 2.4) m dd (11.1, 5.7) dd (11.1, 7.5)

7.2 d (1.8)

6.82 7.09 7.67 6.40

d (8.1) dd (8.1, 1.8) d (15.9) d (15.9)

3.90 s

δC 176.0 75.6 74.4 81.4 67.8 65.9 127.7 111.8 149.4 150.7 116.5 124.2 147.3 115.1 168.8 56.4

a

Coupling constants (J in Hz) in parentheses. bEquivalent to 3′ because of symmetrical structure. cEquivalent to 2′ because of symmetrical structure.

to the existence of high-molecular-weight compounds (over 3000 Da). These results revealed that the main constituents of fraction J are highly oligomeric proanthocyanidins. Then, for the further purification of the high-molecular-weight compounds (proanthocyanidins) in fraction J, ultrafiltration was performed. In our previous study, 3 highly oligomeric proanthocyanidins (over 20-mer) were purified and concentrated by this treatment. As shown in Figure 5, the 1H NMR spectrum of the highly purified oligomeric proanthocyanidins from fraction J showed broad peaks at δ 6.3−7.2 (H-2′, H-5′, and H-6′), 5.7−6.2 (H-6 and H-8), 5.1 (H-2), 4.2−4.8 (H-4 in interflavan linkages), 3.5−4.0 (H-3), and 2.3−2.7 (H-4) that matched catechin or epicatechin units (Figure 5). The 13C NMR spectrum also showed broad peaks at δ 156 (C-5, C-7, and C-8a), 146 (C-3′ and C-4′), 132 (C-1′), 119 (C-6′), 115 (C-2′ and C-5′), 107 (C-6 or C-8 in interflavan linkages), 97 (C-4a, C-6, and C-8 that are not used in interflavan linkages), 77 (C-2), 73 (C-3), 38 (C-4 in interflavan linkages), and 30 (C4), indicating that the proanthocyanidins in the hop bract extract are B-type procyanidins, with flavan-3-ol units that are linked through C-4 to C-8 (or C-6) interflavan bonds (Figure 5).25 In the present LC separation scheme of the hop bract extract, HSCCC using a two-phase solvent system was highly effective, resulting in high-recovery separation and in the considerable reduction of the amount of extract utilized. The comprehensive

Coupling constants (J in Hz) in parentheses.

Figure 3. Key HMBC and 1H−1H COSY correlations of 1.

abundant and hydrophilic compounds in HBP. Further LC separations (HILIC and ODS) of the fraction J constituents were incomplete because both chromatograms showed an unresolved broad peak (data not shown). It was noted that hops contain an abundance of proanthocyanidins with different degrees of polymerization (DP),40 and the hydrophobicity of oligomeric proanthocyanidin significantly decreases as its DP value increases.41 Furthermore, in our preliminary examinations, the acid hydrolysis of the fraction J constituents resulted in the production of a large amount of red pigments (anthocyanidins), and the gel-permeation chromatogram of fraction J showed a broad and large peak, which was attributed

Figure 4. Structures of 1−4. 2203

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Figure 5. 1H and 13C NMR spectra of high-molecular-weight proanthocyanidins in HBP.

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separation and structural analyses resulted in four compounds that have been identified for the first time in plants, and 20 compounds that have not been reported from hops were also identified. Most of the compounds were hydrophilic glycosylated and/or esterified analogues of abscisic acids, hydroxycinnamic acids, flavonols, lignans, hydroxybenzoic acids, or carotenoid. In addition, large amounts of highly oligomeric proanthocyanidins were found in the hop bract extract. They were easily separated from the hydrophilic fraction (fraction J) by the first HSCCC fractionation following ultrafiltration. Furthermore, the 1H and 13C NMR spectra revealed that hop proanthocyanidins have B-type procyanidin structures, which is in agreement with previous studies.24,42 Hereafter, biological studies of all isolated compounds are needed for the elucidation of the various physiological activities of hop compounds and the industrial uses of hop bracts. 1-O-(2′,3′,4′,4′-Tetrahydroabscisoyl)-β-glucopyranose (1). Yellow amorphous powder; [α]D20 + 23° (c 0.2, methanol); UV (methanol) λmax (log ε) 270 (4.42) nm; for 1H and 13C NMR spectroscopic data, see Table 2; TOFMS m/z 431.2273 [M + H]+ (calcd for C21H35O9, 431.2275). (S)-4-Methylene-5-oxopyrrolidine-2-carboxylic acid (2). Yellow amorphous powder; [α]D20 +6° (c 0.3, methanol); UV (methanol) λmax (log ε) 206 (3.56), shoulder at 230 (3.35) nm; TOFMS 142.0497 [M + H]+ (calcd for C6H8NO3, 142.0498). 6-O-p-Coumaroyl-L-galactono-1,4-lactone (3). Yellow amorphous powder; UV (methanol) λmax (log ε) 211 (4.45), 225 (4.45), 312 (4.73) nm; for 1H and 13C NMR spectroscopic data, see Table 4; TOFMS m/z 325.0912 [M + H]+ (calcd for C15H17O8, 325.0917). 6-O-Feruloyl-L-galactono-1,4-lactone (4). Yellow amorphous powder; [α]D20 +8° (c 0.2, methanol); UV (methanol) λmax (log ε) 216 (4.08), 235 (4.01), shoulder at 295 (4.09), 324 (4.25) nm; for 1H and 13C NMR spectroscopic data, see Table 4; TOFMS m/z 355.1007 [M + H]+ (calcd for C16H19O9, 355.1023).

