Humulus (Lupuli flos)

ASSESSMENT REPORT

FOR HERBAL SUBSTANCE(S), HERBAL PREPARATION(S) OR COMBINATIONS

THEREOF WITH TRADITIONAL USE

HUMULUS LUPULUS L., FLOS

BASED ON ARTICLE 16D(1) AND ARTICLE 16F AND 16H OF DIRECTIVE 2001/83/EC AS

AMENDED (TRADITIONAL USE)

Herbal substance(s) (binomial scientific name

of the plant, including plant part)

Humulus lupulus L., flos

Herbal preparation(s)

Comminuted herbal substance

Liquid extracts and tincture

Pharmaceutical forms

Solid or liquid dosage forms for oral use

Rapporteur

Prof. A. J. Vlietinck

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TABLE OF CONTENTS

I.

REGULATORY STATUS OVERVIEW

2

II.

ASSESSMENT REPORT FOR HERBAL SUBSTANCE/HERBAL

PREPARATIONS WITH TRADITIONAL USE

3

II.1.

INTRODUCTION

5

II.1.1.

Description of the herbal substance(s), herbal preparations or

combination thereof

5

II.1.2.

Information on period of medicinal use in the Community regarding the

specified indication

5

II.1.2.1.

Herbal substance

6

II.1.2.2.

Herbal preparations

6

II.2.

PHARMACOLOGY

8

II.2.1.

Pharmacokinetics

8

II.2.1.1.

Phytochemical characterization

8

II.2.1.2.

Pharmacokinetic data on the herbal substance

9

II.2.1.3.

Pharmacokinetic data on the active compound, 8-PN

11

II.2.2.

Pharmacodynamics

12

II.2.2.1.

Sedative effects

12

II.2.2.1.1. In vitro studies

12

II.2.2.1.2. In vivo studies

12

II.2.2.2.

Oestrogenic effects

14

II.2.2.2.1. In vitro studies

14

II.2.2.2.2. In vivo studies

17

II.2.2.3.

Other pharmacological activities of hop strobiles and/or its constituents

20

II.2.2.3.1. Hop essential oil and hop acids

20

II.2.2.3.2. Hop prenylflavonoids

22

II.2.3.

Interactions

23

II.3.

CLINICAL EFFICACY

23

II.3.1.

Clinical studies

23

II.3.1.1.

Sedative activity

23

II.3.1.2.

Oestrogenic activity

24

II.4.

SAFETY

25

II.4.1.

Toxicity

25

II.4.1.1.

Single dose toxicity

25

II.4.1.2.

Subacute and chronic toxicity

26

II.4.1.3.

Genotoxicity

27

II.4.1.4.

Carcinogenicity

28

II.4.1.5.

Reproductive and developmental toxicity

28

II.4.1.6.

Local tolerance

28

II.4.2.

Side effects

28

II.4.3.

Contra-indications, warnings

29

II.4.4.

Interactions

29

II.4.5.

Overdoses

29

II.5.

ASSESSOR’S OVERALL CONCLUSION

30

III.

ANNEXES

III.1.

Community herbal monograph

III.2.

Literature references

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I.

REGULATORY STATUS OVERVIEW 1

MA: Marketing Authorisation;

TRAD: Traditional Use Registration;

Other TRAD: Other national Traditional systems of registration;

Other: If known, it should be specified or otherwise add ’Not Known’

Member State

Regulatory Status

Comments

Austria

MA

TRAD

Other TRAD

Other Specify:

Belgium

MA

TRAD

Other TRAD

Other Specify: Combi + foodsuppl

Bulgaria

MA

TRAD

Other TRAD

Other Specify:

Cyprus

MA

TRAD

Other TRAD

Other Specify:

Czech Republic

MA

TRAD

Other TRAD

Other Specify: Combi

Denmark

MA

TRAD

Other TRAD

Other Specify: Combi

Estonia

MA

TRAD

Other TRAD

Other Specify:

Finland

MA

TRAD

Other TRAD

Other Specify: Combi

France

MA

TRAD

Other TRAD

Other Specify:

Germany

MA

TRAD

Other TRAD

Other Specify: Combi

Greece

MA

TRAD

Other TRAD

Other Specify:

Hungary

MA

TRAD

Other TRAD

Other Specify:

Iceland

MA

TRAD

Other TRAD

Other Specify:

Ireland

MA

TRAD

Other TRAD

Other Specify:

Italy

MA

TRAD

Other TRAD

Other Specify: Food suppl.

Latvia

MA

TRAD

Other TRAD

Other Specify:

Liechtenstein

MA

TRAD

Other TRAD

Other Specify:

Lithuania

MA

TRAD

Other TRAD

Other Specify:

Luxemburg

MA

TRAD

Other TRAD

Other Specify:

Malta

MA

TRAD

Other TRAD

Other Specify: Combi + foodsuppl.

The Netherlands

MA

TRAD

Other TRAD

Other Specify:

Norway

MA

TRAD

Other TRAD

Other Specify: Foodsuppl.

Poland

MA

TRAD

Other TRAD

Other Specify:

Portugal

MA

TRAD

Other TRAD

Other Specify: None

Romania

MA

TRAD

Other TRAD

Other Specify:

Slovak Republic

MA

TRAD

Other TRAD

Other Specify:

Slovenia

MA

TRAD

Other TRAD

Other Specify:

Spain

MA

TRAD

Other TRAD

Other Specify:

Sweden

MA

TRAD

Other TRAD

Other Specify: Combi

United Kingdom

MA

TRAD

Other TRAD

Other Specify: Combi

1 This regulatory overview is not legally binding and does not necessarily reflect the legal status of the products

in the MSs concerned.

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II

ASSESSMENT REPORT FOR HERBAL SUBSTANCE/HERBAL

PREPARATIONS WITH TRADITIONAL USE

II.1.

INTRODUCTION

II.1.1.

Description of the herbal substance(s), herbal preparations or combinations

thereof

Humulus lupulus L., flos (Strobili), (Hops, Hop strobiles)

* Herbal substance

Botanical species

Humulus lupulus L.

Hops, Houblon (Vigne du Nord), Hopfen, Hop, Luppulo, Lupolo, Lupulo

Botanical family

Cannabaceae

Plant part

Flowers (Strobiles) : dried, generally whole, female inflorescences

(Eur. Ph. 01/2005 : 1222)

Common names

(plant part)

- Flores Humuli lupuli, Strobili humuli, Strobili lupuli, Strobuli lupuli

(Latin)

- Hops, Hop strobiles, (English)

- Cônes de Houblon (French)

- Hopfenblüten, Hopfendolden, Hopfenkätzchen, Humulus-lupulus-

Blütenstände (German)

- Hop (Dutch)

Pharmaceutical forms

Dried inflorescences (strobiles)

Glandulae lupuli (Lupulinum) : Hop grains, Lupulin glands, (English) ;

Lupulin (French), Lupulin, Hopfendrüsen, Hopfenmehl (German) ;

Hopklierharen (Dutch)

Other herbal substances

These globular grains serve as rich reservoirs of secondary metabolites

and they are contained in hop cones that are typical of female plants

(up to 40% of the dry mass).

* Herbal preparations

♦ Dried inflorescences (strobiles) : comminuted herbal substance as such or as infusions

♦ Liquid extract (1:1) : prepared with ethanol/water 45% v/v

♦ Liquid extract (1:10) prepared with sweet wine

♦ Tincture : (1:5) prepared with ethanol/water 60% v/v

II.1.2.

Information on period of medicinal use in the Community regarding the specified

In preparing this report, a number of data sources have been taken into account viz . the ESCOP

monographs published respectively in 1997 and 2003, the bibliographic references made available by

ESCOP at the end of 1997, the monograph of the European Pharmacopoeia in 2005 and the results of

several literature searches carried out in 2007.

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indication

II.1.2.1. Herbal substance

This assessment report reviews the available scientific data, particularly the pharmacological and

clinical studies available for Humulus lupulus L., strobiles (Cannabaceae) or hops.

The dried, generally whole female inflorescences of hops ( Lupuli flores ) are described in the European

Pharmacopoeia (Lupuli flos 01/2005:1222) (2005). They are also described in an ESCOP monograph

in 2003 with a therapeutic indication similar to valerian root viz . tenseness, restlessness and sleep

disorders. Hops are referred to in the herbal literature as having various forms of sedative activity

(Chadwick et al., 2006 and references cited therein namely Fluckiger and Hanbury, 1879, Maisch,

1892, Schleif and Galludet, 1907, Greenish, 1909, Wilcox, 1912, Culbreth, 1927, Washburn and

Blome, 1927, Gathercoal and Wirth, 1936, Youngken, 1950, Meyer, 1960, Millspaugh, 1974,

Blumenthal, 2000). They are often combined with more potent sedative herbs such as valerian, passion

flower and lemon balm for the treatment of sleep disturbances (Blumenthal 2000, Schultz et al., 2001).

More than 99% of the world production of hops is intended for breweries, however, since 1973 it has

been claimed that brewing sludge baths containing ca. 30% hop extracts could be used for the

treatment of a variety of gynaecological disorders (Fenselau and Talahay, 1973). References to the

traditional use of hops in the treatment of such disorders have been identified from the US (Donsback,

1977), Romania (Racz et al., 1980), France (Goetz, 1990) and Iran (Zagari, 1992). Additionally, a

number of journal articles (Bednar and Zenisek, 1961, Strenkovskaya, 1968, Fenselau and Talahay,

1973) and patents exist concerning the use of oestrogenic properties of hops, particularly for external

use in cosmetics. Koch and Heim (1953), following up on the folk legend “that women who normally

live a distance from hop gardens regularly begin to menstruate 2 days after arriving to pick hops”,

reported that hops contain the equivalent of 20-300 µg oestradiol/g. While lacking in detail, this one-

page article is apparently the first confirmation that hops may have oestrogenic activity in humans.

Koch and Heim used a version of the Doisy test, an in vivo assay with castrated female infant rodents

(Hensyl, 1990). Chury (1961) reported that the oestrogenic activity for a saponified ethanol extract of

hops was much more active than those of peas, red clover, and cabbage. Several hop-containing

cosmetic preparations have been patented since that time.

It was not until 1988 that the chemical structure of the oestrogenic principle was established (Hänsel

and Schulz, 1988). The report by Milligan et al. (1999), however, may be regarded as the beginning of

the modern, unambiguous understanding of the in vitro oestrogenic activity of hops. They reported

that hops contain a very potent phyto-oestrogen, 8-prenylnaringenin (8-PN), also called hopein, which

belongs to the class of the prenylflavonoids. This finding has been confirmed by a number of

independent research groups, both by in vivo and in vitro studies (Coldham and Sauer, 2001, Zierau et

al., 2002, Takamura-Enya et al., 2003). The high oestrogenic activity of 8-PN was reported for the

first time in 1998, when the compound was isolated from the heartwood of the tropical tree

Anaxagerea benzonensis A. Gray (Annonaceae) (Kitaoka et al., 1998). 8-PN has also been detected in

some beers, in relatively small quantities, however (levels between 20 µg – 100 µg/L) (Rong et al.,

2000, Schaefer et al., 2003). Since then several review articles on the oestrogenic properties of hops in

comparison to other phyto-oestrogens have been published (Cos et al., 2003, Ososki and Kenelly,

2003).

Hop extracts and/or compounds have also been reported to be active as antioxidants, cancer

chemopreventives, antiinflammatory agents, antimicrobials (antibacterials and antifungals), and

cytotoxics (Chadwick et al., 2006). These indications, however, are substantiated mainly by

pharmacological data and not by clinical studies.

