Structural identification and distribution of proanthocyanidins in 13 different hops

Article abstract of DOI:10.1021/jf060395r

Ten newly isolated hop proanthocyanidin oligomers and flavan-3-ol monomers from 13 different hops have been identified as gallocatechin, gallocatechin-(4α→8)-catechin, gallocatechin-(4α→6)- catechin, catechin-(4α→8)-gallocatechin, catechin-(4α→6)- gallocatechin, afzelechin-(4α→8)-catechin, catechin-(4α→8) -catechin-(4α→8)-catechin, epicatechin-(4β→8)-epicatechin- (4β→8)-catechin, catechin-(4α→8)-gallocatechin- (4α→8)-catechin, and gallocatechin-(4α→8)-gallocatechin- (4α→8)-catechin, together with seven previously isolated oligomers, namely, catechin, epicatechin, epicatechin-(4β→8)-catechin, epicatechin-(4β→8)-epicatechin, catechin-(4α→8)-catechin, catechin-(4α→8)-epicatechin, and epicatechin-(4β→8)- catechin-(4α→8)-catechin. These compounds were subjected to acid-catalyzed degradation in the presence of phloroglucinol or by partial or complete acid-catalyzed degradation and reaction with benzyl mercaptan followed by desulfurization. The resultant adducts when compared to authentic samples by high-performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry served to identify the precursors. The composition of proanthocyanidins from 13 different hops was similar, but the concentration of individual compounds showed some differences, which indicated that hop proanthocyanidin profiles are affected by geographic origin and are variable depending on the cultivars.

Full text of DOI:10.1021/jf060395r

4048 J. Agric. Food Chem. 2006, 54, 4048−4056
Structural Identification and Distribution of Proanthocyanidins
in 13 Different Hops
HUI-JING LI AND MAX L. DEINZER*
Department of Chemistry, Oregon State University, Corvallis, Oregon 97331
Ten newly isolated hop proanthocyanidin oligomers and flavan-3-ol monomers from 13 different hops
have been identified as gallocatechin, gallocatechin-(4Rf8)-catechin, gallocatechin-(4Rf6)-catechin,
catechin-(4Rf8)-gallocatechin, catechin-(4Rf6)-gallocatechin, afzelechin-(4Rf8)-catechin, catechin-
(4Rf8)-catechin-(4Rf8)-catechin, epicatechin-(4âf8)-epicatechin-(4âf8)-catechin, catechin-(4Rf8)-
gallocatechin-(4Rf8)-catechin, and gallocatechin-(4Rf8)-gallocatechin-(4Rf8)-catechin, together
with seven previously isolated oligomers, namely, catechin, epicatechin, epicatechin-(4âf8)-catechin,
epicatechin-(4âf8)-epicatechin, catechin-(4Rf8)-catechin, catechin-(4Rf8)-epicatechin, and epi-
catechin-(4âf8)-catechin-(4Rf8)-catechin. These compounds were subjected to acid-catalyzed
degradation in the presence of phloroglucinol or by partial or complete acid-catalyzed degradation
and reaction with benzyl mercaptan followed by desulfurization. The resultant adducts when compared
to authentic samples by high-performance liquid chromatography-atmospheric pressure chemical
ionization tandem mass spectrometry and high-performance liquid chromatography-electrospray
ionization tandem mass spectrometry served to identify the precursors. The composition of
proanthocyanidins from 13 different hops was similar, but the concentration of individual compounds
showed some differences, which indicated that hop proanthocyanidin profiles are affected by
geographic origin and are variable depending on the cultivars.
KEYWORDS: Hops; proanthocyanidins; Humulus lupulus; Cannabinaceae; HPLC/APCI-MS/MS; HPLC/
ESI-MS/MS
INTRODUCTION
ity, understanding the chemistry of this class of polyphenols
has been challenging (9).
Of all the herbs that have been used in beer brewing, only
the hop (Humulus lupulus L., Cannabinaceae) plant is regarded
as an essential raw material in the beer brewing industry (1).
Hops are perennial plants grown on trellises, and different
varieties are derived via breeding programs. The hop plant is
dioecious and cultivated in most temperate zones of the world
for its female inflorescences, commonly referred to as hop cones
or hops. The female flower clusters are partly covered with
lupulin glands, while male flowers have only a few glands in
the crease of their anthers and on their sepals. The resin secreted
by these glands contains bitter acids, essential oils, and
flavonoids (flavonol glycosides, prenylflavonoids, and tannins)
(2). Since hops are used exclusively to give beer its characteristic
aroma, bitterness, foam and light stability, the investigations of
hop oil constituents have continued (2-8). In comparison with
some other constituents, the brewing value of hop proantho-
cyanidins is not well-understood. Proanthocyanidins, also known
as condensed tannins, are flavan-3-ol oligomers and polymers
that give anthocyanidins upon acid depolymerization reactions.
Because of the difficulty of extracting and purifying proantho-
cyanidins, together with their instability and structural complex-
Proanthocyanidins are widely distributed throughout the plant
kingdom. There is a growing body of evidence linking these
compounds with plant defense mechanisms, organoleptic char-
acteristics, and potential health benefits (6-11). Hop proan-
thocyanidins have received special attention in the brewing
industry because they contribute to haze formation (12). They
also stabilize the organoleptic properties and color and contribute
to the astringency and bitterness. The estimated amount of total
hop proanthocyanidins ranges from 0.5 to 5% on a dry weight
basis, depending on the variety, geographic origin, freshness,
and harvesting procedure (6, 7). Previous studies have also
shown that up to 30% of the proanthocyanidins present in beer
is derived from hops. Surprisingly, only a few oligomeric
proanthocyanidins and monomeric flavan-3-ols have been
reported in hops. These include catechin (6), epicatechin (6),
epicatechin-(4âf8)-catechin (procyanidin B1) (13), epicatechin-
(4âf8)-epicatechin (procyanidin B2) (12, 13), catechin-(4âf8)-
catechin (procyanidin B3) (12, 13), and catechin-(4âf8)-
epicatechin (procyanidin B4) (12, 13). Trimeric procyanidins
have also been reported to be present, but their structures were
not elucidated (6). All of the effects of hop proanthocyanidins
on beer values seem to depend on their affinity for proteins.
Since little is known about their structures and composition,
* To whom correspondence should be addressed. Tel.: (541) 737-1773;
fax: (541) 737-4371; e-mail: max.deinzer@oregonstate.edu.
