Oestrogenic activity of the hop phyto-oestrogen, 8-Prenylnaringenin

Abstract

The female flowers of the hop plant (hop cones) are used as a preservative and as a flavouring agent in beer. A novel phyto-oestrogen, 8-prenylnaringenin, was recently identified in hops and this study was undertaken to characterize the oestrogenic activity of this compound using a combination of in vitro and in vivo assays. Natural and semi-synthetic 8-prenylnaringenin showed similar bioactivities both in a yeast screen transfected with the human oestrogen receptor and in oestrogen-responsive human Ishikawa Var-I cells. 8-Prenylnaringenin showed comparable binding activity to both oestrogen receptor isoforms (ER alpha and ER beta). 8-Prenylnaringenin extracted from hops contains similar amounts of both (R)- and (S)- enantiomers, indicating that the compound is normally formed non-enzymatically. Both enantiomers showed similar bioactivity in vitro and similar binding characteristics to ER alpha and ER beta. The oestrogenic activity of 8-prenyl-naringenin in vitro was greater than that of established phyto-oestrogens such as coumestrol, genistein and daidzein. The high oestrogenic activity was confirmed in an acute in vivo test using uterine vascular permeability as an end point. When the compound was given to ovariectomized mice in their drinking water, oestrogenic stimulation of the vaginal epithelium required concentrations of 100 mug ml(-1) (about 500-fold greater than can be found in any beer).

Full text

Introduction
The female flowers (cones) of hops (
Humulus lupulus
L.)
have been used for centuries as a preservative and as a
flavouring agent in beer. Over the years, a recurring
suggestion has been that hops have a powerful oestrogenic
activity and that beer may also be oestrogenic. Hop baths
have been used for the treatment of gynaecological disorders
and hop extracts have been reported to be effective in
reducing hot flushes in menopausal women. In times when
hops were picked by hand, menstrual disturbances among
female pickers were reportedly common. Although the
oestrogenic activity of hops was attributed to xanthohumol,
a prenylated chalcone, this was without any experimental
support (Verzele, 1986). Initial scientific studies of the
oestrogenic activity of hops proved contradictory: some
early investigations (Koch and Heim, 1953; Zenisek and
Bednar, 1960; Hesse
et al
., 1981) reported that the
oestrogenic activity of hops was very high, but other
investigations (Feneslau and Talalay, 1973) found no
indication of oestrogenicity. These discrepancies over the
potential oestrogenic activity of hops are likely to have been
due to the variable nature of the extracts and the variety of
assays used. However, after bioassay-guided fractionation
of hop extracts, Milligan
et al
. (1999) identified a novel
phyto-oestrogen, 8-prenylnaringenin (Fig. 1). Using both
a yeast-based screen to detect oestrogenic activity and a
receptor binding assay using rat uterine cytosol, 8-
prenylnaringenin appeared to be one of the most potent
phyto-oestrogens, and had an oestrogenic activity con-
siderably greater than that of phyto-oestrogens such as
genistein and daidzein (Fig. 1).
There is considerable interest in whether human
exposure to phyto-oestrogens has any health risks or
benefits (Knight and Eden, 1996; Cassidy and Milligan,
1998). The secretions of the lupulin glands present in the
female hop flowers contain predominantly bitter acids
and hop oil constituents, essential in brewing, along with
8-prenylnaringenin and other prenylflavonoids. 8-Prenyl-
naringenin can be found in many beers at concentrations of
up to 0.24 mg l
–1
(Stevens
et al
., 1999; Rong
et al
., 2000).