II.1.2.2. Herbal preparations

The dried inflorescences are used in comminuted form as such or in tea mixtures or they are prepared

as infusions.

Lipophilic extracts are used for the preparation of bath oils whereas hydroalcoholic liquid extracts and

tinctures are prepared for internal use as sedatives, mostly in combinations with other sedative plant

extracts. In several cases, the hydroalcoholic liquid extracts are prepared with sweet wine.

Hop strobiles are described in the DAB10, PFX and the BHP. In the latter (1983) a liquid extract (1:1)

in 45% alcohol and a tincture (1:5) in 60% alcohol are given together with their posology. The

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German E Commission describes a monograph on hop strobiles and hop extracts as sedative in 1984

(BAnz nr. 228, dated 05.12.1984).

In Germany marketing authorisations were granted for HMPs containing:

• Fluid extract (1:12.4-12.6) : ethanol 16% m/m (since 1965)

Daily dosage corresponding to 0.8 g herbal substance (HS) (in divided single dosages, 2-3 x daily)

• Fluid extract (1:94-95), sweet wine (since 1978)

Daily dosage corresponding to 0.4-0.6 g HS (in divided single dosages, 2-3 x daily)

• Fluid extract (1:10), sweet wine (since 1978)

Daily dosage corresponding to approximately 5 g HS (in divided single dosages, 2-3 x daily)

• Dry extract (4-5:1), methanol 50% v/v (since 1993)

Daily dosage corresponding to 1-1.7 g HS (in divided single dosages, 2-3 x daily)

In Germany, the herbal substance is part of many combination products with Valerian, Passion flower

and Melissa in solid and liquid pharmaceutical forms.

Consequently, only hydroalcoholic liquid extracts among them wine and tinctures can be accepted as

traditional drugs.

Assessor’s comments

♦ References concerning the traditional medicinal use of hops as a sedative herb date back to 1879

(Fluckiger and Hanbury). Since then many publications up to the present day have appeared in

which hop strobiles as comminuted herbal substance, alone or in tea mixtures or as infusions

continue to be recommended by proponents of herbal medicine for this indication. Unfortunately,

despite numerous attempts the constituents responsible for the sedative effects of hops have yet to

be established, although some information about the biochemical mode of action of hops as

sedative herb is now available.

Besides the dried inflorescences only liquid extracts prepared with ethanol/water (16% and 45%) and

sweet wine, and tinctures (1:5) prepared with ethanol/water 60% have been used in medicinal products

for more than 30 years. Although the fluid extracts (1:12.4-12.6) and (1:94-95), prepared with ethanol

16% m/m and sweet wine, respectively, have been marketed in Germany since 1978, their daily

dosages corresponding respectively with 0.8 g and 0.4-0.6 g herbal substance are not plausible with

the posology of the other herbal preparations. Consequently, these preparations should not be included

in the Community monographs on hops for traditional use.

Dry extracts have only been on the market since 1993 and are consequently not considered to be

traditional.

♦ References concerning the traditional medicinal use of hops as a phyto-oestrogen date back to

Koch and Heim (1953), who confirmed the folk knowledge that the menstruation cycle of female

hop pickers is influenced by picking hops. In 1961 Chury reported the oestrogenic activity for a

saponified ethanolic extract of hops, where after several hop containing cosmetic preparations

have been patented. In 1973 Fenselau and Talahay stated that brewing baths containing hop

extracts (30%) were taken in Germany for the treatment of a variety of gynaecological disorders.

In 1988 the chemical structure of the active oestrogenic principle was elucidated and in 1999 with

Milligan the modern understanding of the in vitro and in vivo oestrogenic activity of hops began.

Since then, various dried extracts prepared with ethanolic-water mixtures have been standardised

on 8-prenyl-naringenin and sometimes other prenylflavonoids. Several pharmacological,

toxicological and clinical studies with these standardised extracts have been carried out to

investigate their usefulness as drugs to alleviate menopausal discomforts.

Although these studies are of interest, the findings are not yet sufficient to support a marketing

authorisation.

Moreover, as these extracts are enriched in 8-prenylnaringenin and prepared with modern techniques

such as supercritical CO 2 extraction, they cannot be considered to be traditional.

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Consequently, a period of at least 30 years of traditional use of hop strobiles and its preparations is

only fulfilled for their sedative effects. Since the use of dried extracts for this indication has only been

introduced since 1993, only hydroalcoholic liquid extracts including also sweet wine and dried

inflorescences as such or as infusions can be accepted as traditional herbal medicinal products.

II.2.

PHARMACOLOGY

II. 2.1.

Pharmacokinetics

II. 2.1.1. Phytochemical characterization

More than 1000 chemicals have been identified from hops including natural products and their

isomeric derivatives (Verzele and De Keukeleire, 1991; Eri et al., 2000; Farnsworth, 2003, Anonymus,

Beilstein Database, 2003).

The oleo-resin (3% - 20%) consists of various prenylated phoroglucinol derivatives called “bitter

acids”. They are classified either as “α-acids” or “β-acids”, which are distinguished by the fact that the

former ones are precipitated from a crude extract of hops by the addition of lead acetate. The β-acids,

by definition, would remain in solution. The α-acids are humulones (2-12% of dried strobile), e.g.

humulone, cohumulone, prehumulone, posthumulone and adhumulone, whereas the β-acids are called

lupulones (1-10% of dried strobile), e.g. lupulone, colupulone, adlupulone, prelupulone and

postlupulone. The α-acids are regarded as the most important constituents in determining the quality

of hops. They contribute to foam stability as well as imparting antibacterial properties. While regarded

as the principal “bitter acids” from hops, they perhaps paradoxically do not have a bitter taste, even at

concentrations of 100 µg/ml. The hop α-acids isomerize to the corresponding “iso-α-acids” under a

variety of reaction conditions e.g. more favourably at higher pH values. These bitter iso-α-acids are

artifacts and comprise more than 80% of all hop components that occur in typical beers. In the same

circumstances the β-acids are transformed into so-called hulupones (Verzele and De Keukeleire,

1991).

The essential oil (0.5-1.5%) consists mainly of simple oxidized alkanes, monoterpenes and

sesquiterpenes. The primary volatile constituents in all cultivars of hops are the monoterpene myrcene

and the sesquiterpenes β-caryophyllene and humulene (57-82% of the volatile oil) (Eri et al., 2000).

In terms of their traditional economic value, the volatile oil and bitter acids comprise the most

significant classes produced by hops. Candidate sedative compounds in hops have been put forward

viz . the bitter acids humulone and lupulone (Sikorski and Rusiecki, 1938), their degradation product

2’-methyl-3-buten-2-ol (Hänsel et al., 1982) and myrcene (Rao et al., 1990, Lanzotti et al., 1991).

The alcohol, however, is only present in trace amounts in freshly harvested hop strobiles, but it

increases of ± 0.15% of the dry weight up to 20% of the volatiles after two years due to degradation of

the bitter acids (Hänsel et al., 1982 ; Wohlfart et al., 1982a, 1983b ; Hölz, 1992).

Despite numerous attempts to characterize the sedative active constituents in hops, it is believed that

the identity of all compounds responsible for this activity of hops in humans, has yet to be determined

(Schulz et al., 2001).

A third class of compounds consists of flavonoids (0.5-1.5%) including quercetin and kaempferol

glycosides (McMurrough et al., 1981 ; De Cooman et al., 1998) and about 30 prenylated, geranylated,

oxidized and/or cyclized chalcones. The most abundant chalcones are xanthohumol (X) (up to 1% of

dried strobile and 80-90% of total flavonoids) and desmethylxanthohumol (DMX). These chalcones

are readily isomerized to the corresponding flavanones viz . respectively isoxanthohumol (IX) and a

mixture (ca. 3:2) of 6-prenylnaringenin (6-PN) and 8-prenylnaringenin (8-PN) (25-60 mg/kg) (Stevens

et al., 1997, 1999a, 1999b, 2004 ; Rong et al., 2000, Milligan et al., 2000).

The principle oestrogen from hops is the (1:1) racemate of 8-PN or hopein, formed along with

the less active 6-PN (Milligan et al., 1999).

IX is the main prenylated flavonoid in beer, since X is largely isomerized during particular stages

(at elevated temperatures) in the course of brewing (Stevens et al., 1999). These five prenylated

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flavonoids viz . X,DMX,IX, 6-PN, and 8-PN constitute a set of interesting bioactive compounds, since

they have shown their ability to inhibit proliferation of several cancer cell lines in vitro (Miranda et al.,

1999, De Keukeleire et al.,2001,Gerhauser et al., 2002).

Other constituents of hops include amino acids and proteins (15%), polysaccharides (50-60%),

minerals, phenolic acids such as chlorogenic and caffeic acids, lipids and condensed tannins (2-4%)

(Verzele and De Keukeleire et al., 1991, Hölz, 1992).

II. 2.1.2. Pharmacokinetic data on the herbal substance

So far no in vivo pharmacokinetic investigations were performed on hop extracts due not only to the

complexity of the phytochemical composition but also to the many metabolisations of the different

compounds of hops which take place after intake in humans.

Recently, however, several research teams have published their findings on the metabolism of

8-prenylnaringenin (8-PN), xanthohumol (X) and isoxanthohumol (IX). A first indication on the

ADME characteristics of these three compounds can be deduced from CaCo-2 experiments performed

by Bracke et al. (2006). They determined “the apparent permeability coefficient” (P app ) as an important

parameter for the absorption efficacy through the gut epithelium. In this in vitro system.

P app > 1x10 6 cm/s values are correlated with a good absorption, whereas values p app < 1x10 7 cm/s are

correlated with weak absorption (< 1%). For values in between, there is no real relationship between

P app and absorption, but the absorption is considered between > 1% and < 100% (Grès et al., 1998).

8-PN and IX showed P app values of respectively 1,3x10 -6 cm/s and 3,3x10 -6 cm/s, showing that these

compounds could have a good bioavailibity, whereas the P app of X (6,7x10 -7 cm/s) showed that it

might be less bioavailable. Beside the information on the absorption efficacy, the CaCo-2 system also

indicated that phase-II metabolites (glucuronidation and sulphation) are formed. A similar study has

been published by Nikolic et al. (2006). The apparent permeability coefficients were 5.2 ± 0.7 x 10 -5

and 4.9 ± 0.5 x 10 5 cm/s respectively, indicating a good absorption via passive diffusion. According to

these authors, the P app values are similar to those of drugs such as propanolol and testosterone, which

are often used as high permeability standards.

The metabolism of prenylated flavonoids is not very well documented. The biotransformation of

flavonoids is done at several places in the body, but mainly in the liver and the gut lumen, so that the

gut flora might play a significant role as well. As cytochrome P450-enzymes (CYP) are abundantly

present in the liver, they also are important elements in the metabolization process of flavonoids

(Yilmazer et al., 2001a).

Several studies showed that flavonoids are mostly excreted as glucuronides in humans and animals.

Glucuronidation is the most important reaction route for the phase II detoxification process for most of

the xenobiotics. The functionalization is catalysed by the membrane-bound

UDP-glucuronosyltransferase, mainly catalysed in the endoplasmatic reticulum of the liver. In an

in vitro study with rat-derived and human liver microsomes, the glucuronidation of X was studied.

Two important glucuronidates were characterized as respectively C-4’- and C-4-monoglucuronides of

X. It is also important to mention that the conjugated flavonoids still have a significant biological

activity (e.g. antioxidant effects) (Yilmazer et al., 2001b).

