Analysis of the components of hard resin in hops (Humulus lupulus L.) and structural elucidation of their transformation products formed during the brewing process

Article abstract of DOI:10.1021/jf504394h

The resins from hops (Humulus lupulus L.), which add the bitter taste to beer, are classified into two main subfractions, namely, soft and hard resins. α- and β-Acids in soft resin and their transformation during the wort boiling process are well-studied; however, other constituents in resins, especially hard resin, have been unidentified. In this study, we identified humulinones and hulupones as soft-resin components, in addition to 4′-hydroxyallohumulinones and tricyclooxyisohumulones A and B as hard-resin components. These compounds are all oxidation products derived from α- and β-acids. We also investigated compositional changes in the hard resin during the wort boiling process, which has a significant effect on the taste of the beer, by using model boiling experiments. The major changes were identified to be isomerization of 4′-hydroxyallohumulinones into 4′- hydroxyallo-cis-humulinones, followed by decomposition into cis-oxyhumulinic acids. These findings will be helpful in systematically evaluating and optimizing the effect of the hard resin on beer quality.

Full text of DOI:10.1021/jf504394h

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Article
Analysis of the Components of Hard Resin in Hops
(Humulus lupulus L.) and Structural Elucidation of Their
Transformation Products Formed During the Brewing Process
Yoshimasa Taniguchi, Harumi Taniguchi, Makiko Yamada, Yasuko
Matsukura, Hideki Koizumi, Kazuo Furihata, and Kazutoshi Shindo
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504394h • Publication Date (Web): 29 Oct 2014
Downloaded from http://pubs.acs.org on November 5, 2014
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Analysis of the Components of Hard Resin in Hops (Humulus lupulus L.) and
Structural Elucidation of Their Transformation Products Formed During the
Brewing Process
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AUTHORSHIP
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Yoshimasa Taniguchi,*,† Harumi Taniguchi,† Makiko Yamada,† Yasuko Matsukura,†
Hideki Koizumi,† Kazuo Furihata, ‡ and Kazutoshi Shindo§
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†Central Laboratories for Key Technologies, Research & Development Division, KIRIN
Company, Ltd., 1-13-5, Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004,
Japan.
‡Division of Agriculture and Agricultural Life Sciences, The University of Tokyo, 1-1-1,
Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
§Department of Food and Nutrition, Japan Women’s University, 2-8-1, Mejirodai
Bunkyo-ku, Tokyo 112-8681, Japan.
*; Author to whom correspondence should be addressed (telephone +81-45-330-9007; fax
+81-45-782-3657; e-mail Yoshimasa_Taniguchi@kirin.co.jp).
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ABSTRACT:
The resins from hops (Humulus lupulus L.), which add the bitter taste to beer, are
classified into two main sub-fractions; namely soft and hard resins. α-Acids and β-acids
in soft resin and their transformation during the wort boiling process are well-studied,
however, other constituents in resins, especially hard resin, have been unidentified. In this
study, we identified humulinones and hulupones as soft resin components, in addition to
4′-hydroxyallohumulinones and tricyclooxyisohumulones A and B as hard resin
components. These compounds are all oxidation products derived from α-acids or
β-acids. We also investigated compositional changes in the hard resin during the wort
boiling process, which has a significant effect on the taste of the beer, by utilizing model
boiling experiments. The major changes were identified to be isomerization of
4′-hydroxyallohumulinones into 4′-hydroxyallo-cis-humulinones followed by
decomposition into cis-oxyhumulinic acids. These findings will be helpful in
systematically evaluating and optimizing the effect of the hard resin on beer quality.
KEYWORDS: Hop, Humulus lupulus L., soft resin, hard resin, humulinone, hulupone,
4′-hydroxyallohumulinone, 4′-hydroxyallo-cis-humulinone, cis-oxyhumulinic acid, wort
boiling, beer
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INTRODUCTION
Hops (Humulus lupulus L.) are widely used in the brewing industry to add
characteristic bitterness and aroma to beer. The lupulin glands from female
inflorescences of hops are known to accumulate resins and essential oils. The resins are
composed of many different substances and are responsible for the fine bitter taste of beer.
According to the European Brewery Convention and the American Society of Brewing
Chemists, resins in hops can be divided into two main sub-fractions; namely soft resin
(i.e., the fraction soluble in low-boiling paraffin hydrocarbons such as hexane) and hard
resin (i.e., the fraction insoluble in hexane but soluble in ether and cold MeOH).1-4
α-Acids, one of the best studied group of compounds in hops, are found in the soft
resin fraction. These α-acids comprise three main congeners, cohumulone (1a),
n-humulone (1b) and adhumulone (1c), which differ in their acyl side chains. During the
wort boiling process, α-acids thermally isomerize into iso-α-acids via an acyloin-type
ring contraction, resulting in the generation of two epimeric isomers: cis- (3a-c) and
trans-iso-α-acids (4a-c) (Figure 1).5 Iso-α-acids are known to be the major contributors
to the bitter taste of beer,6, 7 and to the stalility of beer foam.8 In addition to α-acids,
β-acids, which consist of three main congeners, colupulone (2a), n-lupulone (2b) and
adlupulone (2c), are also found in the soft resin fraction. The oxidative transformation
products of β-acids generated during the wort boiling process contribute to the bitter taste
of beer.9, 10 There have been numerous published studies that have focused on the soft
resin components and their impact on the properties of beer.
By contrast, however, there is a lack of information concerning the hard resin
components and their effect on beer quality. Xanthohumol (10), a prenyl chalcone, is a
well-known constituent of hard resin.4, 11 However, other components of hard resin have
not, until now, been identified.4, 11 It has been reported that the α- and β-acids undergo
rapid oxidation during the storage of hops, and the amount of hard resin fraction
increases.12, 13 Thus, the hard resin is considered to be mainly composed of the oxidation
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products derived from α-acids and/or β-acids.4, 11
The organoleptic properties and functionality of the hard resin have received
considerable attention over the years. However, opinion concerning the effect of the hard
resin accumulated in stored hops on the bitterness quality of beer remains contentious.6,
14-18 Previous studies using hard resin enriched extracts for brewing proved inconclusive
and failed to explain the effect of the hard resin on the quality of the beer.13, 19 The
apparent inconsistency in the results of these studies could be due to the lack of chemical
analysis of materials used in the experiments. Recently, Alamaguer et al. reported that the
hard resin positively contributed to the foam stability of beer and produced a more
pleasant bitter taste as opposed to the sharper bitterness of iso-α-acids. However, the hard
resin enriched extracts used in the study were not subjected to rigorous chemical
compositional analysis.11 Thus, a suitable method to fully analyze the chemical
composition of the hard resin was needed to properly examine its effect on the properties
of beer. Furthermore, transformation of the hard resin components during the wort
boiling process, which is known to have a significant effect on beer taste, was not
understood, except for the conversion of xanthohumol (10) into isoxanthohumol (11).20
Knowledge of the transformation of hard resin components during the wort boiling
process is also essential to evaluate and optimize the effect of the hard resin on beer
quality.
