Anti-inflammatory acylphloroglucinol derivatives from hops (Humulus lupulus)

Article abstract of DOI:10.1021/np050164z

The polyphenol-enriched fraction of an ethanolic hops extract (Humulus lupulus) was separated to provide four acylphloroglucinol-glucopyranosides (1-4). 1-(2-Methylpropanoyl)phloroglucinol-glucopyranoside 1 has been isolated from hops before, whereas 1-(2-methylbutyryl)phloroglucinol-glucopyranoside 2, known as multifidol glucoside, and 1-(3-methylbutyryl)phloroglucinol- glucopyranoside 3 were found in hops for the first time. 5-(2-Methylpropanoyl) phloroglucinol-glucopyranoside 4 was identified as a new natural product. The compounds were tested for inhibition of COX-1 activity. The aglycon 5, obtained by acid hydrolysis of 1, was equally effective as phloroglucinol, with an IC₅₀ of 3.8 μM. The inhibitory potential of the glucosides was 1 > 2 > 3 and decreased with increasing length of the acyl side chain. Compound 4 was about 2.5-fold less active than 1 (IC₅₀: 23.7 and 58.7 μM, respectively).

Full text of DOI:10.1021/np050164z

J. Nat. Prod. 2005, 68, 1545-1548
1545
Anti-inflammatory Acylphloroglucinol Derivatives from Hops (Humulus
lupulus)
Gregor Bohr,† Clarissa Gerha¨user,‡ Jutta Knauft,‡ Josef Zapp,† and Hans Becker*,†
Fr. 8.2 Pharmakognosie und Analytische Phytochemie der Universita¨t des Saarlandes, D-66041 Saarbru¨cken, Germany, and
Deutsches Krebsforschungszentrum (DKFZ), Abteilung Toxikologie und Krebsrisikofaktoren (C010), Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
Received May 13, 2005
The polyphenol-enriched fraction of an ethanolic hops extract (Humulus lupulus) was separated to provide
four acylphloroglucinol-glucopyranosides (1-4). 1-(2-Methylpropanoyl)phloroglucinol-glucopyranoside 1
has been isolated from hops before, whereas 1-(2-methylbutyryl)phloroglucinol-glucopyranoside 2, known
as multifidol glucoside, and 1-(3-methylbutyryl)phloroglucinol-glucopyranoside 3 were found in hops for
the first time. 5-(2-Methylpropanoyl)phloroglucinol-glucopyranoside 4 was identified as a new natural
product. The compounds were tested for inhibition of COX-1 activity. The aglycon 5, obtained by acid
hydrolysis of 1, was equally effective as phloroglucinol, with an IC50 of 3.8 µM. The inhibitory potential
of the glucosides was 1 > 2 > 3 and decreased with increasing length of the acyl side chain. Compound
4 was about 2.5-fold less active than 1 (IC50: 23.7 and 58.7 µM, respectively).
Hops (Humulus lupulus L., Cannabinaceae) is an im-
portant source of phenolic constituents in beer. The dried
hop cones contain 4-14% polyphenols, mostly phenolic
acids, chalcones, prenylated flavonoids, catechins, and
proanthocyanidins.1-4 Hop products have been used over
centuries almost exclusively in the brewing industry. They
add aroma and bitterness to beer and provide antifungal
and antibiotic properties.5-7 These characteristics are
mainly attributed to their content of R-acids such as
humulone, which are converted during brewing to the
bitter-tasting iso-R-acids. In recent years, hops have gained
considerable interest because of the biological and potential
cancer chemopreventive activities of some of its constitu-
ents (reviewed in refs 8, 9). As an example, the hop-derived
prenylated chalcone, xanthohumol, was identified as a
novel inhibitor of cyclooxygenase-1 (COX-1) by activity-
guided fractionation of beer.10 In continuation of these
studies we here describe results of the phytochemical
analysis and biological testing of a polyphenol-enriched hop
those of known phloroglucinol-glucosides.12 Compound 1
is a known constituent of hops,13 but no NMR data have
been published so far for this compound.
extract, which led to the isolation and structure elucidation
of four monoacylphloroglucinol-glucopyranosides (1-4) and
one aglycon (5) with anti-inflammatory activity.
The NMR data of 2, m/z 371.1421, were very similar to
those of 1, with only the values of the acyl moiety differing
from 1, indicating a 2-methylbutyryl moiety for compound
2 (Tables 1 and 2). The resulting 1-[(2-methylbutyryl)-
phloroglucinyl]-â-D-glucopyranoside had been isolated ini-
tially as “multifidol glucoside” from the latex of Jatropha
multifida L. (Euphorbiaceae) and shows interesting im-
munological activity.12 Its 13C NMR data fit very well with
those of 2, supporting the structure proposed. This is the
first report of the occurrence of 2 in hops.