As for 8-PN, an in vitro study with human liver microsomes has revealed several metabolites. LS-MS

analysis showed that the most important breakdown products are the result of oxidation. In this

process, a hydroxyl group can appear on the prenyl chain, but also a modification of the flavanone

skeleton has been observed. The most frequent metabolites were oxidation products of the prenyl

group, in which the terminal methyl function was hydrolysed. This is in contrast to xanthohumol,

which did not show a similar metabolism (Nikolic et al., 2004 ; Zierau et al., 2004).

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Guo et al. (2006) performed a study on the identification of human hepatic cytochrome P450 enzymes

involved in the metabolism of 8-PN and IX from hops. CYP2C19 was found to catalyze the formation

of both cis- and trans-alcohols (phase I metabolisation) of the prenyl side chain of 8-PN. CYP2C8

converted 8-PN regioselectively to the trans-alcohol of the prenyl group. Finally, CYP1A2 was found

to catalyze the O-demethylation of IX to generate 8-PN. The enzymes CYP1A2, CYP2C19 and

CYP2C8 exhibit polymorphism in humans. The results obtained by Guo et al. suggest that the

oestrogenicity of hop constitutents in vivo will depend in part on metabolic conversion that may show

individual variation.

The possibility that IX would act as a pro-oestrogen was considered by Coldham et al. (2002).

The assumption was based on the extensive biogransformation capacity of the liver, which includes

demethylation. However, the exposure of IX to liver microsomes did not lead to an increase in

oestrogenic activity, from which it was concluded that no 8-PN was produced. In contrast,

Nikolic et al. (2005) describe that liver microsomes can demethylate IX, but not X. However, it was

shown that, besides demethylation, microsomes also modify the prenyl side-chain, finally resulting in

a large variation of minor degradation products. Schaefer et al. (2005) identified low levels of 8-PN in

urine after oral intake of IX by two test persons and attributed this to demethylation by the liver.

Besides the liver, the colon is also an important transformation site in the human body. The human

colon contains ∼ 10 12 micro-organisms/cm 3 (about 400 different species), with an enormous catalytic

and hydrolytic potential. The importance of this microbial community in the metabolism of phyto-

oestrogens in general has been clearly established. Wang et al. (2000) identified two bacteria

responsible for the transformation of lignans and Decroos et al. (2005) recently isolated a microbial

consortium capable of transforming the soy phyto-oestrogen daidzein into equol. Moreover, several

intestinal bacteria were shown to enhance the bioavailability of phyto-oestrogens as they possess

β-glucosidases, which are necessary for the hydrolysis of phyto-oestrogen glycosides

(Rowland et al., 2003). Thus, the gut microbiota are considered to be a factor of utmost importance for

phyto-oestrogen bioavailability (Turner et al., 2003). In view of the importance of the microbial

microflora observed with other well-known phyto-oestrogens, the group of Verstraete (University of

Ghent, Belgium) has launched a study to investigate the most important substances in the hops extract

(IsoX, 8-PN and X) in a SHIME reactor (SHIME or ‘ S imulator of the H uman I ntestinal Mi crobial

E cosystem’). Parts of the results have already been published by Possemiers et al. (2005). The results

obtained showed that certain bacteria in the gut are able to O-demethylate IX, resulting in 8-PN

formation. In an experiment with faecal cultures, this conversion was observed in one-third of the

samples, indicating the importance of interindividual variability in the intestinal microbial community.

These results stress the importance of the gut microbial community toward the final biological activity

of phyto-oestrogens, a factor that should not be neglected when determining the final active dose

inside the body (Possemiers et al., 2006).

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II. 2.1.3. Pharmacokinetic data on the active compound, 8-PN

The pharmacokinetic data which can be found in literature, are mostly on xanthohumol (X). They

show that the bioavailability of X is rather low (Nookandeh et al., 2004 ; Avula et al., 2004). X and its

metabolites are excreted mainly in faeces within 24 h of administration.

Schaefer investigated the pharmacokinetics of 8-PN in rats and dogs in his Ph.D. work (2004). The

full ADME profile and bioavailability profile (using synthetic 8-PN) that he proposes can be found in

the thesis. Briefly, they found that the ingested 8-PN is almost completely absorbed after ingestion.

Still, systemically circulating 8-PN concentrations were only 5% of the ingested amount, indicating a

strong action (presystemic elimination) before the 8-PN could go into the systemic circulation.

The 8-PN in the systemic circulation is also bound to serum proteins (sex hormone binding globulins

in dogs). This binding would be less likely for X, in view of its non-oestrogenic characteristics. Also

Nikolic et al. (2004, 2006) concluded that the bioavailability of 8-PN was reduced significantly by

intestinal and hepatic metabolism.

The comparatively high metabolic stability of 8-PN and its pronounced presystemic elimination via

the bile resulted in enterohepatic recirculation in both rats and dogs. The concentration profile in the

serum after oral uptake showed two maxima: one after 1 h and another (higher concentration) after

2 to 4 h, which is an indication of the enterohepatic concentration. About 50% of the ingested amount

was recovered unchanged in the faeces. The excretion mainly goes via the biliary route, as renal

excretion (unchanged and conjugated 8-PN) is limited, especially after oral administration.

The pharmacokinetic results obtained in rats and dogs were more or less confirmed in a human study

performed by Rad et al. (2006). The study was performed using a randomized, double-blind, placebo-

controlled, dose-escalation design with three groups of eight healthy postmenopausal women. In each

group six subjects received 8-PN and two subjects placebo. 8-PN was given orally in doses of 50, 250

or 750 mg. Drug concentrations in serum, urine and faeces were measured up to 48 h. Serum

concentrations of free 8-PN showed rapid drug absorption and secondary peaks suggestive of marked

enterohepatic recirculation. Independent of the treatment group, approximately 30% of the dose was

recovered in excreta as free compound or conjugates over the 48 h observation period. The first C max

and AUCO-48 h showed dose linearity with ratios of 1:4,5 : 13.6 (C max ) and 1:5,2 : 17.1 (AUC).

Assessor’s comments

The in vitro studies indicated extensive liver biotransformation of X, IX and 8-PN upon absorption.

However, the extent of dietary polyphenol absorption in the small intestine is rather limited (10-20%),

thereby implying that a large proportion reaches the colon. Naringenin, the non-prenylated analogue of

8-PN, showed intensive biotransformation in the intestine, including ring cleavage and

dehydroxylation, followed by absorption and urinary excretion. The extent of degradation strongly

depended on compound concentration and individual composition of the gut microflora of the

different human subjects. On the other hand, when X was fed to rats, it was mainly recovered in

unchanged form from the faeces (89%).

The finding that IX may be 0-demethylated by human hepatic cytochromes P450 to form the potent

phyto-oestrogen 8-PN is of considerable importance. In addition, under the acidic conditions of the

stomach X can be cyclized to form IX and provide yet another route for the formation of 8-PN. Based

on these results, IX and X should be included among the compounds used for standardisation of hop

extracts to be evaluated for oestrogenicity in vitro and in vivo.

The pharmacokinetic profile of 8-PN was studied in rats and dogs and later in human females. It was

shown that 8-PN was rapidly and almost completely absorbed after oral administration. The free

substance is then taken up in the jejunum, enters the portal circulation and goes to the liver. Thereafter,

besides unchanged 8-PN which goes into the systemic circulation, 8-PN undergoes mainly phase II

metabolisation and the hydrophilic conjugates formed are mainly eliminated in the bile.

Bacteria in the gut can deconjugate the phase II metabolites, and lipophilic substances can be taken up

again and go to the enterohepatic circulation. Free substance is mainly excreted via the faeces, but also

conjugates are excreted from the blood with urine.

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In conclusion, the pharmacokinetic profile of 8-PN is characterized by rapid and probably complete

enteral absorption, a pronounced presystemic elimination with subsequent enterohepatic recirculation,

dose linear pharmacokinetics and competively little phase I metabolism. Conjugation to

β-glucuronides or sulphates seems to constitute the main metabolic pathway. Intestinal and hepatic

phase I metabolism plays a minor role in the inactivation of 8-PN. This is in complete contrast to other

natural or synthetic oestrogens, whose decreased oral bioavailability is due to intestinal conjugation

and hepatic metabolic inactivation.

The studies also indicate that 8-PN is transformed into a wide array of metabolites, and some of these

products may have different pharmacological activity than their precursors. It would, therefore, be of

interest to determine the biological activities and potential toxic effects of these metabolites,

particularly since it is known that hydroxylated metabolites of 17-β-oestradiol (mainly the 16-OH

metabolite) are much more active, thereby increasing the risks for oestrogen-dependent cancers.

II. 2.2.

Pharmacodynamics

II. 2.2.1. Sedative effects

II.2.2.1.1. In vitro studies

Over the past decade considerable pharmacological research has been carried out on hop strobiles and

its constituents particularly with respect to oestrogenic activity. However, the publications on in vitro

studies relating to the sedative effects of hops are very scarce.

On the contrary, some spasmolytic effects have been found for hops.

An alcoholic extract of hop strobile (1 g of dried drug in 10 ml of 70% ethanol) produced a strong

spasmolytic effect on isolated smooth muscle from guinea pig intestine with ED 50 values equivalent to

37x10 -6 g of hop strobile per ml for acetylcholine-induced contractions compared to 60x10 -9 g/ml with

atropine, and 39x10 -6 of hop strobile per ml for bariumchloride-induced contractions compared to

57x10 -7 g/ml with papaverine. The extract also inhibited contractions of rat uterus with an ED 50

equivalent to 31x10 -6 g of hop strobile per ml (Caujolle et al., 1969).

Also an effect on the calcium flux has been detected.

A methanolic extract from hop strobile showed strong inhibitory activity on calcium fluxes, inhibiting

depolarization-induced 45 Ca 2+ uptake in clonal rat pituitary cells by 94,7% at 20 µg/ml (p<0.001).

The activity was attributed to prenylated flavonoids, although individual compounds from hop strobile

have not so far been tested in this way (Rauha et al., 1999).

Recently, Meissner and Häberlein (2006) investigated the influence of xanthohumol (X) on the

binding of muscimol-Alexa-Fluor 532 (Mu-Alexa), a fluorescently labeled GABA A receptor agonist

by fluorescence correlation spectroscopy. An incubation of hippocampal neurons with 75 nM of X

increased the specific Mu-Alexa binding with ca. 17%, which was selectivly found in GABA A

receptor Mu-Alexa complexes with hindered lateral motility [D bound2 = (0,11 ± 0,03) µm 2/ s ) ]

as described with midazolam, a benzodiazepine agonist. It was further shown that the modulatory

activity of X on the GABA A receptor was not mediated via an interaction with benzodiazepine

receptors. The authors concluded that X may play an important role for the sedative effects of hop

preparations.

II.2.2.1.2. In vivo studies

Old reports have indicated that preparations of hops have sedative-like activity in frogs (Staven-

Groenberg, 1928 ; Munch et al., 1933 ; Steidle, 1932), pigeons (Sikorski and Rusiecki, 1938), gold

fish (Bouchardy, 1953) and golden carp (Grumback and Mirimanoff, 1955). According to Wohlfart

(1982, 1993), however, these animal tests show many methodological shortcomings.

Only a few animal experiments have been carried out to study the supposed sedating effects of hop

extracts in recent times. Following gavage of different extracts from hops or lupulone, Hänsel and

Wagener (1967) observed no indication of sedation in mice or rats. They used three hop extracts, two

produced with ethanol and another one with methylisobutylketone. Both ethanolic extracts were dried

and administered in oil. Locomotor activity was unaffected up to doses of 500 mg/kg bw and no

antagonistic effect against metamphetamine-induced stimulation was observed. Hexobarbital-induced

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sleeping time remained unchanged. In the Rotarod test up to 200 mg/kg bw caused no impairment of

coordination, and no muscle relaxation was observed.