10.1021/jf060395r CCC: $33.50
© 2006 American Chemical Society
Published on Web 04/28/2006

Hop Oil Proanthocyanidins
J. Agric. Food Chem., Vol. 54, No. 11, 2006 4049
at a flow rate of 100 mL/h. Each fraction (500 mL) was collected and
monitored by HPLC-UV and two-dimensional (2-D) TLC on cellulose
plates developed first with t-butyl alcohol/water/acetic acid (3:1:1, v/v/
v), dried, then developed in the second dimension with 6% aqueous
acetic acid, and visualized with a vanillin-HCl reagent. Fractions that
contained mainly oligomeric proanthocyanidins and monomeric flavan-
3-ols were combined and concentrated by rotary evaporation and
lyophilization to yield 4.2 g of crude proanthocyanidins. The crude
proanthocyanidin mixture (4.2 g) was passed through a 45 cm × 4 cm
column of Sephadex LH-20 preequilibrated with water. The LH-20
column was successively eluted with water (1 L), methanol/water (1
L, 1:1, v/v), methanol (1 L), and finally with acetone/water (1 L, 7:3,
v/v) at a flow rate of 100 mL/h. Each fraction (1 L) was collected and
monitored by 2-D TLC and ESI-MS and then concentrated by rotary
evaporation and lyophilized to give fraction 1 (0.5 g) consisting of
glycosides and other materials, fraction 2 (0.4 g) consisting of
monomeric flavan-3-ols and proanthocyanidin dimers, fraction 3 (0.3
g) consisting of proanthocyanidin oligomers, and fraction 4 (3.0 g)
consisting of proanthocyanidin polymers.
Fractions 2 and 3 were further chromatographed on a 30 cm × 1.5
cm column of Toyopearl TSK HW-40 S using methanol as the eluent
at a flow rate of 1 mL/min. Fractions of 10 mL each were collected
and examined by HPLC-UV at 280 nm. The comparatively pure
constituents of the hop oligomeric proanthocyanidins and monomeric
flavan-3-ols were isolated by semipreparative HPLC using a linear
solvent gradient from 5% B (MeOH) to 40% B in A (1% aqueous
formic acid) over 40 min at a flow rate of 4 mL/min. The UV trace
was recorded at 280 nm. Peak fractions identified by mass spectrometry
were collected manually, the solvents were removed by rotary evapora-
tion, and the remainder was lyophilized to dryness and stored at -15
°C.
The chemical structures of hop proanthocyanidins (Figure 1) consist
of 1 (catechin), 2 (epicatechin), 3 (gallocatechin), 4 (epicatechin-
(4âf8)-catechin, procyanidin B1), 5 (epicatechin-(4âf8)-epicatechin,
procyanidin B2), 6 (catechin-(4Rf8)-catechin, procyanidin B3), 7
(catechin-(4Rf8)-epicatechin, procyanidin B4), 8 (gallocatechin-
(4Rf8)-catechin), 9 (gallocatechin-(4Rf6)-catechin), 10 (catechin-
(4Rf8)-gallocatechin), 11 (catechin-(4Rf6)-gallocatechin), 12 (af-
zelechin-(4Rf8)-catechin), 13 (catechin-(4Rf8)-catechin-(4Rf8)-
catechin, C2), 14 (epicatechin-(4âf8)-catechin-(4Rf8)-catechin), 15
(epicatechin-(4âf8)-epicatechin-(4âf8)-catechin), 16 (catechin-
(4Rf8)-gallocatechin-(4Rf8)-catechin), and 17 (gallocatechin-(4Rf8)-
gallocatechin-(4Rf8)-catechin).
Electrospray ionization tandem mass spectrometry (ESI/MS-
MS) was usually performed on a PE Sciex API III triple-quadrupole
mass spectrometer in the positive ion MS mode. Samples diluted to 10
µg/mL were loop-injected into methanol/0.5% aqueous formic acid (2:
1, v/v) flowing at 8 µL/min into the electrospray source. Ionization
voltage was 5 kV, and the orifice was set at 60 V.
their nutritional significance or complexation ability with
proteins is unclear. The possible sensory properties in beer also
have not been studied.
The aim of this work was to isolate and elucidate the
structures of unknown hop proanthocyanidins and to study their
composition and distribution in 13 different hops. Ten proan-
thocyanidin oligomers and flavan-3-ol monomers isolated from
hops are reported here for the first time in addition to seven
that were previously isolated and identified.
MATERIALS AND METHODS
Materials. The 13 different hops chosen for study include Willamette
hop cones (Oregon, Idaho, and Washington, USA), Vanguard pellet
(USA), Palisade pellet (USA), Tettnang-Hallertauer pellet (Germany),
Hallertauer-Hallertauer pellet (Germany), North American Hallertauer
pellet (USA), Zeus pellet (USA), Cascade pellet (USA), Saaz 36 pellet
(USA), Saaz 72 pellet (USA), and Glacier pellet (USA). Hop pellets
are made from dried hop cones by milling in a hammer mill and then
compressing the hop powder through a 6 mm die to form pellets of
about 10-25 mm in length. The chemistry, content, and brewing value
of these pellets were not changed in any way other than a slight loss
of moisture content. The hops were all commercial samples, and all
commercial hops are female. They were harvested at maturity in 2004
except for the Washington-Willamette hops, which were harvested in
2003.
(+)-Catechin, (-)-epicatechin, (+)-gallocatechin, and (-)-epigal-
locatechin were purchased from Sigma-Aldrich (Milwaukee, WI), and
(+)-afzelechin and (-)-epiafzelechin were gifts kindly provided by Prof.
Tak H. Chan of The Hong Kong Polytechnic University; (+)-taxifolin
for the synthesis of catechin-(4Rf2)-phloroglucinol was also purchased
from Sigma-Aldrich (Milwaukee, WI; grape seeds were kindly provided
by Dr. James A. Kennedy of Oregon State University that were
extracted to prepare epicatechin-(4âf2)-phloroglucinol and epigallo-
catechin-(4âf2)-phloroglucinol; and black currant leaves were kindly
provided by Mrs. Kim Hummer of the USDA ARS National Clonal
Germplasm Repository for tannin extracts that were used to prepare
gallocatechin-(4Rf2)-phloroglucinol.
Hexane, dichloromethane, acetone, ethanol, and methanol were of
HPLC grade and purchased from Fisher Scientific (Santa Clara, CA).
Glacial acetic acid, formic acid, benzyl mercaptan, phloroglucinol, and
Raney nickel were purchased from Sigma (St. Louis, MO). Sephadex
LH-20 and Toyopearl TSK HW-40S were purchased from Amersham
Pharmacia Biotech (Piscataway, NJ). The Synergi C18 column and Luna
phenyl-hexyl C18 column were purchased from Phenomenex (Torrance,
CA). Water was purified to HPLC grade with a Millipore Milli-Q
apparatus (Bedford, MA). All solvent-water mixtures used for column
chromatography contained 0.1% (v/v) formic acid (Fluka brand, Sigma-
Aldrich, Milwaukee, WI) and were degassed by helium sparging prior
to use. Hydrogen (H2), nitrogen (N2), argon (Ar), helium (He), and
sulfur dioxide (SO2) were all high-purity grade.
Preparation of Proanthocyanidins from Hops. Air-dried hop cones
(100.0 g) were briefly immersed in dichloromethane (CH2Cl2) and
stirred for 1 h, and the extract was decanted. The hop cones were further
washed with CH2Cl2 3 times (1.5 L × 3) to extract the resins, pigments,
and lipids, dried in a stream of air in a fume hood, and then ground
with a Wiley mill (sieve no. 20) to obtain hop granules (72.4 g). The
hop granules (72.4 g) were extracted with 1 L of acetone/water (7:3,
v/v). This extraction step was repeated 3 times with 1 L of acetone/
water (7:3, v/v). The combined acetone/water extracts were separated
from the hop granules by filtration and then concentrated on a rotary
evaporator under vacuum at less than 35 °C to remove the acetone.
