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Research Laboratories for Health Science & Food Technologies, Research & Development Division, KIRIN Company, Ltd., 1-13-5,
Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan
Department of Food and Nutrition, Japan Womens University, 2-8-1, Mejirodai Bunkyo-ku, Tokyo 112-8681, Japan
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ABSTRACT: The transformation of -acids [in hops (Humulus lupulus L.)] to iso--acids (in beer) during the brewing process
is well known, but the occurrence and structure of the oxidized -acids during hop storage are not well documented. Because an
understanding of these oxidized compounds is essential to optimize the eects of oxidized hops on the quality of beer, we
investigated the autoxidation products of humulone (a representative congener of -acids) using a simplied autoxidation model.
Among the oxidation products, tricyclooxyisohumulones A (1) and B (2), tricycloperoxyisohumulone A (3),
deisopropyltricycloisohumulone (4), and the hemiacetal 5 of tricycloperoxyhumulone A (5) were isolated, and their structures
were elucidated for the rst time. The occurrence of compounds 14 in stored hops was veried using LC/MS/MS analysis. We
also monitored the levels of compounds 14 during hop storage using LC/MS/MS analysis.
Article
Figure 1. Structures of the -acids (cohumulone, humulone, and adhumulone), cis/trans-iso--acids (cis/trans-isocohumulone, cis/transisohumulone, and cis/trans-isoadhumulone), humulinones (cohumulinone, humulinone, and adhumulinone), and 4-hydroxyallohumulinones (4hydroxyallocohumulinone, 4-hydroxyallohumulinone, and 4-hydroxyalloadhumulinone).
Chart 1
Article
Figure 2. HPLC chromatograms of humulone (A) before and (B) after autoxidation in n-hexane for 144 h at 20 C.
Article
C, type
208.3,
111.9,
196.7,
86.0,
213.3,
36.2,
C
C
C
C
C
CH2
5a
6
6-Me
6-Me
7
56.3,
46.3,
29.5,
17.5,
61.7,
CH
C
CH3
CH3
CH
30.7, CH2
8a
1
2
3
1
2
3
4
5
59.4,
73.3,
30.6,
30.3,
201.5,
48.6,
C
C
CH3
CH3
C
CH2
26.6, CH
23.0, CH3
22.9, CH3
H, mult. (J in Hz)
2.42, dd (18.0,
9.2)
2.22, d (18.0)
2.49, d (9.2)
1.16, s
0.72, s
2.09, m
2.34, dd (13.0,
13.0)
1.98, dd (13.0,
7.2)
1.27, s
1.31, s
2.76, dd (14.6,
6.7)
2.72, dd (14.6,
7.0)
2.11, m
0.94, d (6.6)
0.94, d (6.6)
2
C, type
208.0,
111.7,
197.8,
86.5,
215.7,
37.7,
C
C
C
C
C
CH2
56.1,
46.2,
26.8,
27.6,
60.2,
CH
C
CH3
CH3
CH
30.3, CH2
3
H, mult. (J in
Hz)
C, type
207.8,
111.9,
196.1,
85.9,
212.1,
36.3,
C
C
C
C
C
CH2
1.24, s
0.92, s
1.65, m
56.1,
46.1,
29.2,
18.0,
58.9,
CH
C
CH3
CH3
CH
2.23, m
30.5, CH2
2.42, dd (17.5,
9.6)
2.24, d (17.5)
2.53, d (9.6)
2.11, m
62.0,
74.0,
31.3,
30.7,
200.0,
50.3,
C
C
CH3
CH3
C
CH2
C, type
207.8,
111.7,
196.2,
86.1,
212.2,
36.7,
C
C
C
C
C
CH2
1.16, s
0.71, s
2.32, m
54.4,
44.4,
28.2,
21.8,
43.7,
CH
C
CH3
CH3
CH2
2.32, m
27.9, CH2
2.46, dd (18.0,
9.2)
2.25, d (18.0)
2.52, d (9.2)
2.01, m
59.2,
85.0,
25.3,
24.7,
202.0,
48.1,
1.30, s
1.31, s
2.69, m
C
C
CH3
CH3
C
CH2
2.67, m
26.7, CH
23.1, CH3
23.1, CH3
4
H, mult. (J in
Hz)
2.08, m
0.90, d (6.7)
0.90, d (6.7)
26.5, CH
22.9, CH3
22.8, CH3
H, mult. (J in Hz)
2.48, m
2.27, m
2.48, m
1.09,
0.72,
1.80,
1.65,
2.17,
s
s
ddd (11.8, 11.8, 8.1)
ddd (11.8, 7.3, 2.3)
ddd (13.7, 11.8, 7.3)
1.30, s
1.32, s
2.79, dd (14.9,
7.1)
2.73, dd (14.9,
7.0)
2.13, m
0.95, d (6.6)
0.96, d (6.6)
202.0, C
48.2, CH2
26.5, CH
22.9, CH3
22.8, CH3
2.13, m
0.95, d (6.6)
0.96, d (6.6)
Article
Article
Figure 7. HPLC chromatograms of the isolated compound 3 (A) before and (B) after storage in MeOH for 3 days at ambient temperature.