On the contrary, Bravo et al. (1974) described a reduction in locomotor behaviour of mice following

the intraperitoneal administration of three different extracts of hops (aqueous, ethereous and

alcoholic). The ethereous one was the most active, since it completely inhibited mice motor activity,

20 minutes after injection. Since, however, this effect was only seen in very high doses

(200 mg extract/20 g mouse) it cannot be responsible for the claimed sedative effect in patients. On the

other hand, this study clearly showed that the sedative effect of hops is strongly dependent on the type

of solvent used for the extraction procedure.

Lee et al. (1993a) observed a dose-dependent suppression of spontaneous locomotor activity in mice

after the intraperitoneal administration of 100 mg/kg (p<0.05), 250 mg/kg and 500 mg/kg (p<0.001) of

hop extract (96% ethanolic dry extract). The same authors further investigated the CNS effects of hop

extract, using other behavioural tests such as potentiation of pentobarbital-induced sleep, hypothermic

analysis and anticonvulsant tests (Lee et al., 1993b). Pentobarbital-induced sleeping time increased

dose-dependently ; not significant at 100 mg/kg, by 1,9-fold at 250 mg/kg (p<0,05) and 2,6-fold at

500 mg/kg (p<0,01). In the hot plate test, latency time for licking the forepaws increased with doses of

100 and 250 mg/kg (p<0,01). Rotarod performance decreased by 59% and 65% respectively at

250 mg/kg, and 500 mg/kg (p < 0,001 after 120 minutes). The time to onset of convulsion and survival

time after administration of pentylenetetrazole were significantly lengthened by 500 mg/kg (p<0,001),

but not by 250 mg/kg. A significant and time dependent fall in rectal temperature was observed after a

dose of 500 mg/kg (p<0,001 after 120 minutes). Thus hop strobile extract showed sedative and

hypnotic properties at lower doses (100-250 mg/kg), and at a higher dose of 500 mg/kg it also

produced anticonvulsive and hypothermic effects.

The relevance of these findings remains unclear, since all experiments were carried out after

intraperitoneal injection and no experiments were done following oral application, so that the

bioavailability of the preparation used remains questionable.

In a recent publication (Zanoli et al., 2005), the central effects of a hop CO 2 extract and a fraction

containing the α-acids were studied in animal experiments following oral application of the hop

extract dissolved in Tween 80 (10%) or the α-acid fraction dissolved in peanut oil. Acute effects on

locomotor activity and pentobarbital sleeping time were studied, as well as behavioural parameters in

the elevated maze and the forced swimming test. The authors observed a significant increase of the

pentobarbital-induced sleeping time in rats without affecting the latency to the loss of the righting

reflex. This effect was dose-dependent starting from a minimal dose of 10 mg/kg. Neither the extract

nor its α-acid fraction effected the locomotor activity in the open-field test or excerted an anxiolytic

effect in rats submitted to the elevated plus- maze test.

Interestingly, both hop CO 2 extract and the α-acids containing fraction significantly reduced the period

of immobility in the forced swimming test, when administered three times (24 h, 5 h and 1 h), before

the test indicating an antidepressant activity.

It was concluded that hop CO 2 extract and a hops containing α-acids fraction exert a pentobarbital-

enhancing property without influencing the motor behaviour of rats and an antidepressant activity.

These results seem to show that the α-acids present in hop CO 2 extract can explain the use of hops in

sleep disturbances and that the α-acids could be a new class of compounds for the development of

natural antidepressant agents.

More recently, Schiller et al. (2006) confirmed the sedating effects of the liphophilic extract reported

by Zanoli et al. (2005). They investigated several ethanolic extracts (40% v/v and 90% m/m) and

CO 2 extracts from diverse hop varieties as well as α-acids and β-acids fractions and pure hop oil.

All hop extracts increased ketamine-induced sleeping time in mice. The increase in duration of

ketamine narcosis proved to be a specific central effect and not caused by a pharmacokinetic

interaction, as could be confirmed by a comparable increase in ether-induced sleeping time. In contrast

to the findings of Zanoli et al. they also observed a reduction of locomotor activity. The low doses

Zanoli et al. applied (20 mg/kg/bw as maximum) may be the reason for this discrepancy.

Like Zanoli et al. no anxiolytic effects of hop preparations were found. A decrease in body

temperature induced by all hop extracts as additional parameter was observed, which confirms the

sedating activity of the hop preparations. The results of Schiller et al. are similar to those of Lee et al.

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(2003), who found a marked sedation viz . a reduction of locomotor activity, increase of pentobarbital-

induced sleeping time and antagonistic effects against pentylenetetrazol-induced convulsions

following intraperitoneal injection of a hop extract. On the contrary, the results of Hänsel and

Wagener (1967) are in clear contrast to the findings of Schiller et al. (2006). The latter authors explain

this discrepancy by the use of different raw materials, different conditions of storage by which the

content of α- and β-acids might be reduced significantly and different extraction solvents used.

Finally, Schiller et al. showed that not only the α-acids, but also the β-acids and the hop oil, although

to a lesser extent, exert distinct sedating effects and contribute to the activity of the plants. They also

do not rule out the presence of further sedating components in hops.

A degradation product of the bitter acids, humulones and lupulones, the five carbon olefinic alcohol,

2’-methyl-3-buten-2-ol, given intraperitoneally to mice at high dosage, 800 mg/kg showed central

nervous depressant activity (Hänsel et al., 1980 ; Wohlfart et al., 1983a). Although present only in

small amounts in fresh hops, higher levels of this compound may be generated in vivo by metabolism

of the bitter acids, reaching its maximum concentration after 2 years of storage at room temperature

(Hänsel et al., 1982 ; Wohlfart et al., 1982, Wohlfart, 1983b, Hänsel and Schultz, 1986)). The sedative

effect of this alcohol is comparable, in the same dosage range, to that of the structurally related drug

methylpentynol (Wohlfart et al., 1983a).

Thus, if this compound can fully explain the sedative activity attributed to hops, than it must be

formed in vivo from hop constituents such as bitter acids, that would then be considered as

“pro-drugs” analogous to the case of the oestrogenic activity (Chadwick et al., 2006).

The hypothesis, however that this alcohol can be formed in vivo by metabolisation of α-acids has not

been demonstrated to date (Zanoli et al., 2005). Similarly the proposal that the sedative effect of hops

was due to its content of myrcene, which has shown to have analgesic activity in mice (Hänsel and

Wohlfart, 1980) has not been established.

Recently, Grundmann et al. (2006) showed that the hypothermic effects of hops could be antagonized

with the competitive melatonin receptor antagonist luzindole. Based upon a study in which it was

found that a combination of valerian and hops interacts with serotoninergic 5-HT 4e , 5-HT 6 , 5-HT 7 and

melatoninergic ML 1 and ML 2 receptors (Abourashed et al., 2004 ; Butterweck et al., 2007, Brattström,

2007), these authors evaluated the hypothermic activity of hop extract in mice. In a dosage of

250 mg/kg hops extract significantly decreased body temperature in male BL6/C57 mice (ΔT-1,1°C)

2 h after oral administration. The effects of the plant extract were comparable to melatonin (50 mg/kg,

ΔT-0,8°C, 2 h after i.p. injection). The hypothermic effects of both, melatonin and hop extract could

be antagonised with the competitive melatonin receptor antagonist luzindole.

The authors concluded that this data suggest that the hypothermic effects of hop extract are mediated

through activation of melatonin receptors. Since it is known that melatonin has both hypnotic and

hypothermic effects at physiological levels and that the hypnotic effect may be mediated via the

hypothermic action of melatonin (Zemlan et al., 2005), a similar effect may be suggested for hop

extract. The authors also concluded that neither the α- and β-acids, nor the essential oil were

responsible for these effects (Personal communication by Butterweck).

II. 2.2.2. Oestrogenic effects

II. 2.2.2.1. In vitro studies

II.2.2.2.1.1. Oestrogenic activity of hop strobiles

Circumstantial evidence over many years, including menstrual disturbances reported to be common

among female hop pickers, linked hop strobiles with potential oestrogenic activity (Verzele, 1986 ;

De Keukeleire et al., 1999). In Germany, hop baths were used to treat gynaecologic disorders and hop

extracts have been reported to reduce hot flushes in menopausal women (Goetz, 1990). However,

early studies to confirm this activity experimentally were unconclusive or contradictory due to

methodology of inadequate sensitivity (De Keukeleire et al., 1997, 1999).

In a recent screening of plant drugs for oestrogenic activity, a 50%-ethanolic extract (2 g of hop

strobile to 10 ml) exhibited binding to oestrogen receptors in intact, oestrogen-dependent [ER(+)],

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human breast cancer MCF-7 cells with a potency equivalent to 0,5 µg of oestradiol per 2 g of dried

strobile (for comparison, the potencies of 2 g of thyme or red clover were equivalent to 0,5 or 3 µg of

oestradiol, respectively).

The extract also showed significant ability to stimulate cell proliferation in ER(+)T47D, but not in

ER(-)MDA 468, breast cancer cells (Zava et al., 1998). In contrast, in a different series of experiments,

a similarly-prepared extract of hop strobile at concentrations of 0,01-1,0% V/V was found to

significantly inhibit serum-stimulated growth of ER(+)T47D breast cancer cells (p<0,001)

(Dixon-Shanies and Shaikh, 1999).

Ovarian cells isolated from immature female rats, which 48 hours previously had been injected

(primed) with pregnant mare’s serum gonadotrophin, were incubated with follicle-stimulating

hormone to induce oestradiol secretion. Addition to the culture medium of purified water-soluble

fractions F 1 or F 2 from defatted hop strobile extract reduced the amounts of oestrogen E 2 released from

the ovarian cells (p<0,01) with a probably related decrease in cAMP release (p<0,05)

(Okamato and Kumai, 1992).

In 2005 Overk et al. compared the oestrogenicities of the extracts of hops and red clover ( Trifolium

pratense ) and those of their individual constituents, including respectively prenylated flavanones and

isoflavonoids, using a variety of in vitro oestrogenic assays. The hop extract consisted of a chloroform

partition of a methanolic extract from a previously SF-C0 2 -extracted Nugget hops cultivar and the red

clover extract was an ethanolic extract containing 30% isoflavonoids prepared for a phase II clinical

trial.

The IC 50 values for the oestrogen receptor α and β binding assays (according to Obourn et al. (1993)

and Liu et al. (2001)), were 15 and 27 µg/ml, respectively for hops and 18,0 and 2,0 µg/ml,

respectively for the red clover extract.

Both of the extracts demonstrated also significant activities of transiently transfected ERE-luciferase,

quantitative RT-PCR of an oestrogen-inducible gene, and AP-enzyme induction assays (EC 50 values of

1,1 for hop extracts and 1,9 µg/ml for red clover extracts).

II.2.2.2.1.2. Oestrogenic activity of 8-prenylnaringenin (8-PN)

8-PN, a flavanone occurring in hop strobiles at levels of 25-60 mg/kg (Rong et al., 2000), has been

shown to be a potent phyto-oestrogen with activity greater than that of other established plant

oestrogens (Milligan et al., 1999).