The resulting extract was washed with hexane (0.5 L × 2) 2 times and
subsequently with CH2Cl2 (0.5 L × 2) 2 times to remove more pigments
and nonpolar material, rotary evaporated to remove the residual organic
solvents, and then passed through a 30 cm × 4 cm column of Sephadex
LH-20 preequilibrated with water. The LH-20 column was successively
eluted with water (500 mL), methanol/water (500 mL, 1:3, v/v),
methanol/water (500 mL, 1:1, v/v), methanol/water (500 mL, 3:1, v/v),
methanol (500 mL), and finally with acetone/water (500 mL, 7:3, v/v)
Atmospheric pressure chemical ionization tandem mass spec-
trometry (APCI/MS-MS) was also performed on a PE Sciex API
III triple-quadrupole mass spectrometer in the positive ion MS mode,
and the source was equipped with a heated nebulizer interface kept at
480 °C. Samples were introduced into the mass spectrometer by high-
performance liquid chromatography (HPLC). APCI/MS-MS experi-
ments were carried out with argon-10% nitrogen as the target gas at
a thickness of ca. 1.9 × 1014 atoms per cm2 using a collision energy of
20 V. Analytical HPLC separations were performed on a 250 mm ×
4.6 mm Synergi 4 µm Hydro-RP-80A column with a linear gradient
from 5 to 50% methanol in 1% aqueous formic acid over 50 min at
0.8 mL/min (procedure 1), and semipreparative HPLC was run on a
250 mm × 10 mm, 10 µm Econosil C18 column.
Acid-Catalyzed Degradation of Proanthocyanidins in the Pres-
ence of Phloroglucinol. According to the reported procedure (Figure
2) (14), a solution of proanthocyanidin dimer 1 (0.1 mg), phloroglucinol
18 (2 mg), and acetic acid (2 µL) in the solvent mixture of ethanol/
water (100 µL, 1:3, v/v) was sparged with nitrogen, sealed, and heated
to 100 °C for 20 min. The aliquot of the mixture was then diluted
exactly with ethanol/water (1:3, v/v) to reduce the concentration of
the main phloroglucinol adduct 19 below 0.5 mg/L. This sample was
then analyzed directly by analytical HPLC/APCI-MS/MS on a 250

4050 J. Agric. Food Chem., Vol. 54, No. 11, 2006
Li and Deinzer
Figure 1. Chemical structures of hop proanthocyanidins.
phases containing 1% v/v aqueous acetic acid (mobile phase A) and
methanol (mobile phase B) at a flow rate of 0.8 mL/min. Eluting peaks
were monitored at 280 nm; the gradient was maintained at 5% B for
10 min, then from 5 to 20% B over 20 min, and finally from 20 to
40% B over 25 min. The column was then washed with 90% B for 10
min and reequilibrated with 5% B for 5 min before the next injection
(procedure 2).
The authentic sample of catechin-(4Rf2)-phloroglucinol was pre-
pared from commercial (+)-taxifolin according to a previous report
(14). The solution of (+)-taxifolin (20 mg) and sodium borohydride
(10 mg) in absolute ethanol (4 mL) was sparged with nitrogen and
stirred at room temperature for 1 h, and then phloroglucinol (70 mg)
in 4 mL of hydrochloric acid (0.1 N) was added and further stirred for
30 min. The solution was diluted with 4 mL of water, extracted with
ethyl acetate (4 mL × 3), dried over anhydrous sodium sulfate (Na2-
SO4), filtered, and evaporated under reduced pressure at less than 35
°C. The crude product was purified by a semipreparative HPLC
(procedure 2) and lyophilized to obtain 2.2 mg (62% yield) of dry white
powder, which was characterized by 1H NMR (400 MHz, Bruker) and
LC/APCI-MS (API 300, PE Sciex) operated in the positive mode and
was confirmed as catechin-(4Rf2)-phloroglucinol: APCI-MS, m/z 415
[M + H]+; 1H NMR (CD3OD, 400 MHz) δ (ppm): 4.40 (H-2, d, J )
8.8 Hz), 4.45 (H-4, d, J ) 7.6 Hz), 4.61 (H-3, dd, J ) 8.8, 7.6 Hz),
5.84 (phloroglucinol-2H, d, J ) 2.1 Hz), 6.05 (A-ring, 2H, d, J ) 2.4
Hz), 6.68 (B-ring, 1H, dd, J ) 1.5, 7.8 Hz), 6.72 (B-ring, 1H, d, J )
7.8 Hz), 6.79 (B-ring, 1H, d, J ) 1.5 Hz).
Figure 2. Degradation of proanthocyanidins in the presence of phloro-
glucinol.
mm × 4.6 mm i.d, 4 µm Synergi C18 column protected by a guard
The authentic samples of epicatechin-(4âf2)-phloroglucinol and
epigallocatechin-(4âf2)-phloroglucinol were prepared by acid-
column containing the same material, using a binary gradient of mobile

Hop Oil Proanthocyanidins
J. Agric. Food Chem., Vol. 54, No. 11, 2006 4051
Figure 3. Degradation of proanthocyanidins in the presence of benzyl
mercaptan.
catalyzed degradation of grape seeds. The solution of phloroglucinol
(1 g), ascorbic acid (200 mg), and hydrochloric acid (0.1 N) in methanol
(4 mL) was sparged with nitrogen, and the proanthocyanidin mixture
(50 mg) extracted from grape seed was allowed to react in this solution
at 50 °C for 20 min and then was combined with 5 vol of aqueous
sodium acetate (0.04 M) to stop the reaction. The crude product was
purified by semipreparative HPLC (procedure 2) to give two major
products as follows: epicatechin-(4âf2)-phloroglucinol, white amor-
phous solid (3.5 mg); APCI-MS, m/z 415 [M + H]+; 1H NMR (CD3-
OD, 400 MHz) δ (ppm): 3.93 (H-3, dd, J ) 3.3, 0.9 Hz), 4.54 (H-4,
d, J ) 3.3 Hz), 5.07 (H-2, d, J ) 0.9 Hz), 5.94 (phloroglucinol-2H, d,
J ) 2.0 Hz), 6.07(A-ring, 2H, d, J ) 2.3 Hz), 6.70 (B-ring, 1H, dd, J
) 1.8, 8.2 Hz), 6.78 (B-ring, 1H, d, J ) 8.2 Hz), 7.0 (B-ring, 1H, d,
J ) 1.8 Hz). Epigallocatechin-(4âf2)-phloroglucinol, white amorphous
Figure 4. Partial degradation of proanthocyanidins in the presence of
benzyl mercaptan.