There may also be significant exposure of women to
8-prenylnaringenin from a number of commercially
available herbal preparations, including those claiming
breast enlargements (Sauer and Coldham, 2001). In view of
such exposure of humans to 8-prenylnaringenin and its
apparent high oestrogenic activity, the present study was
undertaken to characterize the nature of the oestrogenic
activity of this compound using a combination of
in vitro
and
in vivo
assays, and to examine the binding of 8-
prenylnaringenin to the two isoforms of the oestrogen
Oestrogenic activity of the hop phyto-oestrogen,
8-prenylnaringenin
S. Milligan
1
, J. Kalita
1
, V. Pocock
1
, A. Heyerick
2
, L. De Cooman
2
,
H. Rong
2
and D. De Keukeleire
2
1
Endocrinology and Reproduction Research Group, School of Biomedical Sciences,
King’s College, Guy’s Campus, London SE1 1UL, UK; and
2
Ghent University,
Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences,
Harelbekestraat 72, B-9000 Ghent, Belgium
Reproduction
(2002) 123, 235–242
Research
The female flowers of the hop plant (hop cones) are used
as a preservative and as a flavouring agent in beer. A
novel phyto-oestrogen, 8-prenylnaringenin, was recently
identified in hops and this study was undertaken to
characterize the oestrogenic activity of this compound
using a combination of
in vitro
and
in vivo
assays. Natural
and semi-synthetic 8-prenylnaringenin showed similar
bioactivities both in a yeast screen transfected with the
human oestrogen receptor and in oestrogen-responsive
human Ishikawa Var-I cells. 8-Prenylnaringenin showed
comparable binding activity to both oestrogen receptor
isoforms (ERα and ERβ). 8-Prenylnaringenin extracted from
hops contains similar amounts of both (
R
)- and (
S
)-
enantiomers, indicating that the compound is normally
formed non-enzymatically. Both enantiomers showed similar
bioactivity
in vitro
and similar binding characteristics
to ERα and ERβ. The oestrogenic activity of 8-prenyl-
naringenin
in vitro
was greater than that of established
phyto-oestrogens such as coumestrol, genistein and daidzein.
The high oestrogenic activity was confirmed in an acute
in vivo
test using uterine vascular permeability as an
end point. When the compound was given to ovariec-
tomized mice in their drinking water, oestrogenic stimula-
tion of the vaginal epithelium required concentrations of
100 µg ml
–1
(about 500-fold greater than can be found in
any beer).
© 2002 Society for Reproduction and Fertility
1470-1626/2002
Email: Stuart.Milligan@kcl.ac.uk

receptor (ERα and ERβ). In addition, as 8-prenylnaringenin
contains a chiral centre at C(2) and can exist in both an (
R
)-
and (
S
)-form, the oestrogenic activity of both enantiomers
was investigated.
Materials and Methods
Compounds
Oestradiol, oestriol, genistein (4’,5,7-trihydroxyisoflavone)
and daidzein (4’,7-dihydroxyisoflavone) were supplied
by Sigma Chemical Co. Ltd, Poole. Coumestrol (3,9-
dihydroxy-6H-benzofuro-[3,2-c][1]benzopyran-6-one) was
from Acros Organics, NJ and formononetin (7-hydroxy-4’-
methoxyisoflavone) from Extrasynthèse, Genay. The anti-
oestrogen ICI 182,780 was a gift from A. Wakeling (Zeneca
Pharmaceuticals, Macclesfield).
Polyphenolic extracts of hops
Dry hop cones (5 g) were extracted consecutively under
reflux and nitrogen with petroleum ether (200 ml, 30 min;
125 ml, 60 min; 50 ml, 60 min) and
n
-hexane (125 ml,
60 min, three times). All fractions were discarded and the
residue was extracted under reflux and nitrogen with 80 ml
methanol:water (3:1; v/v) for 2 h (three times). This extract
was partitioned with petroleum ether (100 ml, twice) and
n
-
hexane (100 ml, twice), and the aqueous methanolic extract
was adjusted to 100 ml. Individual compounds were isolated
by semi-preparative HPLC (Kontron pump system 32X,
photodiode array detector 440, data system 450-MT2/DAD
series) using a Biosil C18 HL 90-10 (Bio-Rad, Nazareth)
10 ⫻ 250 mm (10 µm) column and a gradient of solvent A
(0.05% (v/v) formic acid in water) and solvent B (5% (v/v)
acetonitrile in methanol). Gradient profile: 0–2 min: isocratic
45% B in A; 2–32 min: 45% B in A to 95% B in A; 32–37
min: 95% B in A; 37–45 min: 95% B in A to 45% B in A;
45–47 min: 45% B in A. Retention times of isoxanthohumol,
8-prenylnaringenin, 6-prenylnaringenin and xanthohumol
were 15.1, 19.4, 23.7 and 26.4 min, respectively.