Oestrogenic activity of a much lower order (less than one-hundredth of that of 8-PN) has also been

detected in three other hop flavanones, 6-prenylnaringenin (6-PN), 8-geranylnaringenin (8-GN) and

6,8-diprenylnaringenin (6,8-PN), while xanthohumul (X) and isoxanthohumol (IX) were found to be

inactive (Milligan et al., 2000). EC 50 values for 17β-oestradiol, 8-PN, 6-PN, coumestrol, genistein and

daidzein were 0,3, 40, > 4000, 70, 1200 and 2200 nM, respectively in a screen using oestrogen-

inducible yeast ( Saccharomyces cerevisiae ) expressing the human oestrogen receptor, and

0,8, 4, 500, 30, 200 and 1500 nM respectively, for stimulation of alkaline phosphatase activity in a

human endometrial cell line (Ishikawa Var I). The relative binding affinities of 17β-oestradiol,

8-PN, coumestrol and genistein with rat uterine cytosol containing soluble oestrogen receptor

were 1, 0023, 0008 and 0,003 respectively (Milligan et al., 1999).

The high oestrogenic activity of 8-PN was confirmed by Zierau et al. (2002) using two different

bioassays viz . a yeast based oestrogen receptor assay, containing a stably transfected oestrogen

receptor α (ERα) construct and an expression plasmid carrying oestrogen-responsive sequence

controlling the reporter gene lac-Z encoding the enzyme β-galactosidase and a transactivation assay

using MVLN cells, a bioluminescense MCF-7 derived cell-line.

The same research group showed using a yeast-based androgen receptor assay strong anti-androgen

activities for 8-PN and 6-(1,1-dimethylallyl)naringenin (6-DMA-N), while the parent compound

naringenin was inactive. In an androgen receptor activity-assay based on the analysis of putative

specific antigen (RSA) concentrations in the supernatants of treated PC3(AP)2 cells only

6-DMA-N showed antiandrogenic activity at concentrations of 10 -5 . 8-PN and naringenin had no

detectable antiandrogenic effects.

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6-DMA-N is a structurally related compound, isolated from the African tree Monotes engleri

(Zierau et al., 2003).

The oestrogenic activity of 8-PN was further confirmed in competitive binding assays using purified

human recombinant oestrogen receptors α and β (ER α and ER β ). 8-PN competed strongly with

17β-oestradiol for binding to both receptors with a relative binding affinity of about 0,1, compared to

1,0 for 17β-oestradiol and 0,001 for 8-GN (Milligan et al., 2000).

In another study, involving displacement of [ 3 H ]-17β-oestradiol, 8-PN showed competitive binding

affinity for the oestrogen receptor in bovine uterine cytosol with an IC 50 of 140 nM, compared to

1,0 nM for 17β-oestradiol and 320 nM for genistein. 8-PN also dose-dependently stimulated the

proliferation of cultured, oestrogen-dependent, human breast cancer MCF-7 cells with an EC 50 of

1,9 nM, compared to 0,0032 nM for 17β-oestradiol and 47 nM for genistein, suggesting that it was an

oestrogen receptor agonist (Kitaoka et al., 1998).

The same authors synthetized racemic 8-PN, separated and assayed both enantiomers, and reported

that there was no significant difference in ER binding potency between the 2R and the 2S forms

(Kitaoka et al., 1998).

In an in vitro receptor binding assay, using recombinant human ER α and ER β from cytosolic SF 9 -cell

extracts, Schaefer et al., (2003), showed that 8-PN exhibited > 2-fold higher affinity for ER α than ER β .

Using a mammalian cell-based transactivation assay consisting of U 2 -osteosarcoma cells transient

transfected with either ER α or ER β and a luciferase reporter gene construct these authors demonstrated

that 8-PN is the strongest plant-derived ER α agonist identified so far, being 10-fold more potent than

coumestrol and 100-fold stronger than genistein, but only 70 times weaker than 17β-oestradiol.

The transactivational analysis revealed also a >3,6-fold higher oestrogenic activity of 8-PN at ER α

than ER β , a strong contrast to primarily ERβ-activating coumestrol and genistein.

At the same time they found that the in vivo oestrogenic activity of 8-PN in reproductive tissue was

about 20,000-fold weaker compared to 17β-oestradiol.

As such, 8-PN mimics the effects of the endogenous hormone and could be a natural SERM (selective

oestrogen receptor modulator) with promising potential for the treatment of various oestrogen

deficiency-related conditions (Schaefer et al., 2003).

In 2004 Bovee et al. developed a rapid yeast oestrogen bioassay stably expressing oestrogen receptors

α and β and green fluorescent proteins and tested various phyto-oestrogens. They confirmed the

earlier results of Schaefer et al. that 8-PN is relatively more potent with ER α and less potent than

coumestrol and genistein with ER β .

In 2005 Overk et al. compared the oestrogenicities of the extracts of hops and red clover and those of

their individual constituents viz . 8-PN, 6-PN, IX and X from hops and daidzein, formononetin,

biochanin A and genistein from red clover.

The results of the extracts have been discussed under point II.2.2.2.1.1. Here we give the results of the

individual constituents.

The competitive ER α and ER β binding assays using tritiated 17β-oestradiol are based on the method of

Obourn et al. (1993) with minor modifications (Liu et al., 2001). As already mentioned before the red

clover extract preferentially bound to the ER β receptor nine times greater than to ER α . The hop extract

had nearly a 2-fold preference for the ER α as compared with ER β . For the isolated compounds, the

relative binding affinities for ER α were as follows :

genistein ≈ 8-PN > daidzein > biochanin A > formononetin > IX, while the relative binding affinities

for ER β were genistein > 8-PN ≈ daidzein > biochanin A > formononetin ≈ IX.

Genistein and 8-PN had similar affinities for ER α with IC 50 values of 0,51 and 0,3 µM respectively,

whereas genistein was more selective for ER β as indicated by the IC 50 value of 0,020 and 1,7 µM,

respectively. Both of the extracts, genistein and 8-PN activated the oestrogen response element (ERE)

in Ishikawa cells, while the extracts, biochanin A, genistein, and 8-PN significantly induced

ERE-luciferase expression in MCF-7 cells.

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Hop and red clover extracts as well as 8-PN up-regulated progesterone receptor (PR) m-RNA in the

Ishikawa cell line. In the MCF-7 cell-line, PR m-RNA was significantly up-regulated by the extracts,

biochanin A, genistein, 8-PN and IX. The two extracts had EC 50 values of 1,1 and 1,9 µg/ml

respectively in the alkaline phosphatase induction assay. The EC 50 values of the individual compounds

are consistent with values reported by Milligan et al. (1999) viz . 8 PN >> genistein ≈ daidzein > XI >

biochanin A. None of the compounds had anti-oestrogenic activity.

II.2.2.2.2. In vivo studies

II.2.2.2.2.1. Antigonadotrophic effects of hop strobiles

Purified water-soluble fractions from defatted hop strobile extract were administered subcutaneously

twice daily for 3 days to immature female rats primed with 25 IU of pregnant mare’s serum

gonadotrophin (PMSG). None of the fractions induced a change in uterine weights. However, fractions

F 1 (20 mg/rat) and F 2 (50 mg/rat) significantly suppressed PMSG-induced gain in ovarian weights by

about 25% (p<0,05) compared to controls. Under the same conditions, two further fractions (4 mg/rat)

purified from F 1 suppressed gain in ovarian weights by 42% and 33% (p<0,01) compared to controls

(Kumai and Okamoto (1984). In further experiments on PMSG-primed immature rats, by comparison

with saline-treated control animals, subcutaneously administered fractions F 1 and F 2 reduced the

number of ovulations (p<0,05) ; suppressed levels of serum luteinizing hormone (p<0,001) ;

suppressed thymidine kinase activity in uterine tissue (p<0,01) ; reduced 17β-oestradiol E 2 secretion in

cultures of ovarian cells from the rats (p<0,001) ; and reduced progesterone production in cultures of

luteal cells from the rats (p<0,05 to p<0,001) (Okamato and Kumai, 1992).

To date no other in vivo studies on the oestrogenic effects of hop strobiles have been published.

II.2.2.2.2.2. Oestrogenic effects of 8-prenylnaringenin (8-PN)

In vivo studies with 8-PN are scarce. Only one report highlighted favourable effects of hops on hot

flushes in humans (Goetz, 1990), but preclinical studies aimed at evaluating the efficacy of 8-PN for

alleviating menopausal symptoms and discomforts are still ongoing. Most investigators until now have

focused on animal models. It should be noticed that the relative potency of oestrogenic compounds

in vivo largely depends on a number of factors, including the route of administration and the nature of

the response monitored. Moreover, estimates of oestrogenic activity are markedly affected depending

on the nature of the in vivo bioassay used. Milligan et al. (2002) tested 8-PN in two in vivo assays viz.

an assay based on the rapid response of the uterine vasculature to oestrogenic stimulation and an assay

based on the uterotrophic reponse and mitotic responses of the uterine and vaginal epithelium. In both

assays ovariectomized female Swiss albino mice, about 2-3 months of age, were used.

In the first assay, a quantitative index of the vascular permeability was obtained from the leakage of

radiolabelled albumin from the circulation. Several test substances were subcutaneously administered

to the ovariectomized mice and after 4 hours a significant increase in the vasular permeability was

obtained for oestriol and 17β-oestradiol, but both coumestrol and 8-PN were considerably less potent

(< 1% relative to 17β-oestradiol). The dose-response relationship for 8-PN was similar to that of

coumestrol, and a large stimulatory effect was produced by 100 mmoles 8-PN. 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 of 8-PN

and 17β-oestradiol were blocked completely by prior treatment of the animals with anti-oestrogen

(ICI 182, 780).

In the second assay the oestrogenic potency of the test substances was tested after continuous

administration in the drinking water by monitoring the uterotrophic response and cell mitosis in the

vaginal and uterine epithelia.

Both 8-PN (100 µg/ml) and 17β-oestradiol (100 ng/ml) produced significant increase in vaginal

mitosis after 72 h compared with the negative control (p<0,05 and p<0,005, respectively). However,

although 17β-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-PN treatments.

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They concluded that in addition to its in vitro activity 8-PN shows oestrogenic effects in the two

in vivo test systems. It was noted that 100 µg 8-PN/ml 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-PN on uterine epithelial

mitosis may reflect with the limited amounts given or the temporal differences in mitotic responses

induced by oestrogens in the uterine luminal, uterine glandular and vaginal luminal epithelia of

ovariectomized mice (Finn and Publicover, 1981).

Coldham and Sauer (2001) showed that a dietary supplement for breast enhancement containing hop

extracts was only weakly active in mouse uterotrophic assays following administration in feed or after

subcutaneous injection of the extract at doses of 8-PN up to 250 times higher than that recommended

for women. They concluded that the dietary supplement is unlikely to produce oestrogenic effects

in vivo at the level of the uterus.

In other experiments ovariectomized rats, as a model for oestrogen deficiency induced osteoporosis,

were treated subcutaneously with racemic 8-PN (OVX + 8-PN) at 30 mg/kg/day or with

17β-oestradiol (OVX + OE) at 0,01 mg/kg/day, or with vehicle only (OVX). Another group of rats

was sham-operated, i.e. subjected to ovariectomy surgery without removing the ovaries, and treated

with the vehicle only (sham).

After 2 weeks of treatment, 24-hour urine samples were collected and body weight gain, uterine

weight and bone mineral density were determined. The uterine weights of sham and OVX + 8-PN rats

were found to be 165% higher (p<0,001), and of OVX + OE rats 235% higher (p<0,001), than those of

OVX rats.