1
solid (1.2 mg); APCI-MS, m/z 431 [M + H]+; H NMR (CD3OD,
Partial Acid-Catalyzed Degradation of Hop Proanthocyanidins
in the Presence of Benzyl Mercaptan (17-19). As shown (Figure
4), a solution of hop proanthocyanidin trimer (0.1 mg), benzyl
mercaptan (0.5 µL), sulfur dioxide (SO2, 0.2 µL), and acetic acid (0.3
µL) in a glass capillary tube loaded with ethanol/water (100 µL, 1:1,
v/v) was sparged with nitrogen, sealed, and heated to 60 °C for 0.5-6
h depending on the rate of degradation as detected by analytical HPLC/
APCI-MS/MS. Monomer 1 and dimer 1 were detected by cochro-
matography with authentic samples. The benzyl mercaptan adducts 22
and 23 were isolated by analytical HPLC, collected, lyophilized to
dryness, and then mixed with aqueous Raney nickel (100 µL) in a glass
tube, which was sparged with nitrogen, sealed, shaken several times at
regular intervals over a period of 1 h, and directly analyzed by reverse-
phase HPLC/APCI-MS. The identities of desulfurized products were
established by cochromatography with authentic samples.
400 MHz) δ (ppm): 4.03 (H-3, dd, J ) 2.3, 0.9 Hz), 4.62 (H-4, d, J
) 2.3 Hz), 5.03 (H-2, d, J ) 0.9 Hz), 5.94 (phloroglucinol-2H, d, J )
2.0 Hz), 6.04 (A-ring, 2H, d, J ) 2.3 Hz), 6.50 (B-ring, 2H, s). By the
same method, an authentic sample of gallocatechin-(4Rf2)-phloro-
glucinol was prepared by acid-catalyzed degradation of black currant
leaves (Ribes nigrum Raven) to give a white amorphous solid (2.1 mg),
APCI-MS, m/z 431 [M + H]+; 1H NMR (CD3OD, 400 MHz) δ
(ppm): 4.24 (H-2, d, J ) 9.1 Hz), 4.43 (H-4, d, J ) 8.0 Hz), 4.61
(H-3, dd, J ) 9.1, 8.0 Hz), 5.87 (phloroglucinol-2H, d, J ) 2.4 Hz),
6.01(A-ring, 2H, d, J ) 2.4 Hz), 6.59 (B-ring, 2H, s).
Acid-Catalyzed Degradation of Hop Proanthocyanidins in the
Presence of Benzyl Mercaptan (15). A solution of proanthocyanidin
dimer 1 (0.1 mg), benzyl mercaptan 20 (1 µL), and acetic acid (1 µL)
in a glass capillary tube loaded with ethanol (100 µL) was sparged
with nitrogen, sealed, and heated to 100 °C for 13 h (Figure 3). The
aliquot of the mixture was directly analyzed by analytical HPLC/APCI-
MS/MS and then injected into the semipreparative HPLC column
consisting of 250 mm × 10 mm i.d., 10 µm Econosil C18 column to
isolate the benzyl mercaptan adduct 21 and monomer 1, both of which
were collected and lyophilized to dryness. The mixture of benzyl
mercaptan adduct 21, monomer 1, and aqueous Raney nickel (50 µL)
was added in a glass tube, which was sparged with nitrogen, shaken
several times at regular intervals over a period of 1 h, directly analyzed
by analytical HPLC/APCI-MS/MS, and then injected into the semi-
preparative HPLC to isolate monomer 1 and the desulfurized product,
monomer 2. Monomers 1 and 2 were identified by HPLC/APCI-MS/
MS.
RESULTS AND DISCUSSION
Isolation and Purification of Hop Proanthocyanidins. A
number of chromatographic procedures using Sephadex G25,
Sephahex LH-20, and Toyopearl TSK HW 40 (6, 19, 20) have
been developed for fractionating and isolating proanthocyanidins
on a preparative scale. Various hops were extracted with aqueous
acetone several times, and then the acetone was removed by
evaporation under reduced pressure. The resulting extracts were
washed with hexane to remove nonpolar material and then with
dichloromethane to remove pigment, flavonoids, and lipids.

4052 J. Agric. Food Chem., Vol. 54, No. 11, 2006
Li and Deinzer
Figure 5. HPLC chromatogram of the Oregon−Willamette hop proanthocyanidins.
Identification of Hop Proanthocyanidins. Hop flavan-3-ol
monomers were identified by HPLC/APCI-MS with authentic
samples for comparison using procedure 1. Compounds 1 and
2 (Table 1) showed molecular ions with m/z 291 [M + H]+
and were confirmed as catechin and epicatechin, respectively,
by cochromatography with authentic samples (HPLC/APCI-
MS). Compound 3 ([M + H]+, m/z 307) was identified as
gallocatechin by cochromatography with authentic samples
(HPLC/APCI-MS).
Table 1. HPLC (Procedure 1), Retention Times (Rt), Molecular Ions [M
+
+
H] (APCI), and Mass Spectrometric Fragments of the
Oregon
−
Willamette Hop Proanthocyanidinsa
+
+
compd Rt (min) [M
+
H]
daughter ions of [M
275 (25), 123 (100)
275 (20), 123 (100)
181 (11), 139 (100)
+ H]
1
2
25.31
32.22
17.19
22.06
27.66
20.99
24.58
15.55
16.50
17.74
18.74
26.48
19.68
11.72
28.50
10.25
9.59
291
291
307
579
579
579
579
595
595
595
595
563
867
867
867
883
899
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
561 (11), 427 (100), 409 (95), 291 (34)
561 (12), 427 (100), 409 (87), 291 (25)
561 (6), 427 (100), 409 (92), 291 (28)
Most of the hop proanthocyanidin dimers were identified by
acid-catalyzed degradation in the presence of phloroglucinol.
Since their interflavonoid C-C linkage bonds are easy to cleave
relative to other C-C bonds, it was relatively straightforward
to determine their subunit composition by acid-catalyzed
degradation with phloroglucinol present, which reacts to form
the adduct (14). Hop proanthocyanidin dimers (dimer 1) can
be degraded (Figure 2) to release terminal subunits (i.e., the
flavan-3-ol monomers (monomer 1)) and extension subunits as
intermediate C-4 carbocations and trapped with phloroglucinol
(18) to generate the analyzable phloroglucinol adducts 19 by
HPLC/APCI-MS.
561 (8), 427 (100), 409 (92), 291 (33)
443 (37), 425 (55), 305 (54), 291(75), 287 (100)
443 (24), 425 (35), 305 (41), 291 (60), 287 (100)
427 (51), 409 (51), 307 (78), 289 (75), 247 (100)
427 (43), 409 (41), 307 (65), 289 (70), 247 (100)
427 (100), 411 (68), 291 (32), 273 (45)
715 (45), 579 (100), 427 (20), 409 (41)
715 (55), 579 (100), 427 (30), 409 (47)
715 (40), 579 (100), 427 (31), 291 (10)
731 (46), 605 (14), 595 (18), 593 (35), 579 (100)
747 (22), 731 (46), 609 (24), 595 (100)
a See Materials and Methods for further details.