Article
Figure 8. HPLC chromatograms of the isolated compound 5 (A) before and (B) after storage in EtOH for 24 h at ambient temperature.
C, type
1
2
68.3, C
27.4, CH2
3
4
4-Me
4-Me
5
6
60.8,
48.3,
39.8,
25.7,
55.7,
34.1,
CH
C
CH3
CH3
CH
CH2
86.4,
196.4,
109.5,
204.2,
108.0,
88.2,
29.4,
26.4,
202.2,
46.9,
C
C
C
C
C
C
CH3
CH3
C
CH2
7
8
9
10
11
1
2
3
1
2
3
4
5
27.8, CH
22.9, CH3
23.1, CH3
H, mult. (J in Hz)
2.75, dd (13.9, 8.0)
2.37, d (13.9)
1.98, d (8.0)
1.17,
1.35,
1.94,
2.18,
1.74,
s
s
dd (10.5, 5.1)
dd (13.2, 5.1)
dd (13.2, 10.5)
1.13, s
1.55, s
2.95,
2.89,
2.10,
0.97,
0.98,
dd (13.1, 6.6)
dd (13.1, 7.1)
m
d (6.4)
d (6.4)
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Article
Figure 9. Concentration changes in humulone, TCOIH A (1) and B (2), TCPOIH A (3), and DeITCIH (4) in hops during storage at 40 C over
40 weeks.
1.3). Data processing was performed using TopSpin-NMR software
(version 3.0) (Bruker BioSpin, Rheinstetten, Germany). HRESIMS of
the puried compounds were measured using a Thermo Scientic
LTQ Orbitrap mass spectrometer (Thermo Fisher Scientic, San Jose,
CA, USA). Before measuring the samples, accurate mass calibration
was performed using a polytyrosine-1, 3, 6 calibrant (CS Bio
Company, Menlo Park, CA, USA). Preparative HPLC was conducted
on a Gilson model HPLC system equipped with a 321 pump and a
171 diode array detector (Gilson, Middleton, WI, USA) and on a
Shimadzu Prominence HPLC system (Shimadzu, Kyoto, Japan).
Analytical HPLC was conducted on a Shimadzu Prominence UFLC
system, and LC/MS/MS was conducted on a 4000 Q-Trap mass
spectrometer (AB Sciex, Tokyo, Japan) connected to a Shimadzu
Prominence UFLC system.
Hop Material. Hop pellets (cultivar Hallertau Perle, crop 2009)
were purchased from HopSteiner (Mainburg, Germany) and stored in
a freezer at 20 C prior to use.
Autoxidation of Humulone and Isolation of Compounds 1
5. Humulone was prepared according to a previously reported
method.14 Humulone (8.10 g) was dissolved in n-hexane (810 mL)
and stored at 20 C for 144 h. The supernatant was collected by
decantation and concentrated using a rotary evaporator to aord a
yellow oil (3.70 g). A portion of this product (1.78 g) was repeatedly
subjected to preparative HPLC (column: 150 22 mm i.d., 5 m,
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 75% B in 0 30 min, 75 to 90% B in 30 30.1
min, and 90% B for 30.1 37 min; ow rate: 18.8 mL/min; detector:
270 nm; column temperature: RT), yielding 49 fractions. Fraction 18
(tR 15.215.6 min) and fraction 39 (tR 24.5 25.3 min) contained
compound 3 (140 mg) and compound 5 (330 mg), respectively.
Fraction 15 (tR 13.613.8 min) and fraction 32 (tR 20.620.8 min)
were further puried by a second preparative HPLC (column: 50 20
mm i.d., 5 m, 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; ow rate: 9.5 mL/min;
detector: 270 nm; column temperature: 40 C). Fraction 15-1 (tR 3.8
4.7 min) and fraction 15-2 (tR 5.57.5 min) contained compound 1
(18.9 mg) and compound 2 (4.4 mg), respectively. Fraction 32-1 (tR
6.06.9 min) contained compound 4 (11.0 mg).
EXPERIMENTAL SECTION
Article
ASSOCIATED CONTENT
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AUTHOR INFORMATION
Corresponding Author
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of H. Ozaki.
We also thank members of our research groups for valuable
discussions.
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