Body weight gains of sham, OVX + OE and OVX + 8-PN were significantly lower (p<0,05) than

those of OVX rats. Urinary excretion of hydroxyproline (a conventional marker of bone resorption)

was 1.62 µg/g/day from OVX rats compared to 1,18 and 1,16 µg/g/day for sham and OVX + OE rats

respectively (p<0,01), and 1,01 µg/g/day from OVX + 8-PN rats (p<0,001). The levels of urinary

hydroxypyridinium cross-links (assayed as pyridinoline and deoxypyridoline), which are recognized to

be directly related to bone matrix degradation, were significantly lower in sham rats (p<0,01), and in

OVX + OE and OVX + 8-PN rats (p<0,001), than in OVX rats.

Bone mineral densities of 139,1, 141,9 and 141,9 mg/cm 2 in sham, OVX + OE and OVX + 8-PN rats,

respectively, were significantly higher (p<0,001) than that in OVX rats (132,1 mg/cm 2 ). It was

concluded that 8-PN functions as an oestrogen receptor agonist in reproductive tissues and that the

dosage used had completely prevented ovariectomy-induced bone loss (Miyamoto et al., 1998).

Recently, the influence of 8-PN on oestrogen-related genexpression in liver and uterus tissues was

investigated in rats (Diel et al., 2004). At a 100-fold dose with respect to 17β-oestradiol, 8-PN induced

a qualitatively similar, but less pronounced expression profile. On the other hand, 8-PN was found to

be more potent than 17β-oestradiol in inducing expression of IGFBP-1 (Insulin growth factor binding

protein-1). This factor has been correlated to an improvement of the vascular endothelial function and

blood pressure homeostasis (Laughlin et al., 2004), while higher concentrations of IGFBP-1 are also

associated to a decreased risk of prostate cancer (Ngo et al., 2003).

Schaefer et al. (2003) determined the oestrogenic potency in vivo and in vitro of 8-PN using the classic

uterine growth assay and vagina growth assay in juvenile rats. Their findings confirm the conclusions

of Diel et al. (2004) that 8-PN is a pure oestrogen agonist in vitro , exhibiting an oestrogenic activity

profile comparable to oestron. The in vivo oestrogenic activity of 8-PN in representative tissue,

however, is 20,000-fold weaker compared to 17β-oestradiol. They also suggested that 2S(-)-8-PN has

moderately higher ER affinity and oestrogenic activity, in vitro and in vivo than 2R(+)-8-PN.

In 2005 Hümpel et al. studied 8-PN in adult ovariectomized rats, an established animal model to

mimic hormone dependent osteoporosis in menopausal women. The study demonstrated that 8-PN can

completely protect from ovariectomy induced bone-loss while exhibiting minimal (dose independent)

trophic effects on uterus and endometrium. It is estimated that at equivalent bone protective doses of

17β-oestradiol and 8-PN, the phyto-oestrogen has a 10-fold lower stimulatory effect on uterus and

endometrium. The tissue specific effect of 8-PN was confirmed in a transgenic receptor mouse model

(ERE-LUC mice). Here they also found pronounced oestrogenic activity in prostate.

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In a recent in vivo study using the medaka sex reversal/vtg gene expression assay no oestrogenic

effects of all the naringenin-type flavonoids including also 8-PN were observed. Natural sex steroid

hormones and isoflavonoids such as genistein, on the contrary, can functionally reverse the phenotype

sex of fish (Zierau et al., 2005).

Assessor’s comments

Sedative effects

♦ Although no in vitro studies, directly related to the sedative activity of hop strobiles have been

published, nevertheless several studies in animals have been carried out to investigate the

neuropharmacological properties of Humulus lupulus , traditionally used in the treatment of

different CNS disorders such as insomnia, excitability and restlessness.

One of the studies showed that hop strobile extracts exert sedative and hypnotic properties in mice

at lower doses (100-250 mg/kg) and produce other activities such as anticonvulsive and

hypothermic effects at higher doses of 500 mg/kg.

Hop extracts were able to prolong the pentobarbital sleeping time without affecting the latency to

the loss of the righting reflex in rats, confirming the first study concerning the hypnotic properties

and the traditional observation of sleepiness in hops-pickers. In the same study it was shown that

the α-acids were responsible for the pentobarbital sleep-enhancing properties as well as for the

antidepressant activity found for the hop strobile extract.

In a recent study, carried out with several ethanolic and CO 2 extracts of hops, administered by

gavage to mice, it was clearly shown that all extracts reduced the spontaneous locomotor activity,

increased the ketamine-induced sleeping time and reduced body temperature, confirming a central

sedating effect. No indications of anxiolytic activity were found for any of the test preparations.

The sedating activity could be attributed to three categories of constituents of lipophilic hop

extracts. Though the α-bitter acids proved to be the most active constituents, the β-acids and the

hop oil clearly contributed to the sedating effect of lipophilic hop extracts as well.

The authors also suggested that the contradictory results obtained by different research teams in

the determination of different pharmacological effects was due to the use of different raw

materials, different storage conditions leading to various amounts of active components and the

use of different extraction solvents for the production of the preparations.

Furthermore, candidate molecules for the sedative effects of hop strobiles such as myrcene and

2’-methyl-3-buten-2-ol have been suggested, but their role as sedative agents has not been

substantiated.

Recently, however, it has been shown that one of the chalcones viz . xanthohumol influences the

GABA A receptors and their lateral mobility at hippocampal neurons in a similar way as the

benzodiazepine agonist midazolam without interfering with the benzodiazepine receptors.

Thus, xanthohumol may play an important role for the sedative effect of hop preparations, but this

should be confirmed by in vivo studies.

More recently the in vitro study in which it was suggested that a combination of hops and valerian

interacts with melatoninergic ML 1 receptors, was confirmed by an in vivo investigation on mice.

It was shown that hop extracts (250 mg/kg) had comparable hypothermic effects as melatonin

(50 mg/kg), which could be blocked by the competitive melatonin receptor antagonist luzindole.

Since it is known that the hypnotic effect of melatonin may be mediated via its hypothermic

action, a similar effect for hops may be suggested.

The authors also concluded that this effect was not due to the acids or the essential oil of hops,

since both were absent in the hop extract used in this study.

Oestrogenic effects

♦ In contrast to the studies on the sedative effects, the phyto-oestrogen, responsible for the

oestrogenic activity of hop strobiles has been isolated and identified as the prenylated flavanone,

8-prenylnaringenin or hopein (8-PN). The oestrogenic activity proved to be considerably greater

than that of established phyto-oestrogens such as coumestrol (present in red clover) and genistein

and daidzein (present in soy). The oestrogenicity has been examined in great detail by a number

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of independent research groups worldwide. The other prenylated flavonoids were extremely

weakly oestrogenic or devoid of any oestrogenicity. 8-PN exerts its activity through oestrogen

receptor-mediated mechanisms. It binds very strongly to both oestrogen receptor isoforms

(ER α and ER β ), but in contrast to most known phyto-oestrogens, 8-PN is selective for the

oestrogen receptor-α. As such it mimics the effects of the endogenous 17β-oestradiol. Both have

similar profiles, however, the activity of 8-PN is 5- to 100-fold weaker depending on the test

system and the particular reaction conditions.

8-PN is orally active in ovariectomized mice as a significant increase in vascular permeability was

observed within 4 hours after subcutaneous injection of both 17β-oestradiol and a 100-fold dose of

8-PN. Administration of 17β-oestradiol (100 ng/ml) or 8-PN (100 µg/ml) to the drinking water

showed, after 71 h, a considerable increase in vaginal mitosis. Although 17β-oestradiol caused

also a substantial increase in uterus weight and in epithelial mitosis, significant differences with

8-PN were not observed.

In other experiments, ovariectomized rats were injected with 8-PN over a period of 14 days.

A favourable effect on bone metabolism was observed. Removal of the ovaria normally results in a

drastic increase in levels of bone resorption markers in urine, a decrease in mineral bone density

and a reduction in uterus weight. Treatment with 17β-oestradiol (0,01 mg/kg/day) or 8-PN

(30 mg/kg/day) gave quantitatively comparable effects on bone and uterus showing that 8-PN

functions as an oestrogenic agonist.

The influence of 8-PN on oestrogen-related gen expression in liver and uterus tissues was

investigated in rats. At a 100-fold dose with respect to 17β-oestradiol, 8-PN induced a

qualitatively similar, but less pronounced expression profile. On the other hand a stronger

upregulation of the expression of IGFBP-1 was observed. This growth factor has been correlated

to an improvement of the vascular endothelial function and blood pressure homeostasis, while

higher concentrations of IGFBP-1 are also associated with a decreased risk of prostate cancer.

In conclusion, 8-PN, present in hop strobiles at levels of 25-60 mg/kg, should be considered to be the

active oestrogenic compound of hops. It should be noticed that desmethyl-xanthohumol serves as

pro-oestrogen, since it can be metabolised into a mixture of 8-PN and 6-PN.

II. 2.2.3. Other pharmacological activities of hop strobiles and/or its constituents

In a very comprehensive review article, De Keukeleire and Heyerick (2005) discussed the intriguing

biological activities of hops and its constituents.

II.2.2.3.1. Hop essential oil and hop acids

♦ Antimicrobial activity

Some antimicrobial activity of hop essential oil has been reported (Racz et al., 1980 ; Grange and

Davey, 1990 ; Langezaal et al., 1992 ; Simpson and Smith, 1992 ; Ohsugi et al., 1997 ; Tagashira et

al., 1997 ; Matos et al., 2001) and hops are included in various cosmetic preparations. The lupulones

(β-acids) have a longstanding reputation as antibacterials (against gram-positive bacteria), which had

led to some applications of hop products rich in β-acids, particularly in the sugar industry. It appears

that the three prenyl groups present in lupulone interfere with the building up and the functioning of

the bacterial cell walls leading eventually to leakage of the cell contents.

Resistance of gram-negative bacteria to the resin acids is attributed to the presence of a phospholipid-

containing outer membrane, as lupulones and humulones are inactivated by serum phospholipids

(Teuber and Schmalreck 1973). Structure-activity relationship studies have indicated the requirement

of a hydrophobic molecule and a six-membered central ring for such activity (Schmalreck et al.,

1975).

The acids are thought to possess little activity towards fungi or yeasts. However, antifungal activity

has been documented for the bitter acids towards Trichophyton, Candida, Fusarium and Mucor species

(Mizobuchi and Sato, 1984).

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♦ Antidiabetic activity

Isohumulones are the main bittering principles in beer. Recently, interesting antidiabetic properties of

isohumulones have become apparent (Kondo, 2003). Via a reporter system, it was found that agonistic

effects were exerted on the activity of the nuclear receptors PPARalpha (‘Peroxisome Proliferator-

Activated Receptor-alpha’) and PPARgamma. The PPAR’s are important regulators of the glucose

and fat metabolisms and agonists are applied to treat non-insulin-dependent diabetes (diabetes type II)

and hyperlipidemia. Administration of isohumulones to a mouse model for diabetes type II (KK-A y )

led to a decrease of plasma triglycerides and free fatty acids in a dose-dependent manner. Moreover,

the glucose levels were significantly diminished in KK-A y -mice. The results indicate that intake of

isohumulones may favourably influence conditions of hyperglycemia and hypertriglyceridemia.

Treatment of 20 subjects suffering from mild diabetes with isohumulones (twice a day during

12 weeks), in dosages that were equivalent to only a few glasses of strongly hopped beers, showed a

decline in glucose levels and other parameters, indicating that oral intake of isohumulones ameliorates

insulin sensitivity In patients with diabetes type II.