Preparation of 8-prenylnaringenin
Dry hop cones (300 g) were immersed in dichloro-
methane (2 l) for 2 h. The extract was filtered and the
solvent evaporated. After the addition of 300 ml 70% (v/v)
aqueous methanol, the solution was maintained at –20⬚C
overnight. The precipitate was separated; the filtrate was
concentrated to dryness; the residue was redissolved in
50 ml of 5% (v/v) ethanolic potassium hydroxide; and the
solution was refluxed for 30 min under nitrogen. After
cooling to room temperature, the reaction mixture was
poured into 200 ml 5% (v/v) HCl and extracted twice with
ethyl acetate. The combined extracts were dried (Na
2
SO
4
) and
concentrated. The residue was purified by column chroma-
tography (Silica gel, Merck grade 9385, 60 Å) using ethyl
acetate:cyclohexane (9:1) to give 780 mg of isoxanthohumol
(80%). A cooled solution of boron trichloride (650 µl,
0.65 mmol) in dichloromethane was added to a stirred
solution of isoxanthohumol (15 mg, 0.04 mmol) in acetonitrile
(1 ml) at –78⬚C. The reaction mixture was allowed to warm
up to 0⬚C over a period of 3 h, and was then poured on to
ice and extracted with ethyl acetate. The extract was
washed with water, saturated with NaCl, dried over Na
2
SO
4
and concentrated to dryness. After purification by column
chromatography (Silica gel; ethyl acetate:cyclohexane 8:2),
7.6 mg of 8-prenylnaringenin was obtained (53%).
Separation of (R)- and (S)-8-prenylnaringenin
The enantiomers of the prenylated hop flavanones,
isoxanthohumol, 8-prenylnaringenin, and 6-prenylnaringenin
were separated by chiral liquid chromatography (triacetyl
cellulose (TAC), 4.1 ⫻ 250.0 mm, prepared in the Laboratory
of Separation Sciences, Department of Organic Chemistry,
Ghent University) using methanol as eluent at a flow rate of
0.8 ml min
–1
. UV detection was at 280 nm. The fractions
containing the enantiomers of 8-prenylnaringenin were
collected by semi-preparative chiral HPLC and the solvent
was evaporated. (
R
)-8-prenylnaringenin and (
S
)-8-prenyl-
naringenin were dissolved in methanol (3.4 mg (10 ml)
–1
and 3.3 mg (10 ml)
–1
, respectively) and their CD spectra
were recorded on an Olis DSM 10 CD spectrophotomer
(Olis, Bogart, GA; 1 mmol l
–1
in MeOH).
Determination of oestrogenic bioactivity
in vitro
Hop extracts and pure compounds were diluted in
ethanol. Aliquots (20 µl) were added to individual wells in a
96-well plate and the ethanol was evaporated. Oestrogenic
activity was determined in two separate
in vitro
bioassays
.
The first bioassay used a human endometrial cell line,
Ishikawa Var I (a gift from E. Gurpide, Mount Sinai School
of Medicine, NY) that responds to oestrogenic stimulation
by a marked increase in alkaline phosphatase activity
(Markiewicz
et al
., 1993). Cells (2.5 ⫻ 10
4
cells per well in
100 µl) were plated in 96-well plates in oestrogen-free basal
medium (EFBM; phenol red-free Ham’s F12 and Dulbecco’s
modified eagle’s medium (1:1), and 5% (v/v) charcoal-
236
S. Milligan
et al.
Oestradiol 8-Prenylnaringenin 6-Prenylnaringenin
Genistein Daidzein Coumestrol
HO
O
OOH OH
HO
O
O
OH
OH
O
O
O
HO
OH
CH
3
HO
HO
O
OOH
OH
O
OH
OOH
HO
Fig. 1. Structures of oestradiol and established phyto-oestrogens.

stripped FBS). The anti-oestrogen ICI 182,780 was used in
some wells at a final concentration of 0.16 µmol l
–1
to
evaluate the specificity of the response. Alkaline phosphatase
activity was determined after 72 h by monitoring the
hydrolysis of
p
-nitrophenyl phosphate to
p
-nitrophenol at
405 nm.
Oestrogenic bioactivity was also studied using an
oestrogen-inducible yeast screen (
Saccharomyces cerevisiae
)
expressing the human oestrogen receptor and containing
expression plasmids carrying oestrogen-responsive sequences
controlling the reporter gene lac-Z (encoding the enzyme
β-galactosidase; a gift from J. Sumpter, Brunel University).
Oestrogenic activity was determined from the enzymatic
hydrolysis of chlorophenol red β-
D-galactopyranoside by
monitoring the absorbance at 540 nm (Routledge and
Sumpter, 1996).
Receptor binding
Oestrogen receptor binding activity was studied using
recombinant human oestrogen receptor α and β (ERα and
ERβ) obtained from PanVera Corporation (Madison, WI).