In our previous study, we developed an HPLC method suitable for investigation of
the oxidation products in stored hops, which showed that α-acids are oxidized into
humulinones (5a-c), 4′-hydroxyallohumulinones (6a-c) and tricyclooxyisohumulones A
(7a-c) and B (8a-c), and that β-acids are oxidized into hulupones (9a-c) (Figure 1).21, 22
These oxidation products are thought to be components in the soft or hard resins.
In this study, we analyzed the soft and hard resins derived from stored hops using
our newly developed analytical method and clarified the existence of these oxidation
compounds for the first time. We also investigated the transformation of the hard resin
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components through wort boiling by utilizing model boiling experiments, and
successively isolated and determined the structures of the transformation products. The
concentration changes of these products and their precursors during the boiling process
were also investigated.
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MATERIALS AND METHODS
Chemicals and Materials. The following chemicals were obtained commercially:
ethylenediaminetetraacetic acid (EDTA), H3PO4, MeCN, EtOH, hexane, diethyl ether,
CH2Cl2 (Wako Pure Chemicals, Osaka, Japan); xanthohumol, isoxanthohumol
(Funakoshi, Tokyo, Japan). Deionized water for chromatography was purified by means
of a Milli-Q Gradient A10 system (Millipore, Billerica, MA). Hop pellets, cultivar
Hallertau Perle (HPE), were purchased from Hopsteiner (Mainburg, Germany).
Preparation
Tricyclooxyiso-n-humulones A (7b) and B (8b). Humulinones (5a-c) and hulupones
(9a-c) were prepared according to
protocol reported previously.21
of
Humulinones
(5a-c),
Hulupones
(9a-c),
and
a
Tricyclooxyiso-n-humulones A (7b) and B (8b) were isolated from the autoxidation
products of n-humulone.22
Preparation of 4′-Hydroxyallohumulinones (6a-c and 6c′) from Stored Hops.
Hop pellets (850 g) were stored at 60 °C for 120 h and extracted with H2O (8.5 L) at 50 °C
for 1 h. The extract was filtered and then lyophilized to yield a brown powder (176 g). A
portion of this powder (120 g) was dissolved in H2O (8.1 L) and partitioned with CH2Cl2
(16.2 L) after adding 1 N HCl (810 mL) to the solution. The CH2Cl2 layer containing
4′-hydroxyallohumulinones was collected and dried over anhydrous Na2SO4 and
concentrated to dryness in vacuo to give a yellowish brown oil (22 g). This oil was
dissolved in EtOH and repeatedly subjected to preparative HPLC (column: 150 × 22 mm
id, 5 µm, Alltima C18 column (Systech, Tokyo, Japan); solvent: H2O/H3PO4 (85%),
100/1, (v/v) (solvent A) and MeCN (solvent B), a linear gradient from 30 to 90% B in 0
→ 40 min and 90% B for 40.1 → 45 min; flow rate: 18.8 mL/min; detector: 270 nm;
column temperature: RT), and divided into 11 fractions depending on their elution times.
Compounds 6a, 6b, and 6c/c′ were contained in fractions 3 (tR 8.6 – 9.5 min), 6 (tR 11.0 –
12.1 min) and 7 (tR 12.1 – 12.9 min), respectively. Compound 6a in fraction 3 and
compound 6b in fraction 6 were further purified by repeatedly subjecting them to a
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second preparative HPLC step (column: 50 × 20 mm id, 5 µm, L-column 2 ODS
(Chemicals Evaluation and Research Institute, Tokyo, Japan); solvent: 10 mM
NH4HCO3 (solvent A) and MeCN (solvent B), isocratic elution at 20% B; flow rate: 9.5
mL/min; detector: 270 nm; column temperature: 40 ˚C). Compounds 6a and 6b were
eluted at 3.8 – 4.7 min and at 5.5 – 7.5 min, respectively. Each eluate was immediately
diluted two times with H2O, adjusted to pH 2.0 with 1 N HCl, and partitioned with
CH2Cl2 (1/2 volume of the diluted eluate × 2 times). The respective CH2Cl2 layer was
dried over Na2SO4 and concentrated to dryness to yield pure 6a (814 mg) and 6b (1.2 g).
Similarly, compounds 6c/c′ in fraction 7 were further purified by repeatedly subjecting
them to a second preparative HPLC (column: 150 × 20 mm id, 5 µm, L-column 2 ODS
(Chemicals Evaluation and Research Institute, Tokyo, Japan); solvent: 100 mM
NH4HCO3 (solvent A) and MeCN (solvent B), isocratic elution at 20% B; flow rate: 18.8
mL/min; detector: 270 nm; column temperature: RT). Compounds 6c and 6c′ were eluted
separately at 9.8 – 10.8 min and at 10.8 – 11.8 min, respectively. Pure 6c (40.3 mg) and
6c′ (37.2 mg) were recovered from each eluate in the same way as that described for 6a
and 6b.
4′-Hydroxyalloadhumulinone A (6c): pale yellow oil; [α]20D +4.01 (c 0.4, MeOH);
1H and 13C NMR data, see Table 2; HRESIMS (negative) m/z 393.1916 [M − H]− (calcd
for C21H29O7, 393.1919).
4′-Hydroxyalloadhumulinone B (6c′): pale yellow oil; [α]20D +12.1 (c 0.3,
MeOH); 1H and 13C NMR data, see Table 2; HRESIMS (negative) m/z 393.1914 [M −
H]− (calcd for C21H29O7, 393.1919).
Preparation of Soft and Hard Resins. Soft and hard resins from hops were
prepared according to Analytica EBC3 with some modification.
Hop pellets (100 g) were stored at 60 °C for 48 h, and a portion (10 g) was extracted
with MeOH (100 mL) by stirring at room temperature for 1 h and then filtered. The
filtrate was kept at 4 °C for 1 day, and a waxy precipitate that formed was removed by
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filtration. To a portion of the filtrate (20 mL), MeOH (10 mL), hexane (80 mL) and 1 N
HCl (20 mL) were added, and the two layer solution (hexane/acidic MeOH-H2O) was
partitioned in a separating funnel. The hexane layer, prepared by repeating the above
partition four times (320 mL), was evaporated to dryness to give a yellowish brown oil
(250 mg, soft resin).