The precursors for epicatechin-(4âf2)-phloroglucinol ([M
+ H]+, m/z 415, Rt 27.5 min (procedure 2)) and epigallocat-
echin-(4âf2)-phloroglucinol ([M + H]+, m/z 431, Rt 17.0 min
(procedure 2)) were prepared by acid-catalyzed degradation of
grape seeds in the presence of phloroglucinol (14). Similarly,
the gallocatechin-(4Rf2)-phloroglucinol precursor ([M + H]+,
m/z 431, Rt 15.2 min (procedure 2)) was prepared by acid-
catalyzed degradation of black currant leaves (Ribes nigrum
Raven) (21). The authentic samples of these three phloroglucinol
adducts were confirmed by comparing their spectroscopic and
mass spectrometric data with those reported previously (14, 22).
The authentic sample of catechin-(4Rf2)-phloroglucinol ([M
+ H]+, m/z 415, Rt 26.9 min (procedure 2)) was prepared from
commercial (+)-taxifolin according to a previous report (14).
Various oligomeric proanthocyanidins and monomeric flavan-
3-ols were obtained by column chromatography on Sephadex
LH-20 (2 times) and then on Toyopearl TSK HW-40S. The
hop proanthocyanidin oligomers and flavan-3-ol monomers
(Figure 1) consist of catechin, epicatechin, procyanidin B1,
procyanidin B2, procyanidin B3, procyanidin B4, and epicat-
echin-(4âf8)-catechin-(4Rf8)-catechin, all of which have been
reported previously as being present in hops. The other 10 hop
constituents are reported here for the first time. They are
gallocatechin, gallocatechin-(4Rf8)-catechin, catechin-(4Rf8)-
gallocatechin, afzelechin-(4Rf8)-catechin, gallocatechin-(4Rf6)-
catechin, catechin-(4Rf6)-gallocatechin, epicatechin-(4âf8)-
epicatechin-(4âf8)-catechin, C2, catechin-(4Rf8)-gallocatechin-
(4Rf8)-catechin, and gallocatechin-(4Rf8)-gallocatechin-
(4Rf8)-catechin, which were clearly resolved by HPLC using
procedure 1 (Figure 5) and characterized by HPLC retention
times, mass spectrometric molecular ions, and molecular frag-
ments (Table 1). Moreover, to obtain as pure a compound as
possible, compounds 3, 8-11, and 13 were further chromato-
graphed on a 250 mm × 4.6 mm Luna 5 µm phenyl-Hexyl C18
column.
Compounds 4-7 in Table 1 all showed molecular ions with
m/z 579 [M + H]+, indicating that they were proanthocyanidin
dimers. After acid-catalyzed degradation in the presence of
phloroglucinol, compound 4 yielded epicatechin-(4âf2)-phlo-
roglucinol and catechin ([M + H]+, m/z 291, Rt 37.1 min
(procedure 2)), compound 5 yielded epicatechin-(4âf2)-phlo-
roglucinol and epicatechin ([M + H]+, m/z 291, Rt 46.6 min
(procedure 2)), compound 6 yielded catechin-(4Rf2)-phloro-

Hop Oil Proanthocyanidins
J. Agric. Food Chem., Vol. 54, No. 11, 2006 4053
glucinol and catechin, and compound 7 yielded catechin-
(4Rf2)-phloroglucinol and epicatechin. Therefore, the original
compounds 4-7 were, respectively, epicatechin-catechin, epi-
catechin-epicatechin, catechin-catechin, and catechin-epicat-
echin, and these were finally identified as epicatechin-(4âf8)-
catechin (procyanidin B1), epicatechin-(4âf8)-epicatechin
(procyanidin B2), catechin-(4Rf8)-catechin (procyanidin B3),
and catechin-(4Rf8)-epicatechin (procyanidin B4), respectively,
on the basis of our previously reported spectral characteristics
(6).
catalyst to give the corresponding upper subunits (monomer 2)
and upper central subunits (dimer 2). The structures of hop
proanthocyanidin trimers (trimer 1) could be deduced from the
corresponding upper central subunits (dimer 2) and central-
terminal subunits (dimer 1).
Compound 13 (Table 1) showed a molecular ion with m/z
867 [M + H]+, indicating that it was a proanthocyanidin trimer.
After partial acid-catalyzed degradation with benzyl mercaptan,
compound 13 gave its central-terminal subunit ([M + H]+, m/z
579) as catechin-(4Rf8)-catechin (6) and the benzyl mercaptan
adduct of the upper central subunit ([M + H]+, m/z 701) that
was further desulfurized by hydrogen/Raney nickel to give the
corresponding upper central subunit as catechin-(4Rf8)-cat-
echin. Catechin-(4Rf8)-catechin was confirmed by cochro-
matography with authentic samples, so that compound 13 was
identified catechin-(4Rf8)-catechin-(4Rf8)-catechin. Com-
pound 14 (Table 1) showed a molecular ion with m/z 867 [M
+ H]+, indicating that it was a proanthocyanidin trimer. After
partial acid-catalyzed degradation in the presence of benzyl
mercaptan, compound 14 gave its central-terminal subunit as
catechin-(4Rf8)-catechin and the benzyl mercaptan adduct of
the upper central subunit ([M + H]+, m/z 701) that was further
desulfurized by hydrogen and Raney nickel to give the corre-
sponding upper central subunit ([M + H]+, m/z 579) as
epicatechin-(4âf8)-catechin (4). Epicatechin-(4âf8)-catechin
and catechin-(4Rf8)-catechin were confirmed by cochromatog-
raphy with authentic samples, so that compound 14 was
identified as epicatechin-(4âf8)-catechin-(4Rf8)-catechin.
Compound 15 (Table 1) showed a molecular ion with m/z
867 [M + H]+, indicating that it was a proanthocyanidin trimer.
After partial acid-catalyzed degradation in the presence of benzyl
mercaptan, compound 15 gave its central-terminal subunit as
epicatechin-(4âf8)-catechin and the benzyl mercaptan adduct
of the upper central subunit ([M + H]+, m/z 701) that was
further desulfurized by hydrogen and Raney nickel to give the
corresponding upper central subunit ([M + H]+, m/z 579) as
epicatechin-(4âf8)-epicatechin (5). Epicatechin-(4âf8)-epi-
catechin and epicatechin-(4âf8)-catechin were confirmed by
cochromatography with authentic samples, so that the original
compound 15 was epicatechin-(4âf8)-epicatechin-(4âf8)-
catechin. Compound 16 (Table 1) showed a molecular ion with
m/z 883 [M + H]+, indicating that it was also a proanthocyanidin
trimer. After partial acid-catalyzed degradation and reaction with
benzyl mercaptan, compound 16 gave its central-terminal
subunit ([M + H]+, m/z 595) as gallocatechin-(4Rf8)-catechin
(8) and the benzyl mercaptan adduct of the upper central subunit
([M + H]+, m/z 717) that was further desulfurized by hydrogen
with Raney nickel to give the corresponding upper central
subunit ([M + H]+, m/z 595) as catechin-(4Rf8)-gallocatechin
(10). Catechin-(4Rf8)-gallocatechin and gallocatechin-(4Rf8)-
catechin were confirmed by cochromatography with authentic
samples, so that compound 16 was catechin-(4Rf8)-gallocat-
echin-(4Rf8)-catechin.