Dilutions of the test compounds were incubated in 100 µl
buffer (10 mmol Trizma preset crystals l
–1
(pH 7.5), 10%
glycerol, 2 mmol
DL-dithiothreitol (DTT) l
–1
, 1 mg BSA ml
–1
)
with 15 nmol [2,4,6,7-
3
H]-oestradiol l
–1
(84.0 Ci mmol l
–1
;
Amersham Life Science, Amersham) and ER (1.5 nmol l
–1
).
The mixture was incubated overnight at 4⬚C, and free and
bound hormone were separated using 100 µl 15% (w/v)
hydroxylapatite slurry (in 50 mmol Tris–HCl l
–1
, pH 7.4,
1 mmol EDTA l
–1
). After three washes in buffer (ERα:
40 mmol Tris–HCl l
–1
, pH 7.5, 100 mmol KCl l
–1
, 1 mmol
EDTA l
–1
, 1 mmol EGTA l
–1
; ERβ: 40 mmol Tris–HCl l
–1
,
pH 7.5), the slurry was extracted with two washes of 200 µl
ethanol and the radioactivity in the extracts was
determined.
In vivo
assays
The relative potency of oestrogenic compounds
in vivo
depends on a number of factors, including the route of
administration and the nature of the response monitored.
Estimates of oestrogenic potency are affected markedly
depending on the nature of the
in vivo
bioassay used.
Therefore, two
in vivo
assays were used: the first assay was
based on the rapid response of the uterine vasculature to
oestrogenic stimulation (Arvidson, 1977; Milligan
et al
.,
1998). The second assay was based on the uterotrophic
response and mitotic responses of the uterine and vaginal
epithelium. In both assays, female Swiss albino mice (A.
Tuck & Son Ltd, Battlesbridge, Essex), approximately 2–3
months of age and weighing 20–25 g, were maintained
under constant conditions of lighting (lights on from 06:00 h
to 18:00 h) and temperature (21 ⫾ 1⬚C), and fed on a
pelleted diet (Economy Rodent Maintenance, Essex)
ad
libitum
. Animal care was undertaken according to UK
Home Office guidelines. All experiments were performed
on ovariectomized animals. Ovariectomies were performed
under tribromoethanol anaesthesia at least 2 weeks before
the start of each experiment.
Acute uterine vascular response
In the assay based on the rapid response of the uterine
vasculature to oestrogenic stimulation, a quantitative index
of the vascular permeability was obtained from the leakage
of radiolabelled albumin from the circulation (Arvidson,
1977; Milligan
et al
., 1998). Three and a half hours after s.c.
injection of the test substance (in 0.1 ml saline), 0.5 µCi
125
I-labelled human serum albumin (50 µl) was injected
into the jugular vein of the mice anaesthetized with
tribromoethanol. After 30 min, a blood sample was taken
from the suborbital canthal sinus with a heparinized
capillary pipette and the animals were killed by cervical
dislocation. The blood sample was centrifuged for 5 min at
1000
g
to provide a 100 µl plasma sample. The uteri and a
sample of thigh muscle were removed, washed briefly in
saline and then weighed. The radioactivity in the uterus,
plasma sample and muscle was determined. Previous
studies on the uterus (Arvidson, 1977; Milligan
et al
., 1998)
have indicated that the blood volume of the uterus is very
much smaller than the albumin space (after a circulation
time of 30 min) and that the extravascular albumin space is
the major determinant of the total tissue albumin space. The
tissue-specific extravascular albumin volume (EAV) was
expressed as a ratio of [
125
I] c.p.m. mg
–1
of tissue:[
125
I]
c.p.m. µl
–1
plasma and used as an index of tissue vascular
permeability. Each day s.c. injections of ICI 182,780
(100 µg (100 µl)
–1
arachis oil) were given to some animals
for 4 days before administration of 8-prenylnaringenin or
oestradiol to investigate whether the vascular responses
could be blocked by an anti-oestrogen.
