Fate of Xanthohumol from Hops to Beer
J. Agric. Food Chem., Vol. 47, No. 6, 1999 2427
similar fashion and then hydrolyzed or reduced to
alcohols. To test this hypothesis, a concentrated metha-
nolic extract of yeast, recovered from the raw beer, was
analyzed by LC/MS using single ion monitoring and
daughter ion scanning. Several compounds were de-
tected that had masses consistent with that of oxygen-
ated (iso)xanthohumol (Mr 370); daughter ion mass
spectra allowed these products to be characterized as
prenylated chalcones or flavanones. These compounds
were present only in trace amounts, and some occur
naturally in hops, which left doubt as to whether these
trace constituents are produced by the hops or by the
yeast. At any rate, these xanthohumol derivatives do
not nearly make up for the amount of missing xantho-
humol in the brew trials (14-27%).
Ger a n ylfla von oid s. The geranyl analogue of desm-
ethylxanthohumol, 3′-geranylchalconaringenin, is the
only known geranylchalcone from hops (Stevens et al.,
1997). The fate of this geranylflavonoid could not be
quantified because it and 8-geranylnaringenin were not
available in sufficient amounts at the time the brewing
trials were conducted. However, the chromatographic
profiles indicated that 3′-geranylchalconaringenin be-
haved very similarly to desmethylxanthohumol in both
brewing experiments. After addition of the hops to the
boiling wort, 3′-geranylchalconaringenin was rapidly
converted into two products, which were identified as
8- and 6-geranylnaringenin by LC/MS comparison with
authentic markers (Figure 2).
unaltered. This was taken as proof for H-2′′ being
located trans to the CH3-4′′ group, thus confirming the
presence of a geranyl, not neryl, substituent.
ACKNOWLEDGMENT
We are grateful to Monika Ivancic and Victor L. Hsu
for help with the NMR experiments and to Donald A.
Griffin for technical support.
LITERATURE CITED
Bohm, B. A. Chalcones and aurones. In Methods in Plant
Biochemistry, Vol. 1. Plant Phenolics; Harborne, J . B., Ed.;
Academic Press: London, U.K., 1989; pp 237-281.
Charalambous, G. Involatile constituents of beer. In Brewing
Science; Pollock, J . R. A., Ed.; Academic Press: London,
U.K., 1981; Vol. 2, pp 167-254.
Delcour, J . A. Malt and Hop Flavanoids in Pilsner Beer. In
Modern Methods of Plant Analysis, Vol. 7, Beer Analysis;
Linskens, H. F., J ackson, J . F., Eds.; Springer-Verlag:
Berlin, Germany, 1988; pp 225-240.
Ha¨nsel, R.; Schulz, J . Desmethylxanthohumol: Isolierung aus
Hopfen und Cyclisierung zu Flavanonen. Arch. Pharm.
(Weinheim) 1988, 321, 37-40.
Haslam, E. Natural polyphenols (vegetable tannins) as
drugs: possible modes of action. J . Nat. Prod. 1996, 59,
205-215.
J ain, A. C.; Gupta, R. C.; Sarpal, P. D. Synthesis of lupinifolin,
di-O-methylxanthohumol and isoxanthohumol and related
compounds. Tetrahedron 1978, 34, 3563-3567.
McGuinness, J . D.; Laws, D. R. J .; Eastmond, R.; Gardner, R.
J . The estimation and significance of catechin and dimeric
catechin in beer. J . Inst. Brew. 1975, 81, 237-241.
McMurrough, I. High-performance liquid chromatography of
flavonoids in barley and hops. J . Chromatogr. 1981, 218,
683-693.
McMurrough, I.; Madigan, D.; Smyth, M. R. Adsorption by
polyvinylpolypyrrolidone of catechins and proanthocyanidins
from beer. J . Agric. Food Chem. 1995, 43, 2687-2691.
Miranda, C. L.; Stevens, J . F.; Helmrich, A.; Henderson, M.
C.; Rodriguez, R. J .; Deinzer, M. L.; Barnes, D. W.; Buhler,
D. R. Antiproliferative and cytotoxic effects of prenylated
flavonoids from hops (Humulus lupulus) in human cancer
cell lines. Food Chem. Toxicol. 1999, in press.