Fractionation of an ethanolic hop extract via size exclu-
sion chromatography and preparative HPLC led to the
isolation of four acylphloroglucinol-glucopyranosides (1-
4). Additional phloroglucinol derivatives from hops have
been described recently.11
Compound 1, HRCIMS m/z 358.1278, was identified as
1-[(2-methylpropanoyl)phloroglucinyl]-â-D-glucopyrano-
side. Its 1H NMR spectrum (Table 1) showed in the
aliphatic region signals for a 2-methylpropanoyl moiety at
δ 3.98, sept, 1.14 d (3H), and 1.13 d (3H) and a sugar
moiety. In the aromatic region of the spectrum, two meta-
coupled doublets appeared at δ 5.95 and 6.17, indicating
an asymmetric substituted phloroglucinol moiety. There-
fore, the sugar residue must be attached to C-1 of the
phloroglucinol. The sugar moiety was identified by com-
parison of its 1H and 13C NMR data (Tables 1 and 2) with
Compound 3 again differs from 1 only in the acyl moiety.
In this case, NMR data were supportive of a 3-methylbu-
tyryl moiety (Tables 1 and 2). The HREIMS with m/z
372.1412 supported the structure of 1-[(3-methylbutyryl)-
phloroglucinyl]-â-D-glucopyranoside, which was described
for the first time from strawberry fruit, Fragaria ananassa
Duch.14 Its aglycon, 2-(3-methylbutyryl)phloroglucinol, is
a known ingredient of hops.15
* To whom correspondence should be addressed. Tel: + 49 6894 37175.
The 1H and 13C NMR data of 4 (Tables 1 and 2) suggested
a further acylphloroglucinol-glucoside. In contrast to com-
pounds 1-3, the chemical shifts of C-4 and C-6 (δ 96.6) as
Fax:
+ 49 6894 383935. E-mail: hans.becker@mx.uni-saarland.de or
prof_hans_becker@hotmail.com.
† Universita¨t des Saarlandes.
‡ Deutsches Krebsforschungszentrum (DKFZ).
10.1021/np050164z CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/14/2005

1546 Journal of Natural Products, 2005, Vol. 68, No. 10
Notes
Table 1. 1H NMR Data of Compounds 1-5 (in CD3OD, 500 MHz)a
position
1
2
3
4
5
4
5.95 d (2.5)
6.17 d (2.5)
3.98 sept (7.0)
5.94 d (2.5)
6.17 d (2.5)
3.90 m
5.94 d (2.5)
6.08 s
6.08 s
5.80 s
5.80 s
6
6.17 d (2.5)
2′
3.17 dd (16.0/6.5)
2.88 dd (16.0/7.5)
2.24 sept (7.0)
0.96 d (7.0)
0.93 d (7.0)
5.01 d (8.0)
3.53 dd (9.0/7.5)
3.45 m
3.39 t (9.0)
3.45 m
3.97 sept (7.0)
3.96 sept (7.0)
3′
1.14 d (7.0, 3H)
1.13 d (7.0, 3H)
1.80 m + 1.38 m
0.87 t (7.5, 3H)
1.12 d (7.0, 3H)
5.03 d (7.5)
3.50 dd (9.0/7.5)
3.45 m
1.13 d (7.0, 3H)
1.13d (7.0, 3H)
1.12 d (7.0, 3H)
1.12 d (7.0, 3H)
4′
5′
1′′
5.04 d (7.5)
3.50 dd (9.0/7.5)
3.44 m
3.38 t (9.0)
3.44m
4.91 d (7.5)
3.42 m
2′′
3′′
3.42 m
4′′
3.38 t (9.0)
3.45 m
3.38 m
5′′
3.42 m
6′′ R
6′′ â
3.91 dd (12.3/2.0)
3.71 dd (12.3/5.5)
3.91 dd (12.3/2.0)
3.71 dd (12.3/5.5)
3.91 dd (12.3/2.0)
3.71 dd (12.3/5.5)
3.90 dd (12.3/2.0)
3.70 dd (12.3/5.5)
a Chemical shifts δH (J in Hz).