The residual acidic MeOH- H2O layer was partitioned with diethyl ether (80 mL × 2
times). The diethyl ether layer (160 mL) was washed with sat. NaCl solution (160 mL),
dried over anhydrous Na2SO4, and concentrated to dryness to give a brown solid (200 mg,
hard resin).
Boiling Test for the Hard Resin. To 500 µL of 10 mM citrate buffer (pH 5.5)
pre-heated to 100 °C was added a 25 µL solution of the hard resin in EtOH (5.0 mg/mL).
The combined solution was kept at 100 °C and analyzed by HPLC at 30, 60, 90 and 120
min.
Boiling Test of 4′-Hydroxyallohumulinones (6a-c, c′). To a 500 µL aliquot of 10
mM citrate buffer (pH 5.5) pre-heated to 100 °C was added 25 µL solution of either
4′-hydroxyallocohumulinone
(6a),
4′-hydroxyallo-n-humulinone
(6b),
4′-hydroxyalloadhumulinones A (6c) or B (6c′) (1.0 mg/mL in EtOH). Each combined
solution was kept at 100 °C, and analyzed by HPLC at 30, 60, 90, and 120 min.
Isolation of the Transformation Products Produced by Boiling of
4′-Hydroxyallocohumulinone (6a) and 4′-Hydroxyallo-n-humulinone (6b).
4′-Hydroxyallocohumulinone (6a) (200 mg) was dissolved in EtOH (50 mL) and added
to 10 mM citrate buffer (pH 5.5, 1 L) pre-heated to 100 °C and maintained at this
temperature for 90 min. After cooling to room temperature, the solution was acidified
with 1 N HCl (100 mL) and partitioned with CH2Cl2 (250 mL × 3 times). The CH2Cl2
layer (750 mL) was washed with sat. NaCl solution (400 mL), dried over anhydrous
Na2SO4, and concentrated to dryness to give a yellowish brown oil (150 mg).
The oil was dissolved in EtOH and repeatedly subjected to preparative HPLC
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(column: 150 × 10 mm id, 5 µm, Alltima C18 column (Systech, Tokyo, Japan); solvent:
H2O/H3PO4 (85%), 100/1, (v/v) (solvent A) and MeCN (solvent B), a linear gradient
from 30% B for 0 → 15 min, 30 to 90% B in 15 → 18 min, and 90% B for 18 → 22 min;
flow rate: 4.7 mL/min; detector: 270 nm; column temperature: 40 °C). In this preparative
HPLC, 4′-hydroxyallo-cis-cohumulinone (12a) was eluted at 8.6 – 10.0 min and
cis-oxycohumulinic acid (13a) was eluted at 10.0 – 10.6 min. Each eluate was diluted
with H2O to lower the concentration of MeCN to less than 10%, and extracted with
CH2Cl2 (1/4 volume of the diluted eluate × 3 times). The CH2Cl2 layer was dried over
anhydrous Na2SO4 and concentrated to dryness to yield 12a (33.4 mg) and 13a (4.0 mg).
The transformation products (12b and 13b) of 4′-hydroxyallo-n-humulinone (6b)
were prepared and purified in almost the same way as described for 12a and 13a. In
summary, 4′-hydroxyallo-n-humulinone (6b) (200 mg) was boiled and partitioned
between CH2Cl2 and acidic H2O. The CH2Cl2 layer was concentrated to give an oil (167
mg). The oil was repeatedly subjected to preparative HPLC (column, flow rate, detector,
and column temperature are identical to those used for the isolation of 12a and 13a;
solvent: H2O/H3PO4 (85%), 100/1, (v/v) (solvent A) and MeCN (solvent B), a linear
gradient from 30 to 48% B in 0 → 12 min, 48 to 90% B in 12 → 18 min, and 90% B for 18
→ 21 min). In this preparative HPLC, 4′-hydroxyallo-cis-n-humulinone (12b) and
cis-oxy-n-humulinic acid (13b) were eluted at 9.0 – 9.6 min and at 10.0 – 10.3 min,
respectively. Pure 12b (41.3 mg) and 13b (5.9 mg) were recovered from each eluate in the
same way as described for 12a and 13a.
4′-Hydroxyallo-cis-cohumulinone (12a): pale yellow oil; [α]20D ±0 (c 0.7,
MeOH); 1H and 13C NMR data, see Table 1; HRESIMS (negative) m/z 379.1753 [M −
H]− (calcd for C20H27O7, 379.1762).
4′-Hydroxyallo-cis-n-humulinone (12b): pale yellow oil; [α]20D ±0 (c 0.4, MeOH);
1H and 13C NMR data, see Table 1; HRESIMS (negative) m/z 393.1911 [M − H]− (calcd
for C21H29O7, 393.1919).
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Cis-oxycohumulinic acid (13a): white, amorphous solid; [α]20D ±0 (c 0.1, MeOH);
1H and 13C NMR data, see Table 1; HRESIMS (negative) m/z 267.1230 [M − H]− (calcd
for C14H19O5, 267.1238).
Cis-oxy-n-humulinic acid (13b): pale yellow oil; [α]20D ±0 (c 0.1, MeOH); 1H and
13C NMR data, see Table 1; HRESIMS (negative) m/z 281.1390 [M − H]− (calcd for
C15H21O5, 281.1395).
Preparation of Cis- (13b) and Trans-oxy-n-humulinic acids (14b) by hydrolysis
of n-humulinone (5b). Both cis- and trans-oxy-n-humulinic acids were prepared by
alkaline hydrolysis of n-humulinone (5b) according to the previous report23 with some
modification.
N-humulinone was prepared according to a protocol reported previously.21 To 2 M
NaOHaq (300 mL) pre-heated at 100 ˚C, 30 mL solution of n-humulinone in MeOH (10
mg/mL) was added and mixed well. The temperature was maintained at 100 ˚C for 10 min
before cooling the solution to room temperature, acidifying with 6 N HCl (120 mL) and
partitioning with CH2Cl2 (200 mL × 2 times). The CH2Cl2 layer was dried over
anhydrous Na2SO4 and concentrated to dryness to give a yellow oil (270 mg). The oil
was dissolved in EtOH and repeatedly subjected to preparative HPLC (column: 150 × 22
mm id, 5 µm, Alltima C18 column (Systech, Tokyo, Japan); solvent: H2O/H3PO4 (85%),
100/1, (v/v) (solvent A) and MeCN (solvent B), isocratic elution at 45% B; flow rate: 22.8
mL/min; detector: 270 nm; column temperature: RT). In this preparative HPLC, cis-
(13b) and trans-oxy-n-humulinic acids (14b) were eluted at 5.1 – 5.5 min and at 6.7 – 7.2
min, respectively. Pure 13b (74.6 mg) and 14b (94.6 mg) were recovered from each
eluate in the same way as described for 12a and 13a.