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-297-46-9551. Fax: +81-297-46-1507. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED LC, liquid chromatography; HSCCC, high-speed countercurrent chromatography; HILIC, hydrophilic interaction chromatography; RP, reversed-phase; ODS, octadecylsilyl; NP, normal-phase; COSY, correlation spectroscopy; HMBC, heteronuclear multiple bond correlation; ROE, rotating-frame overhauser enhancement; ESI, electrospray ionization; TOFMS, time-of-flight mass spectrometry; HPLC, high performance liquid chromatography; J-CPC, type-J coil-planet centrifuge; PTFE, polytetrafluoroethylene; HBP, polyphenolrich extract from hop bracts; MTBE, methyl t-butyl ether; TFA, trifluoroacetic acid; UP, upper-phase; LP, lower-phase; DP, degrees of polymerization

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REFERENCES

(1) De Keukeleire, J.; Ooms, G.; Heyerick, A.; Roldan-Ruiz, I.; Van Bockstaele, E.; De Keukeleire, D. Formation and accumulation of αacids, β-acids, desmethylxanthohumol, and xanthohumol during flowering of hops (Humulus lupulus L.). J. Agric. Food Chem. 2003, 51, 4436−4441. (2) Tagashira, M.; Uchiyama, K.; Yoshimura, T.; Shirota, M.; Uemitsu, N. Inhibition by hop bract polyphenols of cellular adherence and water-insoluble glucan synthesis of mutans streptococci. Biosci. Biotechnol. Biochem. 1997, 61, 332−335. (3) Kurumatani, M.; Fujita, R.; Tagashira, M.; Shoji, T.; Kanda, T.; Ikeda, M.; Shoji, A.; Yanagida, A.; Shibusawa, Y.; Shindo, H.; Ito, Y. Analysis of polyphenols from hop bract region using CCC. J. Liq. Chromatogr. Relat. Technol. 2005, 28, 1971−1983. (4) Tanaka, Y.; Honma, D.; Tamura, M.; Yanagida, A.; Zhao, P.; Shoji, T.; Tagashira, M.; Shibusawa, Y.; Kanda, T. New chromanone and acylphloroglucinol glycosides from the bracts of hops. Phytochem. Lett. 2012, 5, 514−518. (5) Shinada, K.; Tagashira, M.; Watanabe, H.; Sopapornamorn, P.; Kanayama, A.; Kanda, T.; Ikeda, M.; Kawaguchi, Y. Hop bract