Table 2. 13C NMR Data of Compounds 1-5 (in CD3OD, 125 MHz)a
position
1
2
3
4
5
1
161.6 (C)
106.2 (C)
167.5 (C)
98.3 (CH)
165.6 (C)
95.3 (CH)
211.9 (C)
40.4 (CH)
20.2 (CH3)
19.5 (CH3)
161.8 (C)
106.8 (C)
167.4 (C)
98.3 (CH)
165.6 (C)
95.3 (CH)
211.8 (C)
47.0 (CH)
28.3 (CH2)
12.0 (CH3)
16.8 (CH3)
101.7 (CH)
74.8 (CH)
78.7 (CH)
71.2 (CH)
78.4 (CH)
62.5 (CH2)
162.2 (C)
107.0 (C)
167.6 (C)
98.3 (CH)
165.8 (C)
95.4 (CH)
207.2 (C)
54.2 (CH2)
26.2 (CH)
23.4 (CH3)
22.9 (CH3)
101.9 (CH)
74.8 (CH)
78.6 (CH)
71.2 (CH)
78.4 (CH)
62.5 (CH2)
165.2 (C)
106.3 (C)
165.2 (C)
96.6 (CH)
164.8 (C)
96.6 (CH)
212.3 (C)
40.3 (CH)
19.5 (CH3)
19.5 (CH3)
165.7 (C)
104.6 (C)
165.7 (C)
95.9 (CH)
165.8 (C)
95.9 (CH)
211.7 (C)
39.9 (CH)
19.6 (CH3)
19.6 (CH3)
2
3
4
5
6
1′
2′
3′
4′
5′
1′′
2′′
3′′
4′′
5′′
6′′
101.5 (CH)
74.8 (CH)
78.7 (CH)
71.2 (CH)
78.4 (CH)
62.5 (CH2)
101.2 (CH)
74.7 (CH)
78.3 (CH)
71.2 (CH)
78.0 (CH)
62.4 (CH2)
a Chemical shifts δC (mult.).
well as of H-4 and H-6 (δ 6.08) were identical. This was
suggestive of symmetrical substitution of the aromatic ring
moiety. The data for the acyl side chain were identical to
those of 1, revealing a 2-methylpropanoyl moiety. There-
fore, 4 was assigned as 5-[(2-methyl-propanoyl)phloroglu-
cinyl]-â-D-glucopyranoside. The HREIMS with m/z 358.1260
supports this structural assignment. Compound 4 is a new
natural product.
Treatment of 1 with hydrochloric acid led to the loss of
the sugar moiety and gave the expected aglycon 5, HRE-
IMS m/z 196.0736. Its NMR data (Tables 1 and 2) were in
good agreement with the structure, 2-(2-methylpropanoyl)-
phloroglucinol. The occurrence of compound 5 has been
previously described for hops,15 but spectroscopic data have
not appeared in the literature.
glucosidases. We speculate that position C-1 is preferred
to position C-5, but these observations require further
biosynthetic studies. For compound 2, the trivial names
“multifidol” for the aglycon and “multifidol glucoside” are
used.12 According to the nomenclature of the hops bitter
acids, we propose new trivial names for compounds 1-4:
“co-multifidol glucoside” for 1, “ad-multifidol glucoside” for
2, and “n-multifidol glucoside” for 3. For compound 4, we
suggest “co-iso-multifidol glucoside” as a trivial name. In
hops, the amount of ad-humulone is fairly constant (15%)
and lower than that of co- and n-humulones (20-50% each
in varying ratios).5 In contrast, with the acylphloroglucinol
derivates, we observed that the “co-” and the “ad-” forms
were more abundant than the “n-” homologue (unpublished
data).