Trans-oxy-n-humulinic acid (14b): white, amorphous solid; [α]20D ±0 (c 0.3,
MeOH); 1H and 13C NMR data, see Table 1; HRESIMS (negative) m/z 281.1389 [M −
H]− (calcd for C15H21O5, 281.1395).
High-Performance Liquid Chromatography (HPLC). A Shimadzu Prominence
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UFLC™ system (Shimadzu, Kyoto, Japan) was used for all HPLC analysis. Data were
processed with LCsolution software (Shimadzu, Kyoto, Japan).
HPLC analyses were performed to investigate the constituents in the soft and hard
resins of hops and to analyze compositional changes in the hard resin components in the
boiling tests using the following conditions21, 22: column: 100 × 2.1 mm id, 3 µm, Alltima
C18 column (Systech, Tokyo, Japan); solvent: H2O/H3PO4 (85%), 1000/0.2, (v/v)
containing EDTA (0.02% w/v) (solvent A) and MeCN (solvent B), a linear gradient from
10 to 52% B in 0 → 26.7 min, 52% B for 26.7 → 30 min, 52 to 75% B in 30 → 32.7 min,
75 to 85% B in 32.7 → 36.7 min, and 85% B for 36.7 → 37.7 min; flow rate: 0.6 mL/min;
detector: 270 nm; column temperature: 40 ˚C. The injection volume was 3.0 µL. In the
boiling tests, transformation products were quantified using isolated reference
compounds. 4′-Hydroxyallo-cis-adhumulinones A (12c) and B (12c′) were quantified
using the calibration curve of 4′-hydroxyallo-cis-n-humulinone (12b) and
cis-oxyadhumulinic acids A (13c) and B (13c′) were quantified using the calibration
curve of cis-oxy-n-humulinic acid (13b).
To separate and check the purity of 4′-hydroxyalloadhumulinones A (6c) and B
(6c′) by HPLC (Figure 7), the following LC conditions were used: column: 100 × 2.1 mm
id, 3 µm, L-column 2 ODS (Chemicals Evaluation and Research Institute, Tokyo, Japan);
solvent: 5 mM HCOONH4 (pH 8.5) (solvent A) and MeCN (solvent B), a linear gradient
from 10 to 36% B in 0 → 39 min, 36 to 80% B in 39 → 44 min, and 80% B for 44 → 52
min; flow rate: 0.25 mL/min; column temperature: 40 ˚C. The injection volume was 3.0
µL.
High-Resolution Electrospray Ionization Mass Spectrometry (HRESIMS).
HRESIMS of the purified compounds was measured using a Thermo Scientific LTQ
Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Before measuring
the samples, accurate mass calibration was carried out using polytyrosine-1,3,6 as a mass
standard (CS Bio Company, Menlo Park, CA).
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Nuclear Magnetic Resonance Spectroscopy (NMR). H, 13C, and 2D NMR
spectra were measured with a Bruker AVANCE400 spectrometer (Bruker BioSpin,
Rheinstetten, Germany). Samples were dissolved in methanol-d4. Chemical shifts were
referenced to the solvent signals (δH/δC 3.30/49.0). Data processing was performed using
TopSpin-NMR software (version 3.0) (Bruker BioSpin). J-Resolved HMBC experiments
were conducted on a Varian INOVA-500 NMR spectrometer (Agilent, Santa Clara,
CA) using the previously reported method.24
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RESULTS AND DISCUSSION
Identification of the Components in the Soft and Hard Resins Derived from
Stored Hops. Recently, we reported the transformation of α-acids to humulinones
(humulinones were further transformed to 4′-hydroxyallohumulinones) and to
tricyclooxyisohumulones A and B due to oxidation.21, 22 During these studies, we also
developed a HPLC method that is suitable for the analysis of α- and β-acid-derived
oxidation compounds.21, 22 Using this HPLC method, we analyzed a MeOH extract of the
stored hops, and soft and hard resins prepared from the MeOH extract. The results are
shown in Figure 2 (2A: MeOH extract of the stored hop, 2B: the soft resin, 2C: the hard
resin).
As shown in Figure 2B, the presence of humulinones (5a-c, c′), hulupones (9a-c) in
addition to α-acids (1a-c) in the soft resin can clearly be observed. This is the first study
reporting the presence of humulinones (5a-c, c′) and hulupones (9a-c) in soft resin.
The compounds contained in the soft resin were barely detectable in the hard resin
(Figure 2C). Indeed, xanthohumol (10) and the hydrophilic α-acid-derived oxidation
products such as tricyclooxyisohumulones A (7a, b) and B (8a, b), and
4′-hydroxyallohumulinones (6a-c, c′) were found only in the hard resin extract. This is
the first study to report the existence of these α-acid-derived oxidation products in hard
resin.
These findings also verify changes in the composition of the soft and hard resin
constituents of hops caused by oxidation, because the concentration changes in α- and
β-acid-derived oxidation products in soft and hard resins detected in this study during
oxidation of hops have been clarified in our previous study.21, 22
Transformation of the Constituents of Hard Resin during the Wort Boiling
Process. There has been little reported data on the compositional changes in hard resin
during the wort boiling process. Thus, we investigated the constituents in the hard resin
after wort boiling. Changes in the chemical composition of hard resin constituents were
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determined by using a model boiling experiment conducted in an aqueous buffer (pH 5.5).
HPLC analysis of the hard resin after 90 min of boiling (Figure 3B) clearly showed the
transformation of xathohumol (10) into isoxanthohumol (11), which was reported
previously.20 In addition, the HPLC analysis also detected a decrease in the concentration
of
4′-hydroxyallohumulinones
(4′-hydroxyallocohumulinone
(6a),
4′-hydroxyallo-n-humulinone (6b) and 4′-hydroxyalloadhumulinone (6c/c′)), and the
appearance of some new compounds (Figure 3B). These findings suggested, for the first
time, further transformation of 4′-hydroxyallohumulinones in the hard resin during the
wort boiling process.