In vivo
assays of vaginal and uterine mitosis and
uterotrophic response
The oestrogenic potency of 8-prenylnaringenin was
tested after continuous administration in the drinking water
by monitoring the uterotrophic response and cell mitoses in
the vaginal and uterine epithelia. Mice were allocated
randomly (
n
= 6 per treatment) to one of the following
treatment groups: (i) 100 ng oestrogen ml
–1
, (ii) 100, 20, 4
and 0.8 µg 8-prenylnaringenin ml
–1
or (iii) a drinking water
control of 1% (v/v) Tween 80 (polyoxyethylenesorbitan
monooleate) in water; the treatment bottles were weighed
and replaced each day. Uterine and vaginal cell proliferation
were assessed 72 h after the start of treatment. Mice were
injected i.p. with 0.1 ml colchicine (BDH, Poole) in saline
(1 mg ml
–1
) 2 h before they were killed. The uterine horns
were removed, blotted on tissue and weighed. The vaginae
and uterine horns were fixed in Bouin’s solution, embedded
in paraffin wax, cut into serial sections (5 µm), and stained
with haematoxylin and eosin. The number of cells under-
going mitosis in the entire uterine luminal epithelium and
random areas of the basal layer of the vaginal epithelium
were counted from four or more sections (at least 1000 cells
Oestrogenic activity of 8-prenylnaringenin
237

counted for each tissue). The mitotic index (% mitosis) was
calculated by the total number of mitoses divided by the
total number of cells counted ⫻ 100.
Statistical analysis
Results were expressed as mean ⫾ SEM and analysed
initially using one-way ANOVA and the Tukey’s multiple
comparison test. Data that did not pass either the normality
test or the equal variance test were analysed using the
Kruskal–Wallis one-way analysis on Ranks followed by
Dunn’s all pairwise multiple comparison test.
Results
Comparison of the oestrogenic activity of natural and
semi-synthetic 8-prenylnaringenin
Natural (purified from hops) and semi-synthetic 8-
prenylnaringenin showed equivalent oestrogenic activity in
both the Ishikawa Var-I assay and the yeast screen (Fig. 2).
The EC
50
values for oestradiol, semi-synthetic 8-prenyl-
naringenin and natural 8-prenylnaringenin were 0.82 ⫾ 0.01,
4.24 ⫾ 0.01 and 4.41 ⫾ 0.02 nmol l
–1
, respectively, in
the Ishikawa assay and 0.33 ⫾ 0.01, 43.7 ⫾ 1.4 and
40.0 ⫾ 1.3 nmol l
–1
, respectively, in the yeast screen. The
responses of Ishikawa Var-I cells were blocked completely
by 10 µmol ICI 182,780 l
–1
(data not shown). The
in vitro
oestrogenic potency of 8-prenylnaringenin was greater than
that of other established phyto-oestrogens in both assays.
The EC
50
values for oestradiol, 8-prenylnaringenin, 6-
prenylnaringenin, coumestrol, genistein and daidzein were
0.8, 4, 500, 30, 200 and 1500 nmol l
–1
, respectively, in the
Ishikawa cell assay, and 0.3, 40, > 4000, 70, 1200 and
2200 nmol l
–1
, respectively, in the yeast screen.
The oestrogenic activity of (R)- and (S)-8-prenylnaringenin
Resolution of 8-prenylnaringenin by chiral HPLC
revealed approximately equal amounts of the enantiomers
in both natural and semi-synthetic 8-prenylnaringenin (Fig.
3a). The circular dichroism spectrum of peak ‘A’ was almost
identical to that reported for (
S
)-naringenin (Gaffield, 1970),
whereas the spectrum of peak ‘B’ was the reflection of ‘A’.
The two enantiomers showed similar oestrogenic activity in
both the recombinant yeast cell and Ishikawa Var-I bioassays
(Fig. 4c). The enantiomers of 8-prenylnaringenin competed
strongly with oestradiol for binding to both ERα and ERβ
with a relative binding affinity of about 0.01 (oestradiol = 1)
(Fig. 4a,b).
The oestrogenic activity of 8-prenylnaringenin
in vivo
The uterine vascular responses to oestradiol, oestriol and
the phyto-oestrogens are shown (Fig. 4d). Oestriol was only
slightly less effective than oestradiol in inducing a rapid
increase in uterine vascular permeability within 4 h of
administration, but both 8-prenylnaringenin and coumestrol
were considerably less potent (< 1% relative to oestradiol).
The dose–response relationship for 8-prenylnaringenin was
similar to that of coumestrol, and a large stimulatory effect
was produced by 100 nmoles 8-prenylnaringenin. The
amount of genistein required to produce the same effect
was at least ten-fold greater. The administration of daidzein
produced no detectable uterine vascular response at the doses
used. The responses to 8-prenylnaringenin and oestradiol
were blocked completely by prior treatment of the animals
with the anti-oestrogen ICI 182,780 (Table 1). There were
no changes in the vascular permeability of the muscle tissue
control in any treatment group.
In vivo
assays of vaginal and uterine mitosis and
uterotrophic response
The addition of 8-prenylnaringenin or oestradiol to the
drinking water of mice is an effective way of providing
continuous, non-invasive oral administration of these
238
S. Milligan
et al.