A small amount of 6-geranylnaringenin was obtained
by alkali treatment of 3′-geranylchalconaringenin iso-
lated from hops. The position of its geranyl substituent
was determined by 2-D NMR: in the 1H-13C HMBC
spectrum, cross-peaks showing interaction between the
chelated 5-hydroxyl and C-6 and between H-1′′ of the
geranyl group and C-6 left no doubt that the geranyl
group was located ortho to the hydroxyl at C-5. Fur-
thermore, the only aromatic A-ring proton showed cross-
peaks with all A-ring carbons but C-5 and was therefore
assigned to H-8.
A larger amount of 6-geranylnaringenin was obtained
by geranylation of naringenin with (()-linalool. The
semisynthetic product was identical to the above 6-gera-
nylnaringenin by UV, LC/MS, and 2D-NMR. It was
treated with alkali to give 3′-geranylchalconaringenin
and an earlier eluting product, which was identified as
8-geranylnaringenin by MS and 2-D NMR spectroscopy.
In the HMBC spectrum of the latter compound, the
aromatic A-ring proton was assigned to H-6 because it
showed cross-peaks with all A-ring carbons but C-9.
Carbon-9 interacted with H-1′′ of the geranyl substitu-
ent at C-8. The identity of carbons 5, 6, and 10 was
confirmed by interactions between these carbons and
the hydrogen-bonded 5-hydroxyl proton. Full assign-
ments of proton and carbon resonances for both gera-
nylnaringenins are listed under Experimental Proce-
dures.
The condensation of naringenin with linalool results
in the formation of a double bond between C-2′′ and C-3′′
of the monoterpenyl substituent (Figure 1). Double-bond
formation may give rise to cis and trans isomers, that
is, neryl- and geranylnaringenins, and this problem was
investigated by means of 1-D NOE experiments. Ir-
radiation of the H-2′′ resonance of 8-geranylnaringenin
resulted in enhancement of the H-1′′ and H-5′′ signals,
whereas the intensity of the CH3-4′′ singlet remained
Moll, M.; Fonknechten, G.; Carnielo, M.; Flayeux, R. Changes
in polyphenols from raw materials to finished beer. Tech.
Q.sMaster Brew. Assoc. Am. 1984, 21 (2), 79-87.
Outtrup, H. Proanthocyanidins, the brewing process, and the
quality of beer. In Plant Polyphenols; Hemingway, R. W.,
Laks, P. E., Eds.; Plenum Press: New York, 1992; pp 849-
858.
Peacock, V. Fundamentals of hop chemistry. Tech. Q.sMaster
Brew. Assoc. Am. 1998, 35 (1), 4-8.
Satoh, T.; Horie, M.; Watanabe, H.; Tsuchiya, Y.; Kamei, T.
Enzymatic properties of squalene epoxidase from Saccha-
romyces cerevisiae. Biol. Pharm. Bull. 1993, 16, 349-352.
Siebert, K. J .; Lynn, P. Y. Comparison of polyphenol interac-
tions with polyvinylpolypyrrolidone and haze-active protein.
J . Am. Soc. Brew. Chem. 1998, 56, 24-31.
Stevens, J . F.; Ivancic, M.; Hsu, V. L.; Deinzer, M. L. Pre-
nylflavonoids from Humulus lupulus. Phytochemistry 1997,
44, 1575-1585.
Stevens, J . F.; Taylor, A. W.; Deinzer, M. L. Quantitative
analysis of xanthohumol and related prenylflavonoids in
hops and beer by liquid chromatography-tandem mass
spectrometry. J . Chromatogr. A 1999, 832, 97-107.
Stewart, G. G.; Russell, I. Yeast Flocculation. In Brewing
Science; Pollock, J . R. A., Ed.; Academic Press: London,
U.K., 1981; Vol. 2, pp 61-92.
Stott, K.; Stonehouse, J .; Keeler, J .; Hwang, T.-L.; Shaka, A.
J . Excitation sculpting in high-resolution nuclear magnetic
resonance spectroscopy: application to selective NOE ex-
periments. J . Am. Chem. Soc. 1995, 117, 4199-4200.