Isolation and Structural Elucidation of the Transformation Products of
4′-hydroxyallocohumulinone (6a) and 4′-hydroxyallo-n-humulinone (6b). To verify
the transformation of 4′-hydroxyallohumulinones into new compounds, isolated
4′-hydroxyallocohumulinone (6a) and 4′-hydroxyallo-n-humulinone (6b) were boiled in
the same buffer. The HPLC analyses clearly showed the transformation of 6a into 12a and
13a (Figure 4A1 and A2) and of 6b into 12b and 13b (Figure 4B1 and B2). We also
boiled tricyclooxyiso-n-humulones A (7b) and B (8b), but the compounds were stable
(i.e., unchanged) under these conditions (data not shown).
To identify the transformation products of 4′-hydroxyallocohumulinone (6a) and
4′-hydroxyallo-n-humulinone (6b), each 200 mg sample of isolated 6a and 6b was
subjected to the boiling test. The compounds in the reaction products were purified using
two phase solvent partition and preparative ODS HPLC, affording 12a (33.4 mg) and 13a
(4.0 mg) from 6a, and 12b (41.3 mg) and 13b (5.9 mg) from 6b.
Compound 12b was obtained as a pale yellow oil. The HRESIMS data showed an
[M − H]− ion at m/z 393.1911, and together with the 13C NMR data, indicated a molecular
1
13
formula of C21H30O7, which is identical to that of 6b. Analysis of the H, C NMR,
1H-1H COSY, HMQC and HMBC spectra of 12b in methanol-d4 established that 12b and
6b possessed the same planar structure, whereas the 13C NMR chemical shifts of C-5 and
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C-1′′ were different between 12b (δ 85.1 (C-5) and δ 36.7 (C-1′′)) and 6b (δ 81.6 (C-5)
and δ 31.3 (C-1′′))21 (Table 1). Furthermore, the NOE observed between H-2′ and H-1′′ in
6b21 was not observed in 12b. From these results, 12b was determined to be the C-5
epimer of 6b, and named 4′-hydroxyallo-cis-n-humulinone, which has not been
previously reported (Figure 5).
Compound 13b was obtained as a pale yellow oil. The HRESIMS data showed an
[M − H]− ion at m/z 281.1390, and together with the 13C NMR data, indicated a molecular
1
13
formula of C15H22O5 (12b − C6H8O2). Analysis of the H, C NMR, 1H-1H COSY,
HMQC, and HMBC spectra of 13b in methanol-d4 established that the
trans-4-hydroxy-4-methyl-2-pentenosyl side chain attached at C-4 in 12b was absent in
13b (Table 1). Thus, the planar structure of 13b was determined as oxy-n-humulinic acid
(Figure 5). To determine relative configurations at C-4 and C-5 in 13b, we prepared cis-
and trans-oxy-n-humulinic acids (epimers at C-5) by alkaline hydrolysis of
n-humulinone (5b)23 as a mixture, and then isolated each isomer using ODS preparative
HPLC. By 1H and 13C NMR analysis (Table 1), one of the two isomeric compounds was
identified to be 13b and thus the other (14b) was determined to be the C-5 epimer of 13b.
In the NOESY experiments on 13b and 14b, the NOE between H-4 and H-1′′ (the prenyl
CH2 attached at C-5) was clearly observed in 14b, but was barely detected in 13b (Figure
S1). Furthermore, J-resolved HMBC experiments24 on 13b and 14b showed that the
13
absolute value of the two-bond C-1H coupling constant between H-4 and C-5 was
2
greater in 13b than in 14b (2J(C5, 4H) = 5.9 Hz in 13b, J(C5, 4H) ≦ 3.0 Hz in 14b)
(Figure S2), indicating a larger dihedral angle between H-4 and 5-OH in 14b than 13b.25
These observations clearly indicated that H-4 and 5-OH were in the α-orientation in 13b,
and that H-4 and the prenyl group attached at C-5 were in the α-orientation in 14b. Thus,
13b was determined as cis-oxy-n-humulinic acid and 14b was determined as
trans-oxy-n-humulinic acid (Figure 5). Although the structures of 13b and 14b were
reported about 50 years ago,23 we present their NMR data for the first time (Table 1).
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Compound 12a was obtained as a pale yellow oil. The HRESIMS data showed an
[M − H]− ion at m/z 379.1753, and together with the 13C NMR data, indicated a molecular
formula of C20H28O7, which was the same as for 6a. Analysis of the 1H, 13C NMR, 1H-1H
COSY, HMQC, and HMBC spectra established that 12a possessed an isobutyryl side
chain at C-2 instead of an isovaleryl side chain in 12b (Table 1). Thus, 12a was identified
as 4′-hydroxyallo-cis-cohumulinone, which has not been previously reported (Figure 5).
Compound 13a was obtained as a white, amorphous solid. The HRESIMS data
showed an [M − H]− ion at m/z 267.1230, and together with the 13C NMR data, indicated
a molecular formula of C14H20O5 (12a − C6H8O2). Analysis of the 1H, 13C NMR, 1H-1H
COSY, HMQC, and HMBC spectra established that 13a also possessed an isobutyryl side
chain at C-2 instead of an isovaleryl side chain in 13b (Table 1). Thus, 13a was identified
as cis-oxycohumulinic acid, which has not been previously reported (Figure 5).
Compounds 12a, 12b, 13a and 13b were determined to be racemates because none
of these compounds showed optical activity. Moreover 12a, 12b, 13a and 13b were
formed via a simple thermal reaction in aqueous solution from the precursors 6a and 6b,
which do exist as racemates.21
Analyses of the Transformation Products of 4′-hydroxyalloadhumulinone
(6c/c′). Adhumulinone, a precursor of 4′-hydroxyalloadhumulinone, is a mixture of two
diastereomers (5c and 5c′) (Figure 6).21 Thus, 4′-hydroxyalloadhumulinone in hard resin
is also a mixture of two diastereomers (6c and 6c′) (Figure 6). Although ODS HPLC
analysis using an acidic buffer could not separate the diastereomers (6c and 6c′) (Figure
4C1 and C2), we successfully separated them through ODS HPLC analysis using a basic
buffer (Figure 7). By employing a mobile phase containing the basic buffer, we isolated
each diastereomer successively, and named the former eluate in analytical HPLC (tR 16.6
min) as 4′-hydroxyalloadhumulinone A (6c) and the latter eluate (tR 17.5 min) as
4′-hydroxyalloadhumulinone B (6c′) (Figure 7). The 1H and 13C NMR data of 6c and 6c′
were almost identical (Table 2), and we could not analyze the respective
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stereochemistries of 6c and 6c′.