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
10
–2
10
–1
10
0
10
1
10
2
10
3
10
4
Concentration (nmol l
–1
)
Absorbance at 405 nm
(a)
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
10
–2
10
–1
10
0
10
1
10
2
10
3
10
4
Concentration (nmol l
–1
)
Absorbance at 540 nm
(b)
Fig. 2. Relative oestrogenic activity of oestradiol (䊉), 8-prenylnaringenin (semi-synthetic; 䊊), 8-prenylnaringenin (natural; 䉲) and other
phyto-oestrogens (coumestrol (䉮), genistein (䊏), daidzein (䊐)) in (a) Ishikawa Var I cells and (b) yeast screen bearing the human oestrogen
receptor. Results are means ⫾
SEM;
n
= 6 wells per point. Where no error bars are visible, the errors were smaller than the symbols.

Oestrogenic activity of 8-prenylnaringenin
239
50
40
30
20
10
0
0.0 2.5 5.0 7.5 10.0 12.5 15.0
Time (min)
mAU
(a)
A
B
17.5
8-Prenylnaringenin
HO
O
OOH
OH
HO
OH O
O
OH
(
R
)-8-Prenylnaringenin(
S
)-8-Prenylnaringenin
(b)
Fig. 3. (a) Enantioseparation of (
S
)-8-prenylnaringenin (peak A) and (
R
)-8-prenylnaringenin (peak B). (b) Absolute structures of the
enantiomers.
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
10
–2
Concentration (nmol l
–1
)
Absorbance at 540 nm
(c)
10
–1
10
0
10
1
10
2
10
3
30
25
20
15
10
5
0
10
–12
Moles injected
Uterine vascular permeability (µl mg
–1
)
(d)
10
–11
10
–10
10
–9
10
–8
10
–6
10
–7
10
–5
120
100
80
60
40
20
0
10
0
Concentration (nmol l
–1
)
[
3
H]Oestradiol bound (%)
(a)
10
1
10
2
10
3
10
4
10
5
120
100
80
60
40
20
0
Concentration (nmol l
–1
)
[
3
H]Oestradiol bound (%)
(b)
10
0
10
1
10
2
10
3
10
4
10
5
ERα ERβ
Fig. 4. Competitive displacement of [2,4,6,7-
3
H]oestradiol from isolated (a) oestrogen receptor α (ERα) and (b) ERβ by oestradiol (䊉) and
(
R
)-8-prenylnaringenin (䊊) and (
S
)-8-prenylnaringenin (䉲). (c) The relative oestrogenic activity of oestradiol, (
R
)-8-prenylnaringenin and
(
S
)-8-prenylnaringenin in the yeast screen bearing the transfected human oestrogen receptor (
n
= 4; mean ⫾ SEM) and (d) the oestrogenic
activity of oestradiol (䊉), oestriol (䊊), 8-prenylnaringenin (䉲), coumestrol (䉮), genistein (䊏) and daidzein (䊐), and the control (䉬) on
uterine vascular permeability in ovariectomized mice 4 h after s.c. administration (
n
= 6–10 per treatment). Vascular permeability was
expressed as (⫻ 100) [
125
I] c.p.m. mg
–1
of tissue/[
125
I] c.p.m. µl
–1
plasma (mean ⫾ SEM).

compounds. There was little variation in the amount of
liquid consumed between treatment groups (3.72 ⫾ 0.26 ml
per mouse per day) and on average, 15.9 mg 8-prenyl-
naringenin per kg per day was ingested by the treatment
group that consumed the highest concentration of 8-
prenylnaringenin. Both 8-prenylnaringenin (100 µgml
–1
)
and oestradiol (100 ng ml
–1
) produced significant increases
in vaginal mitosis after 72 h compared with the negative
control (
P
< 0.05 and
P
< 0.005, respectively; Fig. 5).
However, although oestradiol also produced significant
increases in uterine mass and in epithelial mitosis, there
were no significant differences in the mice exposed to any
of the 8-prenylnaringenin treatments (data not shown).
Discussion
Results from the present study confirm that 8-prenylnaringenin
is one of the most potent phyto-oestrogens known to date
(Milligan
et al
., 1999). The results from the
in vitro
and
in
vivo
bioassays and oestrogen receptor binding assays in the
present study and from that of Kitaoka
et al
. (1998) are
consistent with its oestrogenic activity being considerably
greater than that of genistein or daidzein. In the
in vitro
assays, the oestrogenic activity of 8-prenylnaringenin also
slightly exceeded that of coumestrol. The apparent differences
in potency of 8-prenylnaringenin between the yeast screen
and the Ishikawa cell assays are consistent with similar
observations comparing other compounds in these assays
(Le Gueval and Pakdel, 2001).