2204

dx.doi.org/10.1021/jf405544n | J. Agric. Food Chem. 2014, 62, 2198−2206

Journal of Agricultural and Food Chemistry

Article

polyphenols reduced three-day dental plaque regrowth. J. Dent. Res. 2007, 86, 848−851. (6) Yaegaki, K.; Tanaka, T.; Sato, T.; Murata, T.; Imai, T.; Tagashira, M.; Akazome, Y.; Hirai, N.; Ohtake, Y. Hop polyphenols suppress production of water-insoluble glucan by Streptococcus mutans and dental plaque growth in vivo. J. Clin. Dent. 2008, 19, 74−78. (7) Inaba, H.; Tagashira, M.; Kanda, T.; Ohno, T.; Kawai, S.; Amano, A. Apple- and hop-polyphenols protect periodontal ligament cells stimulated with enamel matrix derivative from Porphyromonas gingivalis. J. Periodontol. 2005, 76, 2223−2229. (8) Inaba, H.; Tagashira, M.; Honma, D.; Kanda, T.; Kou, Y.; Ohtake, Y.; Amano, A. Identification of hop polyphenolic components which inhibit prostaglandin E2 production by gingival epithelial cells stimulated with periodontal pathogen. Biol. Pharm. Bull. 2008, 31, 527−530. (9) Kou, Y.; Inaba, H.; Kato, T.; Tagashira, M.; Honma, D.; Kanda, T.; Ohtake, Y.; Amano, A. Inflammatory responses of gingival epithelial cells stimulated with porphyromonas gingivalis vesicles are inhibited by hop-associated polyphenols. J. Periodontol. 2008, 79, 174− 180. (10) Morinaga, N.; Iwamaru, Y.; Yahiro, K.; Tagashira, M.; Moss, J.; Noda, M. Differential activities of plant polyphenols on the binding and internalization of cholera toxin in Vero cells. J. Biol. Chem. 2005, 280, 23303−23309. (11) Yahiro, K.; Shirasaka, D.; Tagashira, M.; Wada, A.; Morinaga, N.; Kuroda, F.; Choi, O.; Inoue, M.; Aoyama, N.; Ikeda, M.; Hirayama, T.; Moss, J.; Noda, M. Inhibitory effects of polyphenols on gastric injury by Helicobacter pylori VacA toxin. Helicobacter 2005, 10, 231− 239. (12) Choi, O.; Yahiro, K.; Morinaga, N.; Miyazaki, M.; Noda, M. Inhibitory effects of various plant polyphenols on the toxicity of Staphylococcal α-toxin. Microb. Pathog. 2007, 42, 215−224. (13) Ito, Y.; Bowman, R. L. Countercurrent chromatography: Liquidliquid partition chromatography without solid support. Science 1970, 167, 281−283. (14) Ito, Y. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J. Chromatogr., A 2005, 1065, 145−168. (15) Alpert, A. J. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J. Chromatogr. 1990, 499, 177−196. (16) Bohr, G.; Gerhaüser, C.; Knauft, J.; Zapp, J.; Becker, H. Antiinflammatory acylphloroglucinol derivatives from hops (Humulus lupulus). J. Nat. Prod. 2005, 68, 1545−1548. (17) Fuchs, C.; Spiteller, G. Rapid and easy identification of isomers of coumaroyl and caffeoyl-D-quinic acid by gas chromatography/mass spectrometry. J. Mass Spectrom. 1996, 31, 602−608. (18) Struijs, K.; Vincken, J.-P.; Verhoef, R.; van Oostveen-van Casteren, W. H. M.; Voragen, A. G. J.; Gruppen, H. The flavonoid herbacetin diglucoside as a constituent of the lignan macromolecule from flaxseed hulls. Phytochemistry 2007, 68, 1227−1235. (19) Ossipov, V.; Nurmi, K.; Loponen, J.; Haukioja, E.; Pihlaja, K. High-performance liquid chromatographic separation and identification of phenolic compounds from leaves of Betula pubescens and Betula pendula. J. Chromatogr., A 1996, 721, 59−68. (20) Nakamura, K.; Ogasawara, Y.; Endou, K.; Fujimori, S.; Koyama, M.; Akano, H. Phenolic compounds responsible for the superoxide dismutase-like activity in high-brix apple vinegar. J. Agric. Food Chem. 2010, 58, 10124−10132. (21) Ge, F.; Ke, C.; Tang, W.; Yang, X.; Tang, C.; Qin, G.; Xu, R.; Li, T.; Chen, X.; Zuo, J.; Ye, Y. Isolation of chlorogenic acids and their derivatives from Stemona japonica by preparative HPLC and evaluation of their anti-AIV (H5N1) activity in vitro. Phytochem. Anal. 2007, 18, 213−218. (22) Kazuma, K.; Noda, N.; Suzuki, M. Malonylated flavonol glycosides from the petals of Clitoria ternatea. Phytochemistry 2003, 62, 229−237.