The yield of isolated 6c and 6c′ was too low to enable the separation of their
transformation products in the boiling tests and perform structural determination by NMR
analyses. Thus, we tentatively identified 12c (main transformed product of 6c) as
4′-hydroxyallo-cis-adhumulinone A and 13c (minor transformed product of 6c) as
cis-oxyadhumulinic acid A, by comparison with the transformation products from
4′-hydroxyallocohumulinone (6a) and 4′-hydroxyallo-n-humulinone (6b) (Figure 4C3).
Similarly, 12c′ and 13c′ (transformed products of 6c′) were tentatively identified as
4′-hydroxyallo-cis-adhumulinone B and cis-oxyadhumulinic acid B, respectively (Figure
4C4).
The HPLC analysis of isolated 6c demonstrated that it did not contain 6c′ (Figure
7B). Moreover, HPLC analysis of isolated 6c′ also showed that it did not contain 6c
(Figure 7C). However, the boiling test on 6c afforded a small amount of 12c′ and 13c′ in
the HPLC analysis (Figure 4C3). Asmall quantity of 12c and 13c was also obtained in the
boiling test of 6c′ (Figure 4C4). These results suggested that the configuration at C-2′′′ in
6c, 6c′, 12c, 12c′, 13c and 13c′ may convert through proton exchange of H-2′′′ during the
boiling process. Figure 8 shows a plausible thermally induced epimerization of 12c into
the enantiomer of 12c′ and of 12c′ into the enantiomer of 12c.
Quantitative Analyses of 4′-Hydroxyallohumulinones and Their
Transformation Products during the Boiling Process. We aimed to quantitatively
evaluate the changes in 4′-hydroxyallohumulinones and their transformation products
during boiling. Thus, boiling solutions of 6a, 6b, and 6c/c′ were sampled at 30, 60, 90 and
120 min and analyzed by HPLC. Figure 9 shows the time dependent decrease in
4′-hydroxyallohumulinones, and compositional changes in the transformation products.
The degree of conversion of 4′-hydroxyallo-cis-humulinones (12a, 12b, and 12c/c′)
increased until around 60 min and then slowly decreased thereafter. By contrast, the
amount of cis-oxyhumulinic acids (13a, 13b, and 13c/c′) increased in inverse proportion
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to the decrease in the amount of 4′-hydroxyallo-cis-humulinones.
These results suggested that during boiling, 4′-hydroxyallohumulinones (6a-c, c′)
primarily isomerized into 4′-hydroxyallo-cis-humulinones (12a-c, c′), which then
decomposed to cis-oxyhumulinic acids (13a-c, c′). The total amount of generated
4′-hydroxyallo-cis-humulinones and cis-oxyhumulinic acids accounted for 40 to 50% of
the decomposed 4′-hydroxyallohumulinones. Our analysis indicates that under these
experimental conditions about 10-15% of ad-congener substrates (6c and 6c′) are
epimerized through proton exchange at C-2′′′ (calculated from the ratio of 12c and 12c′
generated from 6c (Figure 9C) and of 12c′ and 12c generated from 6c′ (Figure 9D)). This
is the first study to report time-dependent changes of α-acid-derived oxidation products
in the hard resin during boiling. We believe these findings will be extremely valuable
when evaluating effects of the hard resin generated through the oxidation of hops on beer
properties.
In conclusion, we have clarified the main constituents, other than α-acids and
β-acids, of the soft and hard resins from stored hops. Furthermore, we investigated the
compositional changes in the hard resin through wort boiling using model experiments,
and found 4′-hydroxyallohumulinones (main constituents of the hard resin) were
transformed to 4′-hydroxyallo-cis-humulinones and cis-oxyhumulinic acids. We believe
our results will help to systematically evaluate the hard resin in order to optimize
the effects of the hard resin on beer quality.
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ACKNOWLEDGEMENT
We thank members of our research groups for valuable discussions, especially
Mikio Katayama and Yasuji Kawachi.
Supporting Information Available: The NOESY spectra and J-HMBC spectra of
compounds 13b and 14b. This material is available free of charge via the Internet at
http://pubs.acs.org.
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REFERENCES
(1) CURRENT REVIEW. J. Inst. Brew. 1957, 63, 281-293.
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
(2) Krauss, G.; Cook, A. H.; Verzele, M. Recommendations concerning nomenclature of
hop resin components. J. Inst. Brew., London 1969, 75, 340-342.
(3) Methods: 7.5 Bitter substances in hops and hop products: Lead conductance value and
total resin, soft resin and hard resin, In Analytica-EBC, European Brewery Convention,
Carl Getränke-Fachverlag: Nürnberg, Germany, 2000.
(4) Palamand, S. R.; Aldenhoff, J. M. Bitter tasting compounds of beer. Chemistry and
taste properties of some hop resin compounds. J. Agr. Food Chem. 1973, 21, 535-543.
(5) Urban, J.; Dahlberg, C. J.; Carroll, B. J.; Kaminsky, W. Absolute Configuration of
Beer's Bitter Compounds. Angew. Chem., Int. Ed. 2013, 52, 1553-1555.
(6) Kowaka, M.; Kokubo, E. Composition of bitter substances of hops and characteristics
of beer bitterness. J. Am. Soc. Brew. Chem. 1977, 35, 16-21.
(7) De Keukeleire, D. Fundamentals of beer and hop chemistry. Quim. Nova 2000, 23,
108-112.
(8) Bishop, L. R.; Whitear, A. L.; Inman, W. R. Scientific basis for beer foam formation
and cling. J. Inst. Brew., London 1974, 80, 68-80.
(9) Haseleu, G.; Intelmann, D.; Hofmann, T. Structure determination and sensory
evaluation of novel bitter compounds formed from β-acids of hop (Humulus lupulus L.)
upon wort boiling. Food Chem. 2009, 116, 71-81.
(10) Haseleu, G.; Intelmann, D.; Hofmann, T. Identification and RP-HPLC-ESI-MS/MS
Quantitation of Bitter-Tasting β-Acid Transformation Products in Beer. J. Agric. Food
Chem. 2009, 57, 7480-7489.
(11) Almaguer, C.; Gastl, M.; Arendt, E. K.; Becker, T. Contributions of hop hard resins
to beer quality. BrewingSci. - Monatsschr. Brauwiss. 2012, 65, 118-129.
(12) Burton, J. S.; Stevens, R. Evaluation of hops. XI. The hard resin and presence of
hulupinic acid. J. Inst. Brew. 1965, 71, 51-56.