Compounds 8 and 10 (Table 1) both showed molecular ions
with m/z 595 [M + H]+, indicating that they were also
proanthocyanidin dimers. After acid-catalyzed degradation and
reaction with phloroglucinol, compound 8 gave gallocatechin-
(4Rf2)-phloroglucinol and catechin, and compound 10 gave
catechin-(4Rf2)-phloroglucinol and gallocatechin ([M + H]+,
m/z 307, Rt 17.4 min (procedure 2)). Compounds 8 and 10 were,
thus, gallocatechin-catechin and catechin-gallocatechin, respec-
tively, and were further confirmed as gallocatechin-(4Rf8)-
catechin and catechin-(4Rf8)-gallocatechin according to pre-
vious reports (23, 24). The method used here would also be
helpful for the identification of 4 f 6 linked hop proanthocya-
nidins. Compounds 9 and 11 (Table 1) both showed molecular
ions with m/z 595 [M + H]+, again indicating that they were
proanthocyanidin dimers. After acid-catalyzed degradation and
reaction with phloroglucinol, compound 9 gave gallocatechin-
(4Rf2)-phloroglucinol and catechin, and compound 11 gave
catechin-(4Rf2)-phloroglucinol and gallocatechin. Thus, the
original compounds 9 and 11 were identified as gallocatechin-
catechin and catechin-gallocatechin, respectively, and were
tentatively deduced as gallocatechin-(4Rf6)-catechin and cat-
echin-(4Rf6)-gallocatechin as they eluted later on the HPLC
column and required a higher collision energy for fragmentation
than did the corresponding 4 f 8 linked analogues.
One hop proanthocyanidin dimer (compound 12, Table 1)
was identified by acid-catalyzed degradation in the presence of
benzyl mercaptan followed by desulfurization with hydrogen
and Raney nickel. The acid-catalyzed degradation of hop
proanthocyanidin dimers (dimer 1) in the presence of benzyl
mercaptan (20, Figure 3) yielded the terminal subunits as flavan-
3-ol monomers (monomer 1) and the extended subunits as
intermediate C-4 carbocations that could be trapped by benzyl
mercaptan to give adducts 21, which were reductively desulfu-
rized to generate the analyzable monomer 2. Compound 12
showed a molecular ion with m/ z 563 [M + H]+, indicating
that it was a proanthocyanidin dimer. After acid-catalyzed
degradation and reaction with benzyl mercaptan, compound 12
released catechin and the benzyl mercaptan adduct of (epi)-
afzelechin that was desulfurized by hydrogen with Raney nickel
to give afzelechin ([M + H]+, m/z 275, Rt 44.5 min (procedure
2)). Compound 12 was characterized as afzelechin-catechin and
was tentatively identified as afzelechin-(4Rf8)-catechin by
comparison of the specific rotation ([R]26 -186.2° (c ) 0.2,
D
acetone)) with that from the literature (ref 17: [R]28D -189.6°
(c ) 0.5, acetone)).
Compound 17 (Table 1) showed a molecular ion with m/z
899 [M + H]+, indicating that it was a proanthocyanidin trimer.
After partial acid-catalyzed degradation and reaction with benzyl
mercaptan, compound 17 gave gallocatechin-(4Rf8)-catechin
(8, central-terminal subunit) and catechin (1, terminal subunit),
both of which were confirmed by cochromatography with
authentic samples and the benzyl mercaptan adducts that were
further desulfurized by hydrogen/Raney nickel to give gallo-
catechin (3, upper subunit) and gallocatechin-(4Rf8)-gallocat-
echin (upper central subunit, [M + H]+, m/z 611) that were
identified according to the previous report (25, 26). Compound
The hop proanthocyanidin trimers were identified by partial
acid-catalyzed degradation and reaction with benzyl mercaptan
and desulfurization by hydrogen with Raney nickel. As shown
(Figure 4), partial acid-catalyzed degradation in the presence
of benzyl mercaptan (21) gave the terminal subunits (monomer
1), the benzyl mercaptan adducts of the upper central subunits
22, the central-terminal subunits (dimer 1), and the benzyl
mercaptan adducts of the upper subunits 23. Compounds 23
and 22 were desulfurized by hydrogen with Raney nickel

4054 J. Agric. Food Chem., Vol. 54, No. 11, 2006
Li and Deinzer
Table 2. Proanthocyanidin Oligomer Profiles of the 13 Different Hopsa
1#
compd 1a# 1b# 1c# 2#
3# 4# 5# 6# 7# 8# 9# 10# 11# av
1
2
21.7 16.5 7.7 12.8 14.7 29.5 11.4 17.4 13.2 23.6 32.1 17.7 9.8 17.6
20.8 22.7 8.7 12.0 18.3 19.0 17.5 13.8 14.5 12.8 5.5 10.4 15.3 14.7
3
4
5
2.5 1.7 3.2 1.6
12.2 13.5 20.7 18.3 14.2 12.0 11.5 10.7 20.0 12.0 19.5 11.3 16.1 14.8
8.2 12.0 13.1 5.4 8.7 6.3 4.0 2.2 5.3 7.7 6.6 5.7 8.0 7.2
1.2 1.9 1.8 2.2 2.9 2.2 2.0 4.2 2.2
2.3
6
7
12.8 11.3 18.9 24.7 14.8 6.5 22.7 19.8 15.2 14.9 11.7 4.7 20.2 15.2
5.4 6.1 10.0 12.1 18.0 6.0 15.2 20.7 6.8 11.3 4.8 23.4 20.3 12.3
8
9
1.7 1.2 0.9 1.0
2.2 1.9 3.0 1.0
2.1 1.8 1.0 0.8
2.1 0.9 2.5 1.5
2.1 4.4 4.3 3.7
1.2 0.9 1.6 0.8
2.3 2.6 1.2 0.9
2.2 1.9 2.6 3.1
0.3 0.2 0.3 0.1
1.3 4.4 1.8 2.1 2.6 1.7 4.5 2.2 1.1
0.9 2.4 2.7 2.0 4.0 1.7 1.4 2.8 1.0
0.4 0.7 1.3 1.7 2.3 0.9 0.8 1.9 0.5
0.2 1.0 1.7 3.0 2.1 0.9 1.2 7.5 0.3
0.6 3.1 2.5 0.8 0.7 1.0 2.3 0.7 0.6
0.7 1.1 1.4 1.7 1.9 1.5 1.8 2.8 1.0
2.2 2.5 0.5 0.4 2.1 2.7 1.4 1.3 0.6
3.7 3.2 3.7 1.0 5.9 4.8 3.8 3.0 3.0
2.0
2.1
1.2
1.9
2.1
1.4
1.6
3.2
10
11
12
13
14
15
16
17
0.1 0.2 0.1 0.1 0.3 0.3 0.2 0.1 0.04 0.2
0.4 0.2 0.4 0.02 0.1 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.04 0.2
a Abundance (mol %) of the total hop proanthocyanidins determined by HPLC
(Procedure 1) at 280 nm. 1# (Willamette) [1a# (Oregon Willamette), 1b# (Idaho
Willamette), and 1c# (Washington Willamette)]; 2# (Vanguard); 3# (Palisade); 4#
(Tettnang Hallertauer); 5# (Hallertauer Hallertauer); 6# (Zeus); 7# (Idaho
Hallertauer); 8# (Cascade); 9# (Saaz 36); 10# (Saaz 72); and 11# (Glacier).