The acyl side chains of compounds 1-5 are identical to
those of the hops R-acids co-humulone, ad-humulone, and
n-humulone. Therefore, by analogy with the findings of
Zuurbier et al.,16,17 we propose that 1-5 are formed from
phloroisovalerophenone or phloroisobutyrophenone inter-
mediates, which are generated by the enzyme phlorisov-
alerophenone synthase (VPS) during the biosynthesis of
the hops bitter acids.18 Accordingly, condensation of three
malonyl-CoA units and one isobutyryl-CoA unit gives rise
to phloroisobutyrophenone (5) as a precursor of co-humu-
lone and compounds 1 and 4, respectively. Condensation
of three malonyl-CoA units and one isovaleryl-CoA unit
generates phoroisovalerophenone as a precursor of n-
humulone and compound 3, and finally three malonyl-CoA
units and one 2-methylbutyryl-CoA unit are condensed to
the precursor of ad-humulone and compound 2, respec-
tively. The glycosylation of compounds 1-4 is mediated by
The compounds were tested for inhibition of COX-1
activity using microsomes of ram seminal vesicles. COX-1
contains two catalytic sites, the cyclooxygenase site, which
exists along a hydrophobic channel within the core of the
protein, and a peroxidase site containing a heme moi-
ety.21,22 Using a Clark-style oxygen microelectrode, the
oxygen consumption was measured to follow the bis-
dioxygenation of arachidonic acid to yield prostaglandin
G2.20 Dose-response curves for all acylphloroglucinol de-
rivatives in comparison with phloroglucinol (6) are given
in Figure 1. The aglycon 5 was as equally effective as
phloroglucinol in inhibiting oxygen consumption, with a
half-maximal inhibitory concentration (IC50) of 3.8 µM. The
insertion of a glucose moiety at position C-1 or C-5 to yield
compounds 1 and 4, respectively, reduced the COX-1
inhibitory potential about 6- and 15-fold, with IC50 values
in the range of 23.7 and 58.7 µM. Elongation of the acyl

Notes
Journal of Natural Products, 2005, Vol. 68, No. 10 1547
Extraction and Isolation. The aqueous extract (500 g) was
partitioned with n-hexane (6 × 0.5 L) to remove residues of
hop bitter acids, such as humulones and lupulones. Afterward,
the aqueous layer was partitioned with ethyl acetate (EE) (6
× 0.5 L). The yield of the EE fraction (EE00) after evaporating
the solvent was 4.42 g. EE00 (3.00 g) was separated by column
chromatography using Sephadex LH-20 by isocratic elution
with 100% methanol. Thereby, 12 fractions were obtained:
EE01 (83 mg), EE02 (662 mg), EE03 (142 mg), EE04 (1303
mg), EE05 (102 mg), EE06 (129 mg), EE07 (88 mg), EE08 (179
mg), EE09 (50 mg), EE10 (17 mg), EE11 (48 mg), EE12 (50
mg). HPLC separation and UV spectra indicated the presence
of acylphloroglucinols in fraction EE04. Therefore, this fraction
(450 mg) was further separated by preparative HPLC.
HPLC was performed on an RP-18ec column (VP 250/10
Nucleodur 100-5 C18 ec, Macherey-Nagel, Du¨ren, Germany)
using acetonitrile/water (25:75) with 0.05% TFA. The solvent
delivery system was a Waters M-45 (Waters, Milford, MA).
Peaks were detected with an RI-Detector (RI-Detector 8110,
Bischoff, Leonberg, Germany) and collected to yield 207 mg
of compound 1, 30 mg of 2, 10 mg of 3, and 7 mg of 4.
1-[(2-Methylpropanoyl)phloroglucinyl]-â-D-glucopyra-
noside (1): white powder; [R]2D0 -55.6° (MeOH); IR (KBr) νmax
3383 (OH), 1600 (CdO) cm-1; 1H and 13C NMR data, see Tables
1 and 2; HRCIMS m/z 358.1278 (calcd for C16H22O9, 358.1264).
1-[(2-Methylbutyryl)phloroglucinyl]-â-D-glucopyrano-
side (2): white powder; [R]2D0 -54.3° (MeOH); IR (KBr) νmax
3386 (OH), 1601 (CdO) cm-1; 1H and 13C NMR data, see Tables
1 and 2; HRCIMS m/z 372.1421 (calcd for C17H24O9, 372.1420).
1-[(3-Methylbutyryl)phloroglucinyl]-â-D-glucopyrano-
side (3): white powder; [R]2D0 -58.8° (MeOH); IR (KBr) νmax
3396 (OH), 1600 (CdO) cm-1; 1H and 13C NMR data, see Tables
1 and 2; HREIMS m/z 372.1412 (calcd for C17H24O9, 372.1420).
5-[(2-Methylbutyryl)phloroglucinyl]-â-D-glucopyrano-
side (4): beige powder; [R]2D0 -17.4° (MeOH); IR (KBr) νmax
3391 (OH), 1608 (CdO) cm-1; 1H and 13C NMR data, see Tables
1 and 2; HREIMS m/z 358.1260 (calcd for C16H22O9, 358.1264).
Acid Hydrolysis of 1 to Generate 5. A 125 mg aliquot of
compound 1 was dissolved in a mixture of 20 mL of water and
20 mL of hydrochloric acid (2 N) and heated under reflux for
30 min. On cooling, the mixture was partitioned with ethyl
acetate (5 × 20 mL), dried over Na2SO4, and filtered over a
paper filter that was wet with ethyl acetate. Evaporation of
solvent yielded 60 mg of a yellowish oil, which was then
purified using HPLC as described above. The yield of 5 was
28 mg.