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493
(13) Laws, D. R. J. Hops resins and beer flavor. V. The significance of oxidized hop resins
in brewing. J. Inst. Brew. 1968, 74, 178-182.
(14) Ono, M.; Kakudo, Y.; Yamamoto, R.; Nagami, K.; Kumada, J. Simultaneous analysis
of hop bittering components by high-performance liquid chromatography. II. Evaluation
of hop deterioration. J. Am. Soc. Brew. Chem. 1987, 45, 61-69.
(15) Foster, R. T., II; Weber, K.; Jangaard, N. O. The effect of hop acids transformation on
kettle utilization and finished beer flavor and aroma. Tech. Q. - Master Brew. Assoc. Am.
1981, 18, 109-115.
(16) Aitken, R. A.; Bruce, A.; Harris, J. O.; Seaton, J. C. The bitterness of hop-derived
materials in beer. J. Inst. Brew., London 1970, 76, 29-36.
(17) Whitear, A. L. Changes in resin composition and brewing behavior of hops during
storage. J. Inst. Brew. 1966, 72, 177-183.
(18) Howard, G. A.; Martin, P. A. Bittering power of stored hops. J. Inst. Brew. 1964, 70,
424-439.
(19) Rigby, F. L. The practical significance of recent developments in the chemical
analysis of hops. The Brewers Digest 1958, 50-59.
(20) Stevens, J. F.; Taylor, A. W.; Clawson, J. E.; Deinzer, M. L. Fate of Xanthohumol and
Related Prenylflavonoids from Hops to Beer. J. Agric. Food Chem. 1999, 47, 2421-2428.
(21) Taniguchi, Y.; Matsukura, Y.; Ozaki, H.; Nishimura, K.; Shindo, K. Identification
and quantification of the oxidation products derived from α-acids and β-acids during
storage of hops (Humulus lupulus L.). J. Agric. Food Chem. 2013, 61, 3121-3130.
(22) Taniguchi, Y.; Taniguchi, H.; Matsukura, Y.; Kawachi, Y.; Shindo, K. Structural
Elucidation of Humulone Autoxidation Products and Analysis of Their Occurrence in
Stored Hops. J. Nat. Prod. 2014, 77, 1252-1261.
(23) Dierckens, J.; Verzele, M. Oxidation products of humulone and their
stereoisomerism. J. Inst. Brew., London 1969, 75, 453-456.
(24) Furihata, K.; Seto, H. J-Resolved HMBC, a new NMR technique for measuring
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heteronuclear long-range coupling constants. Tetrahedron Lett. 1999, 40, 6271-6275.
(25) Morvai, M.; Nagy, T.; Kocsis, A.; Szabo, L. F.; Podanyi, B. Effect of oxygen
substituents on two- and three-bond carbon-proton spin-spin coupling constants. Magn.
Reson. Chem. 2000, 38, 343-359.
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FIGURE CAPTIONS
Figure 1. Structures of α-acids: cohumulone (1a), n-humulone (1b), adhumulone (1c)
and β-acids: colupulone (2a), n-lupulone (2b), adlupulone (2c) and cis-iso-α-acids:
cis-isocohumulone (3a), cis-iso-n-humulone (3b), cis-isoadhumulone (3c) and
trans-iso-α-acids:
trans-isocohumulone
(4a),
trans-iso-n-humulone
(4b),
trans-isoadhumulone (4c) and humulinones: cohumulinone (5a), n-humulinone (5b),
adhumulinone (5c) and 4′-hydroxyallohumulinones: 4′-hydroxyallocohumulinone (6a),
4′-hydroxyallo-n-humulinone
tricyclooxyisohumulones
tricyclooxyiso-n-humulone
tricyclooxyisohumulones
(6b),
A:
4′-hydroxyalloadhumulinone
(6c)
and
tricyclooxyisocohumulone
A
(7a),
A
(7b), tricyclooxyisoadhumulone
A
(7c) and
(8a),
B:
tricyclooxyisocohumulone
B
tricyclooxyiso-n-humulone B (8b), tricyclooxyisoadhumulone B (8c) and hulupones:
cohulupone (9a), n-hulupone (9b), adhulupone (9c) and xanthohumol (10) and
isoxanthohumol (11).
Figure 2. HPLC chromatograms of the MeOH extract of hops stored at 60 °C for 48 h
(A), the soft resin fraction prepared from the MeOH extract (B), and the hard resin
fraction prepared from the MeOH extract (C). Structures of the compounds are given in
Figures 1 and 6.
Figure 3. HPLC chromatograms of the hard resin (A) before and (B) after boiling in an
aqueous buffer (pH 5.5) for 90 min. Structures of the compounds are given in Figures 1,
5, and 6.
Figure 4. HPLC chromatograms of the isolated compounds before and after boiling in an
aqueous buffer (pH 5.5) for 90 min. 4′-Hydroxyallocohumulinone (6a) (A1) before and
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553
(A2) after boiling; 4′-hydroxyallo-n-humulinone (6b) (B1) before and (B2) after boiling;
4′-hydroxyalloadhumulinones A (6c) and B (6c′) (C1 and C2, respectively) before and
(C3 and C4, respectively) after boiling.
Figure 5. Chemical structures of 4′-hydroxyallo-cis-cohumulinone (12a),
4′-hydroxyallo-cis-n-humulinone
(12b),
cis-oxycohumulinic
acid
(13a),
cis-oxy-n-humulinic acid (13b), and trans-oxy-n-humulinic acid (14b).
Figure 6. Chemical structures of adhumulinone diastereomers (5c and 5c′) and
4′-hydroxyalloadhumulinones A and B (6c and 6c′).
Figure 7. HPLC chromatograms of 4′-hydroxyalloadhumulinones A and B (6c and 6c′,
respectively) in the mobile phase using a basic aqueous buffer before (A) and after (B and
C) their isolation.
Figure 8. Proposed epimerization of 4′-hydroxyallo-cis-adhumulinones Aand B (12c and
12c′) through the proton exchange at C-2′′′ as a result of boiling the sample.
Figure 9. Concentration changes in 4′-hydroxyallocohumulinone (6a),
4′-hydroxyallo-cis-cohumulinone (12a), and cis-oxycohumulinic acid (13a) (A) and in
4′-hydroxyallo-n-humulinone (6b), 4′-hydroxyallo-cis-n-humulinone (12b), and
cis-oxy-n-humulinic acid (13b) (B) and in 4′-hydroxyalloadhumulinone A (6c),
4′-hydroxyallo-cis-adhumulinones A and B (12c and c′), and cis-oxyadhumulinic acids A
and B (13c and c′) (C) and in 4′-hydroxyalloadhumulinone B (6c′),
4′-hydroxyallo-cis-adhumulinones A and B (12c and c′), and cis-oxyadhumulinic acids A
and B (13c and c′) (D) during boiling in an aqueous buffer (pH 5.5).