−
−
−
−
−
−
17, therefore, was deduced as gallocatechin-(4Rf8)-gallocat-
echin-(4Rf8)-catechin.
Relative Amounts of Individual Proanthocyanidins in 13
Different Hops. Thirteen different hops were examined to
determine whether the composition of their proanthocyanidin
oligomers had any value in hop variety identification. The
compositions of the 13 different hop proanthocyanidin oligomers
(Table 2) were similar, consisting mostly of three flavan-3-ol
monomers, nine proanthocyanidin dimers, and five proantho-
cyanidin trimers, but the concentrations of these individual
compounds showed some differences.
The percent (mol %) compositions of proanthocyanidins were
based on a comparison of peak integrations at 280 nm. The
detector response of proanthocyanidin dimers and trimers was
estimated using molar absorption coefficients relative to the
monomers. Molar absorptivities of three representative com-
pounds were measured: monomer (catechin, ꢀ280: 3975); dimer
(procyanidin B1, ꢀ280: 6725); and trimer (epicatechin-(4âf8)-
epicatechin-(4Rf8)-catechin, ꢀ280: 11360), so the relative molar
response ratios of monomers, dimers, and trimers were 1:1.69:
2.86. Although other factors such as environment and harvesting
procedure were not taken into account, the results (Table 2)
provide some useful information for the understanding of the
relative composition of the samples and the profiles of different
hop varieties.
On the whole, proanthocyanidins (88.0%) were dominant in
all samples, and small quantities of prodelphinidins (9.9%) and
propelargonidin (2.1%) were also present (Table 2). The
reported hop catechin (1, 17.6%), epicatechin (2, 14.7%),
procyanidin B1 (4, 14.8%), procyanidin B2 (5, 7.2%), procya-
nidin B3 (6, 15.2%), and procyanidin B4 (7, 12.3%) together
with the newly identified procyanidin trimer epicatechin-
(4âf8)-epicatechin-(4âf8)-catechin (15, 3.2%) were the major
hop proanthocyanidin oligomers, and the other 10 hop proan-
thocyanidin oligomers amounted to 15.0% on the total (Table
2).
Figure 6. (A) Proanthocyanidin oligomer profiles of Saaz 36 and Saaz
72 Hops. (B) Proanthocyanidin oligomer profiles of Willamette hops grown
in Oregon (2004), Idaho (2004), and Washington (2003). (C) Proantho-
cyanidin oligomer profiles of Hallertauer hops grown in Tettnang (Germany,
2004), Hallertauer (Germany, 2004), and Idaho (2004).
respectively), but the Washington-Willamette and Glacier hop
proanthocyanidin oligomers were exactly the opposite, 7.7 and
9.8%, respectively (Table 2). With regard to proanthocyanidin
dimers, procyanidin B3 (6, 15.2%) and procyanidin B1 (4,
14.8%) were dominant, followed by procyanidin B4 (7, 12.3%)
and procyanidin B2 (5, 7.2%). The procyanidin B3 content in
Vanguard, Hallertauer-Hallertauer, and Glacier hop proantho-
cyanidin oligomers was 24.7, 22.7, and 20.2%, respectively,
but Saaz 72 hop proanthocyanidin oligomers contained only
Catechin (1, 17.6%) was the dominant flavan-3-ol monomer,
followed by epicatechin (2, 14.7%) and gallocatechin (3, 2.3%).
Saaz 36 and Tettnang-Hallertauer hop proanthocyanidin oli-
gomers had the highest content of catechin (i.e., 32.1 and 29.5%,

Hop Oil Proanthocyanidins
J. Agric. Food Chem., Vol. 54, No. 11, 2006 4055
LITERATURE CITED
4.7% procyanidin B3 (Table 2). Prodelphinidin dimers (com-
pounds 8-11) and the propelargonidin dimer (afzelechin-
(4Rf8)-catechin, 12) were generally present in hop proantho-
cyanidin oligomers. Tettnanger-Hallertauer and Saaz 36 hop
proanthocyanidin oligomers contained gallocatechin-(4Rf8)-
catechin (8) at 4.4 and 4.5%, respectively. Saaz 72 hop
proanthocyanidin oligomers contained 7.5% catechin-(4Rf6)-
gallocatechin (11), and the compound 12 content in Idaho-
Willamette and Washington-Willamette hop proanthocyanidin
oligomers was 4.4 and 4.3%, respectively (Table 2). Proantho-
cyanidin trimers included epicatechin-(4âf8)-epicatechin-
(4Rf8)-catechin (15, 3.2%), C2 (13, 1.4%), and epicatechin-
(4âf8)-catechin-(4Rf8)-catechin (14, 1.6%). Idaho-Hallertauer
and Cascade hop proanthocyanidin oligomers contained com-
pound 15 at 5.9 and 4.8% levels, respectively, but Zeus hop
proanthocyanidin oligomers contained only 1.0%. Catechin-
(4Rf8)-gallocatechin-(4Rf8)-catechin (16, 0.2%) and gallo-
catechin-(4Rf8)-gallocatechin-(4Rf8)-catechin (17, 0.2%) were
very limited in hop proanthocyanidin oligomers (Table 2).
(1) Moir, M. Hopssa millennium review. J. Am. Soc. Brew. Chem.
2000, 58, 131-146.
(2) Stevens, J. F.; Taylor, A. W.; Nickerson, G. B.; Ivancic, M.;
Henderson, M. C.; Haunold, A.; Deinzer, M. L. Prenylflavonoid
variation in Humulus lupulus: distribution and taxonomic
significance of xanthogalenol and 4′-O-methylxanthohumol.
Phytochemistry 2000, 53, 759-775.
(3) Neve, R. A. Hops; Chapman and Hall: London, 1991.
(4) 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.
(5) Overk, C. R.; Yao, P.; Chadwick, L. R.; Nikolic, D.; Sun, Y.;
Cuendet, M. A.; Deng, Y.; Hedayat, A. S.; Pauli, G. F.;
Farnsworth, N. R.; Van, R. B. Comparison of the in vitro
estrogenic activities of compounds from hops (Humulus lupulus)
and red clover (Trifolium pratense). J. Agric. Food Chem. 2005,
53, 6246-6253.
(6) 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.
(7) Callemien, D.; Jerkovic, V.; Rozenberg, R.; Collin, S. Hop as
an interesting source of resveratrol for brewers: Optimization
of the extraction and quantitative study by liquid chromatogra-
phy/atmospheric pressure chemical ionization tandem mass
spectrometry. J. Agric. Food Chem. 2005, 53, 424-429.