Figure 1. Inhibition of COX-1 activity by acylphloroglucinol deriva-
tives, measured by inhibition of oxygen consumption during in vitro
prostaglandin formation by COX-1. Dose-dependent inhibition by 1 (1);
2 (9); 3 (0); 4 (]); and 5 (b) in comparison with phloroglucinol (6) (4).
side chain with a methyl group in 2 further reduced the
anti-inflammatory activity (IC50 ) 131.3 µM), whereas the
phloroisovalerophenone derivative 3 was basically inactive,
with only 12% inhibition at a 100 µM concentration (IC50
> 100 µM). From these data, it was concluded that (i)
phloroglucinol (6) is a good COX-1 inhibitor, (ii) substitu-
tion of compound 5 with the short 2-methylpropanoyl
moiety at position C-2 does not reduce the inhibitory
potential, (iii) further addition of a sugar moiety in position
C-1 reduces the IC50 value of 5 about 6-fold, (iv) glycosy-
lation at position C-5 reduces the inhibitory potential more
than addition of a sugar moiety at position C-1, and (v)
modifications of the acyl side chain severely lower the
COX-1 inhibitory potential.
The anti-inflammatory potential of phloroglucinol de-
rivatives has been reported before. For example, hyperforin,
the major lipophilic constituent in Hypericum perforatum
L. (St. John’s wort), was described as a dual inhibitor of
COX-1 and of lipoxygenase-5.23 Szewczuk and Penning
recently identified resorcinol as the minimum structure
necessary for inactivation of COX-1.24 The proposed mech-
anism involved inhibition of the peroxidase activity of COX-
1, which is required to initiate the cyclooxygenase activity.
Partial oxidation of the m-hydroquinone moiety within the
compounds would then inactivate the cyclooxygenase activ-
ity.25 Access to the cyclooxygenase site within the hydro-
phobic channel might discriminate the derivatives depend-
ing on their molecular structure and overall size. This could
explain the lower activity of the glycosides tested in
comparison with the aglycon 5 or with phloroglucinol (6).
In conclusion, we have isolated and structurally char-
acterized acylphloroglucinol derivatives from hops, which
were identified as novel inhibitors of COX-1. Our investiga-
tions provide some suggestions with respect to a structure-
activity relationship; however, for a precise study, more
derivatives should be tested.
2-(2-Methylpropanoyl)-1,3,5-benzenetriol (5): yellowish
oil; [R]2D0 (0.0° (MeOH); IR (KBr) νmax 3319 (OH), 1601 (CdO)
cm-1; 1H and 13C NMR data, see Tables 1 and 2; HREIMS m/z
196.0736 (calcd for C10H12O4, 196.0735).
Inhibition of Cyclooxygenase-1 Activity. Inhibition of
cyclooxygenase-1 (COX-1) activity was measured by monitor-
ing oxygen consumption during the conversion of arachidonic
acid to prostaglandins using a Clark-type O2-electrode (Han-
satech Ltd., Kings Lynn, U.K.).10,20 The reaction mixture
contained approximately 0.5 U COX-1 in a 100 µL microsome
fraction, prepared from ram seminal vesicles as a crude source
of COX-1 (specific activity 0.2-1 U/mg protein). For calcula-
tion, the rate of O2 consumption was compared to a DMSO
control (100% activity). Phloroglucinol (6) was tested as a
reference compound.
Experimental Section
General Experimental Procedures. Optical rotations
were measured with a Perkin-Elmer 241 polarimeter at 20 °C.
IR spectra were recorded on a Bio Rad FTS3000 Excalibur
S-Series infrared spectophotometer. NMR spectra were re-
corded on a Bruker Avance 500 and a Bruker Avance DRX
500 spectrometer in CD3OD. Mass spectra were measured on
a Finnigan MAT 90 mass spectrometer. Analytical HPLC was
performed on a Waters Alliance 2690 separation module with
a PDA-detector (Waters, Milford, MA) using an acetonitrile/
water gradient. Column: RP-18ec (EC 250/4 Nucleosil 100-5
C18 Hop, Macherey-Nagel, Du¨ren, Germany).
Acknowledgment. We thank D. Kaltner from Simon H.
Steiner Hopfen GmbH for providing the extract and financial
support of this work. Also, financial support of the Wissen-
schaftsfo¨rderung der Deutschen Brauwirtschaft e.V. for our
work on beer and hops constituents is highly acknowledged.
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Steiner Hopfen GmbH, D-84048 Mainburg.

1548 Journal of Natural Products, 2005, Vol. 68, No. 10
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