♦ Activity on osteoporosis

Humulone, the major constituent of the mixture of α-acids, has been shown to inhibit bone resorption

using an in vitro “pit formation assay” (formation of pits on dentine slices incubated with mouse bone

cells). Xanthohumol and humulone have been identified as inhibitors of bone resorption at

concentrations at or above 10 -6 and 10 -11 , respectively (p<0,01).

Humulone showed high inhibitory activity with an IC 50 of 5,9x10 -9 M (Tobe et al., 1997a). These

findings indicate that hops may be active against osteoporosis.

It should be noted, however, that xanthohumol and especially humulone, have no oestrogenic

activities, which may indicate that the inhibition of bone resorption of hops is not associated with

oestrogenicity (De Keukeleire et al., 1999).

♦ Antinflammatory activity

Humulone also proved to be a potent inhibitor of the expression of cyclooxygenase-2 (COX-2) via

interaction with NFκB, which translates into pronounced anti-iflammatory activity (Yamamoto et al.,

2000). Also in vivo anti-inflammatory effects have been seen for humulone, which seemed to be the

active anti-inflammatory agent of hop strobiles.

A dry methanolic extract of hop strobile, applied topically at 2 mg/ear, inhibited

12- 0 -tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema in mice by 90% (p<0,01) six hours

after TPA treatment. Humulone, isolated from hop strobile by bioassay-guided fractionation and

identified as an anti-inflammatory constituent, inhibited the oedema with a ID 50 of 0,2 mg/ear

(ID = inhibitory dose).

Topically-applied humulone also inhibited arachidonic acid-induced inflammatory ear oedema in mice

with an ID 50 of 2,2 mg/ear (p<0,01 against controls) compared to 0,4 mg/ear (p<0,01) for indometacin

(Yasukawa et al., 1993).

♦ Antiproliferative activity

Humulone has been found to inhibit angiogenesis (formation of new blood vessels, which is essential

for tumour growth), as well as proliferation (uncontrolled growth) of endothelial cells (Shimamura

et al., 2001). The latter effect has been confirmed by an in vivo test, in which tumour promotion could

be inhibited.

Humulone applied topically at 1 mg/mouse to the backs of mice markedly inhibited the tumour-

promoting effect of TPA on 7,12-dimethylbenz[ a ]-anthracene-initiated skin tumour formation. In the

control group 100% of mice developed tumours (first tumour appeared in week 6), compared to only

7% in the humulone-treated group (first appearance in week 16). Humulone treatment resulted in a

99% reduction in the average number of tumours per mouse at week 18 (p<0,01) (Yasukawa et al.,

1995).

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II.2.2.3.2. Hopprenylflavonoids

♦ Cancer chemopreventive activity

8-Prenylnaringenin (8-PN) is effective in the aggregation of MCF-7/6 breast cancer cells, which

indicates that the compound may inhibit metastasis (Rong et al., 2001). Moreover, it was shown that

8-PN inhibits angiogenesis in vitro and in vivo (Pepper et al., 2004). Various tests using different

cancer cell lines showed no toxicity of 8-PN at levels below 50 µg, while antiproliferative activities

were also observed (Tokalov et al., 2004).

Xanthohumol (X) and to a lesser extent dehydrocycloxanthohumol (DHX) and isoxanthohumol (IX)

show a broad spectrum of inhibitory mechanisms at the initiation, promotion and progression stage of

carcinogenesis (Gerhauser et al., 2002).

On examining proliferation of human breast cancer cells (MCF-7 and MDAMB 231), colon cancer

cells (HT-29), prostate cancer cells (LNCaP and PC-3) and cancer cells of the ovaries (A-2780), a

dose-dependent inhibition of the growth of all cancer cells was noted on addition of low

concentrations of X (low micromolar range) (Miranda et al., 1999 ; Gerhauser et al., 2001a ; 2002).

The activity is correlated with inhibition of DNA polymerase-alpha, the only eukaryotic enzyme

capable of initiating DNA synthesis de novo . In the presence of X , many cancer cells undergo

apoptosis and programmed cell death. An additional antiproliferative mechanism was found in HL-60

leukemia cells (Gerhauser et al., 2002). Terminal cell differentiation is induced whereby cancer cells

start to behave normally again. In these tests, X was 10-fold more active than genistein present in soy.

Inhibition of carcinogenesis ex vivo was also demonstrated for X using so-called mouse mammary

gland organ cultures. In this model system, breast tissue is removed from mice and small cancer

lesions are caused by addition of a potent carcinogen. Extremely low concentrations of X (low

nanomolar range) completely inhibited formation of these lesions (Gerhauser et al., 2002).

Interestingly, resveratrol, a stilbenoid-type polyphenol held responsible for a number of health-

beneficial effects of red wine, was 210-fold less active in this assay.

X showed an inhibitory activity on specific cytochrome P450 enzymes that mediate the conversion of

procarcinogens to carcinogens (Miranda et al. 2000a ; Henderson et al., 2000). Also, it was observed

that X induces the activity of the enzyme quinone reductase in vitro , which is indicative of

detoxification of a variety of carcinogens (Miranda et al., 2000b ; Dietz et al., 2005). The compound

was able to scavenge reactive oxygen species and to interfere with formation of radicals. On trapping

of hydroxyl and peroxy radicals, X was 9- and 3-fold, respectively, more active than a well-known

reference, Trolox (Gerhauser et al., 2001b), while inhibition of oxidation of low-density lipoproteins

(LDL) was more effective than that caused by alpha-tocopherol (Miranda et al., 2000c).

Interestingly, studies have shown that X has anti-inflammatory properties by inhibition of the activities

of cyclooxygenases (COX-1 and COX-2) that account for the production of prostaglandins

(Gerhauser et al., 2002).

♦ Effects on the synthesis of triglycerides

Xanthohumol (X) considerably interferes in the synthesis of triglycerides. The activity of

diacylglycerol transferase, a liver enzyme which is involved in the formation of glycerides, was

substantially inhibited (Tabata et al., 1997). This interaction is associated with positive effects on

hypertriglyceridemia including a decreased risk for associated diseases such as atherosclerosis and

diabetes. It was very recently found, using HepG2 cells as model system, that X decreased

apolipoprotein B secretion in a dose-dependent manner under both basal and lipid-rich conditions

(Casaschi et al., 2004). Furthermore, X inhibited the synthesis of triglycerides in the microsomal

membrane and the transfer of newly synthesized triglycerides to the microsomal lumen indicating that

triglycerides availability is a determining factor in the regulation of apolipoprotein B secretion. The

inhibition of triglycerides synthesis is caused by a reduction in diacylglycerol transferase activity

suggesting that X is a potent inhibitor of apolipoprotein B secretion.

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Assessor’s comments

Besides the most potent phyto-oestrogen currently known with possible health-beneficial properties

(8-PN), hops also contains xanthohumol, the quantitatively most important prenylchalcone (up to

1,3% m/m), which displays potential therapeutic properties (Gerhauser and Frank, 2005)

This compound shows a wide spectrum of inhibition mechanisms at all stages of carcinogenesis. Low

concentrations of X dose-dependently inhibit the growth of cancer cells among them breast cancer

cells (MCF-7 and MDAMB-231) and cancer cells of the ovaries (A-2780).

Consequently, if hop-based phytotherapeuticals would be developed as potential alternatives to

hormone replacement therapy for menopausal women, not only the high oestrogenic potential of 8-PN

should be taken into account, but also the cancer chemopreventive activity of xanthohumol.

Standardisation of both active components in hop-derived extracts is therefore recommended.

The studies described in this assessment report may be considered as a rationale for further studies and

trials to establish efficacy.

II. 2.3.

Interactions

Limited evidence from one animal study suggests that hops may potentiate the effects of sedative

drugs (Lee et al., 1993b).

Interactions with other phyto-oestrogens such as alfalfa, black cohosh, bloodroot, burdock, kudzu,

licorice, red clover, soy and others, are theoretically possible. It should be noted, however, that like

17β-oestradiol, 8-PN is an agonist, whereas phyto-oestrogens containing isoflavonoids as active

oestrogenics are known to be mixed agonists/antagonists. Soy isoflavonoids act as agonists when no

endogenous oestrogens are available, but become antagonists when high endogenous levels of

oestrogens are available.

Laboratory research shows that oestrogen-like substances in hops may have effects on oestrogen-

sensitive parts of the body. It is not clear what interactions may occur when hop extracts are used

together with other hormonal therapies such as birth control pills, hormone replacement therapy,

tamoxifen, or aromatase inhibitors such as letrozole, because no studies have been concluded to

confirm this. Hops may interfere with the way the human body processes certain drugs using the

liver’s ‘cytochrome P450’ enzyme system, so that the levels of certain drugs may be decreased in the

blood, but they have not been confirmed by studies. Clinical cases of drug interactions with hops have

not been reported to date.

II.3.

CLINICAL EFFICACY

II. 3.1.

Clinical studies

II. 3.1.1. Sedative activity

No clinical studies have been conducted to date with hops or hop preparations as single component

products for the treatment of restlessness or insomnia.

Four non-controlled and three placebo or reference-controlled, double-blind clinical studies in patients

suffering from non-organic sleep disorders have been conducted with a fixed extract combination

(Ze 91019) of valerian root and hop strobile extracts. Both extracts were prepared with 45% methanol

m/m with a dry extract ratio of 5-3:1 (valerian) and 6.6:1 (hops), respectively. (Brattström, 1996,

Lataster and Brattström, 1996, Flesch, 1997, Füssel et al., 2000, Notter et al., 2003, Rodenbeck and

Hajak, 1998, Schellenberg et al., 2004 and Koetter et al., 2007).

In addition, one non-controlled and four placebo or reference-controlled double-blind clinical studies

are reported using a related fixed combination product consisting of dry extracts of valerian root and

hops (Wegener et al., 2003, Schmitz and Jäckel, 1998, Morin et al., 2005, Leathwood et al., 1982,

Müller-Limmroth and Ehrenstein, 1977).

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Assessor’s comments

Preclinical and clinical studies are available for combinations of hops with other sedative plants such

as valerian, passion flower and lemon balm. Consequently, separate monographs on combinations will

be elaborated.

II. 3.1.2. Oestrogenic activity

As described in previous sections, preclinical in vitro and in vivo tests suggested that hop extracts

exert an oestrogenic activity.

Before the publication in 1999 by Milligan et al., only one small clinical trial specifically related to the

oestrogenic properties of hops had been identified. In this placebo controlled study, 20 patients

experiencing hot flushes due to ovarian insufficiency (15 in menopausal phase and 5 following

ovariectomy), were treated with a dry aqueous extract of hop strobile (5:1), initially at 1,6-2,6 g/day,

later in some cases reduced to 1,2-1,6 g/day. Five other patients received placebo. Assessment was

based on scores calculated by multiplying the intensity of hot flushes (scale 1 to 3) by their frequency

(scale 1 to 9). In verum patients, the initial average score of 22,7 decreased to 8,2 after 30 days of

treatment, whereas in placebo group, the initial score of 20 decreased only to 18. Compared to the

placebo group, 76% of the verum patients achieved a statistically significant improvement in scores

and 7 out of 20 patients achieved a reduction of at least 15 points (Goetz, 1990). While the author

reported that the formulation was effective in treating hot flushes, it was neither chemically nor

biologically standardized by any modern standards (Chadwick et al., 2006).