In addition to its
in vitro
activity, 8-prenylnaringenin
shows oestrogenic effects
in vivo
. 8-Prenylnaringenin
induces the rapid uterine vascular response typical of
oestrogens and this response is blocked by prior treatment
with an anti-oestrogen. The compound has been reported to
induce uterine growth and to suppress ovariectomy-induced
bone loss in ovariectomized rats when administered each
day s.c. at a dose of 30 mg per kg per day (Miyamoto
et al
.,
1998). In the present study, it was noted that 100 µg 8-
prenylnaringenin ml
–1
in the drinking water (equivalent to
an intake of about 15 mg per kg per day) was able to induce
the characteristic oestrogenic mitotic response in the vaginal
epithelium of ovariectomized mice. The apparent lack of
effect of 8-prenylnaringenin on uterine epithelial mitoses
may reflect either the limited amounts given or the temporal
differences in the mitotic responses induced by oestrogens
in the uterine luminal, uterine glandular and vaginal luminal
epithelia (Finn and Martin, 1973; Finn and Publicover, 1981).
Although it is difficult to compare the continuous exposure
of mice to 8-prenylnaringenin (via oral intake) experimentally
with human exposure to the compound via beer consump-
tion, it has to be noted that the oral concentrations required
to produce an oestrogenic effect in mice are about 500-fold
greater than can be found in any beer (Stevens
et al
., 1999;
Rong
et al
., 2000). In addition, many beers are now made
using extracts from hops or hop products rather than whole
hops: depending on the nature of the hop product used, these
beers have even lower concentrations of 8-prenylnaringenin
or contain no 8-prenylnaringenin at all (Stevens
et al
., 1999;
Rong
et al
., 2000).
A number of phyto-oestrogens (for example, coumestrol,
genistein) have a stronger binding affinity for ERβ than for
240
S. Milligan
et al.
Table 1. The ability of the anti-oestrogen ICI 182,780 to inhibit the increases in uterine vascular permeability (expressed as (⫻ 100) [
125
I]
c.p.m. mg
–1
of tissue per [
125
I] c.p.m. µl
–1
plasma) induced by oestradiol and 8-prenylnaringenin
Treatment Uterus Muscle
Control (arachis oil) 4.70 ⫾ 0.84 0.53 ⫾ 0.02
ICI 182,780 pre-treatment 4.20 ⫾ 0.27 0.47 ⫾ 0.04
Oestradiol (10
–10
mol l
–1
) 12.53 ⫾ 1.17 0.53 ⫾ 0.07
ICI 182,780 pre-treatment + oestradiol (10
–10
mol l
–1
) 5.83 ⫾ 1.73*** 0.61 ⫾ 0.03
8-Prenylnaringenin (10
–7
mol l
–1
) 22.19 ⫾ 1.9 0.54 ⫾ 0.02
ICI 182,780 pre-treatment + 8-prenylnaringenin (10
–7
mol
–1
) 5.96 ⫾ 0.58*** 0.64 ⫾ 0.06
Values are mean ⫾ SEM.
***Significantly different compared with treatment without ICI 182, 780 (
P
< 0.001;
n
= 6 mice per treatment).
10
8
6
4
2
0
Negative
control
Treatments in drinking water
Mitosis in vaginal epithelium (%)
0.8 µg ml
–1
8PN
4 µg ml
–1
8PN
20 µg ml
–1
8PN
100 µg ml
–1
8PN
100 ng ml
–1
E
2
**
*
Fig. 5. Mitotic response of the vaginal luminal epithelium of
ovariectomized mice to 8-prenylnaringenin (8PN) or oestradiol (E
2
)
administered for 72 h in drinking water. Data are mean ⫾
SEM
(
n
= 6 per treatment). Significant differences are denoted by
*
P
< 0.05, **
P
< 0.005 (Kruskal–Wallis test on ranks and Dunn’s
multiple comparison test).

ERα (Kuiper
et al.
, 1997; Casanova
et al.