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Table 1. NMR Spectroscopic Data (400 MHz, Methanol-d4) for Compounds 12a, 12b, 13a, 13b, and 14ba
12a
12b
13a
13b
14b
pos.
δC, type
δH, mult. (J in Hz)
δC, type
δH, mult. (J in Hz)
δC, type
δH, mult. (J in Hz)
δC, type
δH, mult. (J in Hz)
δC, type
δH, mult. (J in Hz)
1
2
200.9, C
113.3, C
195.4, C
92.0, C
-
201.6, C
114.1, C
196.0, C
92.3, C
-
199.6, C
-
200.2, C
-
200.5, C
-
-
-
111.6, C
-
112.6, C
-
112.8, C
-
3
-
-
197.8, C
-
198.3, C
-
199.8, C
-
4
-
-
80.6, CH
4.35, s
80.9, CH
4.35, s
74.2, CH
4.14, s
5
84.9, C
-
85.1, C
-
82.2, C
-
-
-
-
-
-
-
82.4, C
-
-
-
-
-
-
-
77.7, C
-
-
-
-
-
-
-
1'
2'
3'
4'
5'
6'
198.2, C
121.9, CH
155.9, CH
71.4, C
-
198.3, C
121.9, CH
156.0, CH
71.4, C
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6.78, d (15.5)
6.92, d (15.5)
-
6.79, d (15.5)
6.92, d (15.5)
-
29.1, CH3
29.1, CH3
1.27, s
29.1, CH3
29.1, CH3
1.28, s
1.29, s
1.29, s
2.63, dd (13.9, 8.7)
2.48, dd (13.9, 6.6)
2.62, dd (14.0, 8.5)
2.45, dd (14.0, 6.7)
2.61, dd (13.5, 9.2)
2.45, dd (13.5, 6.4)
2.60, dd (13.6, 9.1)
2.43, dd (13.6, 6.4)
2.54, dd (14.0, 7.7)
2.48, dd (14.0, 6.9)
1'' 36.79, CH2
36.7, CH2
34.8, CH2
34.8, CH2
34.6, CH2
2''
3''
4''
5''
1'''
119.1, CH
136.6, C
5.10, m
119.0, CH
136.6, C
5.10, m
119.3, CH
136.5, C
5.03, m
119.2, CH
136.4, C
5.02, m
118.3, CH
137.6, C
5.03, m
-
-
-
-
-
17.9, CH3
26.2, CH3
203.5, C
1.52, s
1.60, s
-
17.9, CH3
26.2, CH3
197.8, C
1.53, s
1.61, s
-
17.8, CH3
26.1, CH3
205.2, C
1.52, s
1.58, s
-
17.9, CH3
26.1, CH3
199.9, C
1.52, s
1.58, s
-
18.1, CH3
26.1, CH3
201.9, C
1.65, s
1.68, s
-
2.74, dd (13.6, 7.2)
2.69, dd (13.6, 7.2)
2.77, dd (13.8, 7.0)
2.64, dd (13.8, 7.1)
2.78, dd (14.5, 7.0)
2.73, dd (14.5, 6.9)
2'''
36.75, CH
3.50, sep (6.9)
46.6, CH2
37.3, CH
3.49, sep (6.9)
47.7, CH2
48.4, CH2
3'''
4'''
5'''
18.0, CH3
18.6, CH3
-
1.10, d (6.9)
1.14, d (6.9)
-
27.6, CH
22.86, CH3
22.94, CH3
2.11, m
17.8, CH3
18.7, CH3
-
1.09, d (6.9)
1.12, d (6.9)
-
27.1, CH
22.8, CH3
22.9, CH3
2.11, m
26.7, CH
22.8, CH3
22.9, CH3
2.13, m
0.95, d (6.7)
0.97, d (6.7)
0.94, d (6.2)
0.96, d (6.2)
0.96, d (6.7)
0.96, d (6.7)
a Arbitrary numbering according to structures 12a, 12b, 13a, 13b and 14b shown in Figure 5.
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Table 2. NMR Spectroscopic Data (400 MHz, Methanol-d4) for Compounds 6c and
6c′a
6c
6c′
pos.
δC, type
201.8, C
111.3, C
198.9, C
90.1, C
δH, mult. (J in Hz)
δC, type
201.6, C
111.3, C
199.0, C
89.9, C
δH, mult. (J in Hz)
-
-
1
2
3
-
-
-
-
-
-
4
81.6, C
-
81.6, C
-
5
200.0, C
121.4, CH
155.8, CH
71.4, C
-
200.0, C
121.4, CH
155.8, CH
71.4, C
-
1'
2'
3'
4'
5'
6'
7.03, d (15.7)
6.87, d (15.7)
-
7.04, d (15.7)
6.87, d (15.7)
-
29.3, CH3
29.4, CH3
1.31, s
29.3, CH3
29.4, CH3
1.31, s
1.31, s
1.31, s
2.47, dd (14.8, 4.7)
2.42, dd (14.8, 8.9)
2.47, dd (15.1, 5.5)
2.41, dd (15.1, 8.5)
31.0, CH2
31.1, CH2
1''
118.9, CH
135.5, C
5.30, m
118.9, CH
135.5, C
5.30, m
2''
3''
4''
5''
1'''
-
-
18.1, CH3
26.1, CH3
204.1, C
1.41, s
1.62, s
-
18.1, CH3
26.1, CH3
204.2, C
1.41, s
1.62, s
-
42.6, CH
16.0, CH3
27.3, CH2
11.9, CH3
3.46, ddq (6.8, 6.8, 6.8)
42.7, CH
15.9, CH3
27.3, CH2
12.0, CH3
3.44, ddq (6.8, 6.8, 6.8)
2'''
3'''
4'''
5'''
1.13, d (6.8)
1.09, d (6.8)
1.64-1.74, m
1.36-1.46, m
1.70-1.80, m
1.40-1.50, m
0.87, dd (7.4, 7.4)
0.92, dd (7.4, 7.4)
a Arbitrary numbering according to structures 6c and 6c′ shown in Figure 6.
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Journal of Agricultural and Food Chemistry
Figure 1
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Figure 2
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Journal of Agricultural and Food Chemistry
Figure 3
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