(8) Jerkovic, V.; Callemien, D.; Collin, S. Determination of stilbenes
in hop pellets from different cultivars. J. Agric. Food Chem.
2005, 53, 4202-4206.
(9) Merghem, R.; Jay, M.; Brun, N.; Voirin, B. Qualitative analysis
and HPLC isolation and identification of procyanidins from Vicia
faba. Phytochem. Anal. 2004, 15, 95-99.
(10) Maatta-Riihinen, K. R.; Kahkonen, M. P.; Torronen, A. R.;
Heinonen, I. M. Catechins and procyanidins in berries of
Vaccinium species and their antioxidant activity. J. Agric. Food
Chem. 2005, 53, 8485-8491.
(11) Zhou, Z. H.; Zhang, Y. J.; Xu, M.; Yang, C. R. Puerins A and
B, two new 8-C substituted flavan-3-ols from Pu-er tea. J. Agric.
Food Chem. 2005, 53, 8614-8617.
(12) Moll, M.; Fonknechten, G.; Carnielo, M.; Flayeux, R. Changes
in polyphenols from raw materials to finished beer. MBAA Tech.
Q. 1984, 21, 79-87.
(13) McMurrough, I. High -performance liquid chromatography of
flavonoids in barley and hops. J. Chromatogr. 1981, 218, 683-
693.
(14) Kennedy, J. A.; Jones, G. P. Analysis of proanthocyanidin
cleavage products following acid catalysis in the presence of
excess phloroglucinol. J. Agric. Food Chem. 2001, 49, 1740-
1746.
(15) Kennedy, J. A.; Matthews, M. A.; Waterhouse, A. L. Changes
in grape seed polyphenols during fruit ripening. Phytochemistry
2000, 55, 77-85.
(16) Thompson, R. S.; Jacques, D.; Haslam, E.; Tanner, R. J. N. Plant
proanthocyanidins. Part I. Introduction: the isolation, structure,
and distribution in nature of plant procyanidins. J. Chem. Soc.,
Perkin Trans 1 1972, 1387-1399.
(17) Hsu, F.-L.; Nonaka, G.; Nishioka, I. Tannins and related
compounds. XXXI. Isolation and characterization of proantho-
cyanidins in Kandelia candel (L.) druce. Chem. Pharm. Bull.
1985, 33, 3142-3152.
(18) Foo, L. Y.; Karchesy, J. J. Procyanidin dimers and trimers from
Douglas fir inner bark. Phytochemistry 1989, 28, 1743-
1747.
Clearly, there were differences in relative amounts of
compounds 1, 5, and 7 in Saaz 36 and Saaz 72 hop proantho-
cyanidin oligomers (Figure 6A). This was surprising because
these two hops were supposedly genetically identical, and they
were grown in the same location in Idaho. These clones were
established many years apart, and it has often been observed
that clonal selections from varieties established long ago in a
given locality might be different. Anecdotally, they were
reported to have different brewing characteristics as well.
Willamette hops (Oregon, Idaho, and Washington) and
Hallertauer hops (Germany Tettnang, Germany Hallertauer, and
Idaho) were selected to study the effect of geographic origin
on their proanthocyanidin profiles (Table 2 and Figure 6B,C).
It is evident that most hop constituents are affected by
geographic origin. For example, the relative percentage of
compound 1 in Willamette hop proanthocyanidin oligomers,
from Oregon, Idaho, and Washington, was 21.7, 16.5, and 7.7%,
respectively (Table 2 and Figure 6B). The relative percentages
of compound 6 in Hallertauer hop proanthocyanidin oligomers,
from Tettang (Germany, 2004), Hallertauer (Germany, 2004),
and Idaho (2004) were 6.5, 22.7, and 15.2%, respectively (Table
2 and Figure 6C). These results suggest clearly that the
proanthocyanidin profiles of the 13 different hops are affected
by geographic origin.
In summary, this study on the composition and distribution
of hop proanthocyanidins in 13 different hops gives a clear
picture of proanthocyanidin profiles and further showed that
hop proanthocyanidin profiles were affected by geographic
origin and were variable depending on the cultivars. Seventeen
hop proanthocyanidin oligomers and flavan-3-ol monomers were
identified by chemical degradation, HPLC/APCI-MS/MS and
HPLC/ESI-MS/MS. As far as we know, the prodelphinidins
containing gallocatechin units and propelargonidin containing
afzelechin units have now been identified in hops for the first
time. This study will provide useful information for determining
the impact of hop proanthocyanidins on beer flavor and stability
and may also pave the way to the discovery of the real
interaction between proanthocyanidins and proteins in beer.
ACKNOWLEDGMENT
We thank the Mass Spectrometry Facility of the Environmental
Health Sciences Center at Oregon State University. We also
thank Drs. J. F. Stevens and James A. Kennedy for helpful
comments and advice.
(19) Ricardo Da Silva, J. M.; Rigaud, J.; Cheynier, V.; Cheminat,
A.; Moutounet, M. Procyanidin dimers and trimers from grape
seeds. Phytochemistry 1991, 30, 1259-1264.

4056 J. Agric. Food Chem., Vol. 54, No. 11, 2006
Li and Deinzer
(20) 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 chroma-
tography. J. Agric. Food Chem. 2003, 51, 4101-4110.
(21) Foo, L. Y.; Porter, L. J. The structure of tannins of some edible
fruits. J. Sci. Food Agric. 1981, 32, 711-716.
(22) Punyasiri, P. A. N.; Tanner, G. J.; Abeysinghe, I. S. B.; Kumar,
V.; Campbell, P. M.; Pradeepa, N. H. L. Exobasidium Vexans
infection of Camellia sinensis increased 2,3-cis isomerization
and gallate esterification of proanthocyanidins. Phytochemistry
2004, 65, 2897-2994.
(25) Santos, S. C.; Waterman, P. G. Polyphenols from Eucalyptus
oVata. Fitoterapia 2001, 72, 316-318.
(26) McMurrough, I.; Madigan, D.; Smyth, M. R. Semipreparative
chromatographic procedure for the isolation of dimeric and
trimeric proanthocyanidins from barley. J. Agric. Food Chem.
1996, 44, 1731-1735.
Received for review February 9, 2006. Revised manuscript received
March 28, 2006. Accepted March 30, 2006. Financial support from the
Anheuser-Busch Companies and the Hop Research Council is gratefully
acknowledged. This publication was made possible in part by Grant
P30 ES00210 from the National Institute of Environmental Health
Sciences, NIH.
(23) Goupy, P.; Hugues, M.; Boivin, P.; Amiot, M. J. Antioxidant
composition and activity of barley (Hordeum Vulgare) and malt
extracts and of isolated phenolic compounds. J. Sci. Food Agric.
1999, 79, 1625-1634.
(24) Sun, D.; Herbert, W.; Foo, L. Y. Procyanidin dimers and
polymers from Quercus dentata. Phytochemistry 1987, 26,
1825-1829.
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