, 1999), and ERβ
may be an important mediator for the action of phyto-
oestrogens. This study shows that 8-prenylnaringenin can
compete with oestradiol for binding with the ERα and ERβ,
although there was no marked difference in binding affinity
for the two receptors. The relative ligand binding affinities,
tissue distribution and developmental pattern of expression
of ERα and ERβ may help to explain the action of ER
agonists, such as phyto-oestrogens (Brandenburger
et al
.,
1997; Tetsuka
et al
., 1998). (
R
)- and (
S
)-prenylnaringenin
showed comparable activities both in the
in vitro
bioassays
and in the competitive binding assay to ERα and ERβ. The
enantiomers have also been shown to behave similarly in a
competitive binding assay with uterine oestrogen receptors
(Kitaoka
et al
., 1998).
Many natural flavanones prevail in the (
S
)-configuration
and are formed from the corresponding chalcones as a
consequence of the action of chalcone isomerase. However,
the lupulin glands of hops lack chalcone isomerase and the
presence of equal amounts of (
R
)- and (
S
)-8-prenylnaringenin
indicates that the compound may be derived from
xanthohumol or desmethylxanthohumol via a non-enzymatic
route (for example, thermal Michael-type cycloaddition).
However, it is also possible that the precursors are
converted to racemic 8-prenylnaringenin during drying,
storage and extraction of hops (Stevens
et al
., 1999; Rong
et al.
, 2000). The same may also be true for some other hop-
derived prenylated flavanones, as isoxanthohumol and
6-prenylnaringenin also occurred in both (
R
)- and (
S
)-forms
(data not shown). Further conversions and decompositions
may occur during the brewing process and such reactions
may account for the variable amounts of 8-prenylnaringenin
and other prenylflavonoids found in beer (Stevens
et al
.,
1999; Rong
et al
., 2000).
Although 8-prenylnaringenin has also been isolated from
a crude Thai drug derived from the heartwood of
Anaxagorea luzonensis
A. Gray (Annonaceae) (Kitaoka,
1998), hops probably represent the most significant source
of exposure of humans to the compound. In the past, hop
workers would have been exposed to this oestrogenic
compound via inhalation of hop dust or through
transcutaneous absorption rather than via dietary intake and
such exposure may provide an explanation for the
menstrual disturbances in female hop workers (Verzele,
1986). The reported ability of hop extracts to reduce hot
flushes in post-menopausal women (Goetz, 1990) is also
consistent with the presence of oestrogenic activity.
Although hops are used to impart flavour, bitterness and
other properties to beer, the amount of hops used in
brewing is relatively small and, therefore, 8-
prenylnaringenin is not a significant dietary component.
However, it is important to note that hops are now
incorporated into a number of herbal preparations for
women, including some claiming ‘breast enlargement’.
Claims for the effectiveness of at least some such ‘breast
enlargement’ preparations are partly based on their phyto-
oestrogen content. The concentration of 8-prenylnaringenin
in one type of these ‘breast-enhancing’ preparations is
10.9 µggm
–1
, which would result in a daily intake of
approximately 130 µg per day (Sauer and Coldham, 2001).
This value should be compared with the fact that some
beers have been reported to contain concentrations of
240 µg 8-prenylnaringenin l
–1
(although most are below
30 µgl
–1
; Stevens
et al
., 1999). Therefore, although moderate
beer intake could provide a daily intake of 8-prenylnaringenin
in the same range as that provided by some ‘breast
enhancement’ products, it should be noted that there are
no reports of clinical trials demonstrating either the
effectiveness or oestrogenic activity of such herbal
preparations in humans. Indeed, Sauer and Coldham (2001)
found that the supplement tested was inactive in a mouse
uterotrophic assay after administration either in the feed or
by s.c. injection. In addition, the present study showed that
the concentrations of 8-prenylnaringenin required to
produce oestrogenic responses in mice are 100 µgml
–1
,
that is, at least 400-fold greater than found in any beer.
Finally, although there may be interactions between alcohol
intake and the risk of breast cancer (Hulka and Moorman,
2001), there is no evidence linking beer intake with
breast enhancement. Other biological activities of 8-
prenylnaringenin have been described, including anti-
proliferative effects on breast and colon cancer cell lines,
inhibition of cytochrome P450-mediated activation of
procarcinogens, inhibition of bone resorption and inhibition
of diacylglycerol acyltransferase activity (Miranda
et al
.,
1999; Henderson
et al
., 2000). The biological effects, if any,
of consumption of 8-prenylnaringenin via beer or herbal
preparations remain to be clarified.
The authors are grateful to The Leverhulme Trust and to The
Royal Society for supporting this work.
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Received 13 September 2001.
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242
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