(23) Wald, B.; Wray, V.; Galensa, R.; Herrmann, K. Malonated flavonol glycosides and 3,5-dicaffeoylquinic acid from pears. Phytochemistry 1989, 28, 663−664. (24) Stevens, J. F.; Miranda, C. L.; Wolthers, K. R.; Schimerlik, M.; Deinzer, M. L.; Buhler, D. R. Identification and in vitro biological activities of hop proanthocyanidins: inhibition of nNOS activity and scavenging of reactive nitrogen species. J. Agric. Food Chem. 2002, 50, 3435−3443. (25) Shoji, T.; Mutsuga, M.; Nakamura, T.; Kanda, T.; Akiyama, H.; Goda, Y. Isolation and structural elucidation of some procyanidins from apple by low-temperature nuclear magnetic resonance. J. Agric. Food Chem. 2003, 51, 3806−3813. (26) Kato, T.; Tomita, N.; Hoshikawa, M.; Ehara, K.; Shima, J.; Takahashi, N.; Sugiyama, H. In vitro oxidation of indoleacetic acid by crude enzyme from rice husk - An aspect of preharvest sprouting. Heterocycles 1998, 47, 497−500. (27) Tilstra, L.; Sattler, M. C.; Cherry, W. R.; Barkley, M. D. Fluorescence of a rotationally constrained tryptophan derivative, 3carboxy-1,2,3,4-tetrahydro-2-carboline. J. Am. Chem. Soc. 1990, 112, 9176−9182. (28) Simat, T.; Meyer, K.; Steinhart, H. Synthesis and analysis of oxidation and carbonyl condensation compounds of tryptophan. J. Chromatogr., A 1994, 661, 93−99. (29) Chiba, M.; Hisada, S.; Nishibe, S.; Thieme, H. 13C NMR analysis of symplocosin and (+)-epipinoresinol glucoside. Phytochemistry 1980, 19, 335−336. (30) Ono, M.; Oda, E.; Tanaka, T.; Iida, Y.; Yamasaki, T.; Masuoka, C.; Ikeda, T.; Nohara, T. DPPH radical-scavenging effect on some constituents from the aerial parts of Lippia triphylla. J. Nat. Med. 2008, 62, 101−106. (31) Park, J.-H.; Yeon, S.-W.; Cho, J.-G.; Lee, D.-Y.; Kim, Y.-S.; Baek, N.-I. Lignans from silkworm droppings and their promotional activities on heme oxygenase-1 (HO-1). J. Appl. Biol. Chem. 2010, 53, 734−739. (32) Hisamoto, M.; Kikuzaki, H.; Nakatani, N. Constituents of the leaves of Peucedanum japonicum Thunb. and their biological activity. J. Agric. Food Chem. 2004, 52, 445−450. (33) Andersson, R.; Lundgren, L. N. Monoaryl and cyclohexenone glycosides from needles of Pinus sylvestris. Phytochemistry 1988, 27, 559−562. (34) Fayos, J.; Bellés, J. M.; López-Gresa, M. P.; Primo, J.; Conejero, V. Induction of gentisic acid 5-O-β-D-xylopyranoside in tomato and cucumber plants infected by different pathogens. Phytochemistry 2006, 67, 142−148. (35) Strack, D.; Heilemann, J.; Wray, V.; Dirks, H. Structures and accumulation patterns of soluble and insoluble phenolics from norway spruce needles. Phytochemistry 1989, 28, 2071−2078. (36) Hegde, V. R.; Pu, H.; Patel, M.; Jachens, A.; Gullo, V. P.; Chan, T.-M. A new antitumor compound from the plant Oryctanthus sp. as a VEGF receptor binding inhibitor. Bioorg. Med. Chem. Lett. 2005, 15, 4907−4909. (37) Lamb, N.; Shaw, A. C.; Abrams, S. R.; Reaney, M. J. T.; Ewan, B.; Robertson, A. J.; Gusta, L. V. Oxidation of the 8′-position of a biologically active abscisic acid analogue. Phytochemistry 1993, 34, 905−917. (38) Bertoli, A.; Fanfoni, L.; Felluga, F.; Pitacco, G.; Valentin, E. Chemoenzymatic synthesis of optically active α-methylene-γ-carboxyγ-lactams and γ-lactones. Tetrahedron Asymmetry 2009, 20, 2305− 2310. (39) Walaszek, Z.; Horton, D. Conformational studies on aldonolactones by N.M.R. spectroscopy. conformations of D-glucono-, D-mannono-, D-gulono- and D-galactono-1,4-lactone in solution. Carbohydr. Res. 1982, 105, 131−143. (40) Taylor, A. W.; Barofsky, E.; Kennedy, J. A.; Deinzer, M. L. Hop (Humulus lupulus L.) proanthocyanidins characterized by mass spectrometry, acid catalysis, and gel permeation chromatography. J. Agric. Food Chem. 2003, 51, 4101−4110. (41) Yanagida, A.; Murao, H.; Ohnishi-Kameyama, M.; Yamakawa, Y.; Shoji, A.; Tagashira, M.; Kanda, T.; Shindo, H.; Shibusawa, Y. 2205

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Journal of Agricultural and Food Chemistry

Article

Retention behavior of oligomeric proanthocyanidins in hydrophilic interaction chromatography. J. Chromatogr., A 2007, 1143, 153−161. (42) Li, H.-J.; Deinzer, M. L. Structural identification and distribution of proanthocyanidins in 13 different hops. J. Agric. Food Chem. 2006, 54, 4048−4056.

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on February 26, 2014, with Figures 3 and 4 incorrectly cited, and an error to Figure 5. The corrected version was reposted on February 28, 2014.

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