Recently, a first prospective, randomized, double-blind placebo-controlled study on the use of a hydro-

alcoholic standardized hop extract to alleviate menopausal discomforts was published (Heyerick et al.,

2006). The hop extract was standardized on 8-PN (100 or 250 µg), which may seem a small amount,

but comparison of 8-PN with established phyto-oestrogens indicated that 8-PN had several 100-fold

more potent oestrogenic activity than e.g. soy isoflavones such as genistein and dadzein, which have

been administered in daily doses of 50 to 100 mg (Milligan et al., 1999). It was therefore deduced that

amounts of 100 µg of 8-PN might have considerable efficacy. The hop-based capsules contained hop

extract that was obtained by an aqueous ethanolic extraction of spent hops following extraction of

hops using SC-C0 2 (food supplement, Menohop R ).

In this three-armed clinical trial 67 patients experiencing mild to severe menopausal discomforts

(more specifically at least 2-5 hot flushes per day corresponding to a score of at least 2 for the item

“hot flushes” on the modified Kupferman-index (KI) during several weeks), were treated with the hop

extract standardized on 100 µg 8-PN (n:20), the hop extract standardized on 250 µg 8-PN (n=20) or

placebo (n=26) over 12 weeks. Assessment was based on the responses obtained using a modified KI

and a patients questionnaire.

All groups, including placebo, showed a significant reduction of the KI both after 6 and after

12 weeks. The hop extract at 100 µg 8-PN was significantly superior to placebo after 6 weeks

(p=0,023) but not after 12 weeks (p=0,086). No dose-response relationship could be established, as the

higher dose (250 µg) was less active than the lower dose both after 6 weeks and after 12 weeks. Still, a

trend for a more rapid decrease of KI was noticed for both active groups as compared to placebo.

In particular, the decrease in hot flushes score (isolated from KI) was found significant for both

treatment groups after 6 weeks (p<0,01) with respect to placebo. Results of the patient’s questionnaire

were consistent with those of the KI, with the most pronounced effects being observed for the 100 µg

treatment. The authors concluded that a daily intake of a hop extract, standardized on 8-PN, exerted

favourable effects on vasomotor symptoms and other menopausal discomforts. Since, however, no

dose-response relationship could be established, further clinical studies are needed to confirm the

present observation.

A randomized, double-blind cross-over study is in preparation (Erkolla et al.).

More recently a study has been published in which a medical device in form of gel was tested intra-

vaginally in women with genital atrophy. This study was designed as an open non-controlled clinical

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study to assess the efficacy and safety of the gel, containing 1% of a mother tincture hop extract

(according to the French Pharmacopoeia X), hyaluronic acid, liposomes formed by cholesterol and

lecithin, all considered to be active ingredients.

The authors conclude that the results showed a marked effect of the tested product on the vaginal

dryness and other menopausal symptoms and that no treatment related adverse events were

complained by the patients (Morali et al., 2006).

Although the results of this study are interesting, it is difficult to draw conclusions, since the hop

extract was not standardized on 8-PN and the relative contribution of different other ingredients cannot

be deduced from the results obtained.

Assessor’s comments

To date only two small placebo-controlled clinical studies have been published using hop extracts, on

20 and 67 patients, respectively. A standardized hop extract (on 8-PN) was used in the latter study

only. The fact that in the three-armed clinical trial no clear dose-response relationship was found is of

concern. The relatively small numbers of patients may have contributed to the absence of a clear

dose–response relationship; a larger follow-up study is required to demonstrate clinical efficacy. The

data available are not sufficient to support a well-established use indication for hops in the treatment

of menopausal symptoms.

II.4.

SAFETY

II. 4.1.

Toxicity

Hops have been used in the brewing industry for centuries, without any known adverse effect to the

health of consumers. Thus, given the history of long-term and present use in humans with no

significant adverse effects, it is considered that hops are safe (Chadwick et al., 2006).

II. 4.1.1. Single dose toxicity

The following toxicological data are given in Hagers Handbuch (1993).

Ethanolic hop extract : LD 50 3,500 mg/kg/bw p.o., 1,200 mg/kg/bw s.c. (mice) :

LD 50 2,700 mg/kg/bw p.o. (rats).

Methylisobutylketone-hop extract : LD 50 2,700 mg/kg/bw p.o. (mice) ; LD 50 415 mg/kg/bw p.o. (rats).

Lupulone : LD 50 525 mg/kg/bw p.o. ; LD 50 1,200 mg/kg/bw s.c. : 600 mg/kg/bw i.m. (mice) :

LD 50 1,800 mg/kg/bw p.o. ; LD 50 330 mg/kg/bw i.m. (rats).

Humulone : LD 50 1,500 mg/kg/bw p.o. ; 600 mg/kg/bw i.m. (rats).

A standardized hydro-alcoholic hop extract enriched in 8-PN, and consisting of 0,22% 8-PN,

0,2% 6-PN, 5,5% xanthohumol and 1,7% isoxanthohumol (Menohop ® ) was tested for acute toxicity

and mutagenicity in rats (Publication in preparation).

The starting dose used in the acute toxicity studies was based upon the dose, which was used in the

clinical trial viz . 2,6 mg/kg/day and calculated via a multiplication factor of 6,66 from man to rats

(Notox ® , 2005). Three female Wistar rats in four groups each were treated with escalating doses of 25,

250, 1000 and 2500 mg hop extract/kg body weight (bw) per day by oral gavage during

2 consecutive days in order to determine the maximum tolerated dose (MTD). Clinical signs, body

weight and food consumption were determined, as well as clinical pathology prior to microscopy and

macroscopy at termination.

Based on the data from this dose escalation phase the dose level for MTD was selected to be

2500 mg/kg/day. Although a real MTD was not reached, this dose level represents a 100-fold expected

human dose, so that the safety margin is expected to be sufficiently high. During the MTD-phase

10 rats (5 males and 5 females) were given one dose of 2500 mg/kg daily for 5 consecutive days by

oral gavage. No mortality or clinical signs were noted during the whole MTD-phase and finally no

treatment related findings in haematology and clinical biochemistry parameters were observed and no

findings were noted after macroscopic and microscopic examination. It was concluded that repeated

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oral administration of 2500 mg/kg/bw in male and female Wistar rats on 5 consecutive days were well

tolerated and that no clear signs indicative of test substance induced toxicity were found.

These results obtained in rats were confirmed recently in humans by Rad et al. (2006), who

investigated the kinetics and systematic endocrine effects of 8-PN after single oral doses.

It was found that single oral doses of up 50-75,0 mg 8-PN (ca. 3500-fold the intended amount of

Menohop ® in clinical studies) were well tolerated by postmenopausal women and that no drug-related

adverse events were recorded.

Recently, toxicity and cell cycle effects of synthetic racemic 8-PN in human cells was studied in

comparison to quercetin and related flavonoids. These investigations were done on different cell lines

viz . the promyeloid leukemia cell line HL 60 and the adherent breast cancer cell line MCF 7. A low

toxicity and weak cytostatic properties of 8-PN and related naringenin derivatives were found

(Tokalov et al., 2004).

Besides the oestrogenic effects, Effenberger et al. (2005) also studied the cytostatic effects of the most

prominent prenylflavonoids present in hops. It was shown that only at high concentration (> 10 -4 M)

prenylflavonoids such as 8-PN, 6-PN, X and IX displayed cytostatic effects on V79 cells (Chinese

hamster fibroblasts).

II. 4.1.2. Sub-acute and chronic toxicity

In their investigations on the oestrogenic effects of 8-PN on bone metabolism, Miyamoto et al. (1998)

found no overt signs of toxicity after administration of 8-PN at a dose of 30 mg/kg/drug

subcutaneously for 2 weeks.

Recently, Christoffel et al. (2006) have published a study on the effects of 8-PN on the

hypothalamo-pituitary-uterine axis in rats after a 3-month treatment. Therefore, a number of

oestrogen-related parameters, in the hypothalamus, pituitary and uterus were chosen to compare the

putative oestrogenic effects of 8-PN with those of 17β-oestradiol. Two doses of 8-PN and

17β-oestradiol-3-benzoate (E 2 B) respectively 6,8 and 68,4 mg/kg/bw of 8-PN and 0,17 and

0,7 mg/kg/bw of E 2 B and their effects were compared on uterine weight, pituitary hormones (LH, FSH

and prolactin) and the expression of oestogen-regulated genes and of oestrogen receptor (ER) α and

ER β in the hypothalamus, pituitary and uterus.

Both doses of E 2 B and the high dose of 8-PN suppressed serum LH and FSH, and stimulated serum

prolactin levels, uterine weight, and progesterone receptor, insulin-like growth factor I and

complement protein C 3 mRNA transcripts.

In the preoptic and the mediobased areas of the hypothalamus, all treatments had negligible effects on

ER α and ER β and gonadotrophin-releasing hormone (GnRH) receptor gene-expression, while ERβ and

GnRH receptor transcripts in the anterior pituitary were reduced under both E 2 B doses and the high

8-PN dose. The mRNA concentrations of the LH α and LH β subunits in the pituitary were suppressed

by E 2 B and 8-PN.

In summary, 8-PN had very similar though milder effects than E 2 B on all tested parameters. The

authors concluded that inhibition of climacteric complaints by E 2 B takes place in the hypothalamus,

where it inhibits the overactive GnRH pulse generator. 8-PN may be used to inhibit climacteric

symptoms effectively. They proposed to carry out human pharmacological studies in order to show

whether the stimulatory effect of 8-PN on the uterus would require the concomitant administration of

progesterone to prevent endometrial over stimulation.

Up till now, no data are available on the long-term toxicology of hop extracts when used by

menopausal women. It should, however, not been neglected that hop extracts are rich in xanthohumol,

which is considered to possess antiproliferative effects on both cancer cell lines of human breast and

ovaries by inhibitory mechanisms at the initiation, promotion and progression stages (Gerhauser et al.,

2002; Gerhauser and Frank, 2005 ; Van Hoecke et al., 2005).

It is being questioned whether dietary and/or environmental exposure to phyto-oestrogens could

impose health risks such as endocrine disruption. In case of hop prenylflavonoids, beer is the main

dietary source. The average beer consumption in the United States was calculated at about 225 mL of

beer per capita per day in 2001 (USDA, 2003). When assumed that this amount was consumed as US

major brand lager/pilsner beers (500-1000 µg prenylflavnoids/l beer), the daily intake of

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prenylflavonoids would be about 0,14 mg (Stevens et al., 1999b). However, the concentrations

detected in beer (and therefore average intake) strongly depend on the brewing process, as strong ales

contain up to 4 mg prenylflavonoids/l. Although xanthohumol (X) is the predominant prenylflavonoid

present in hops (0,1-1% of dry weight), most of it is transformed into isoxanthohumol (IX) by thermal

isomerisation during wort boiling. Therefore IX is the major prenylflavonoid found in beer and is

present in concentrations from 500 µg/l (lager/pilsner) up to 4 mg/l (strong ale) (Stevens et al., 1999b ;

Rong et al., 2000). Similarly, desmethylxanthohumol (DMX) is converted into 8-PN resulting in final

concentrations in beer of up to 100 µg 8-PN/l. But despite the high activity of 8-PN, the total

oestrogenic activity in beer is still 500 to 1000 times lower than the concentration needed for harmful

in vivo activity (~ 100 mg/l) (Milligan et al., 2002). Moreover, many beers are now made using hop

extracts instead of whole hops, giving lower concentrations of 8-PN or no 8-PN at all. Therefore, it is

generally agreed that based on the current knowledge, no detrimental health effects can be attributed to

phyto-oestrogens upon moderate beer consumption (Milligan et al., 1999, 2002 ; Stevens et al., 2004).

Hop extracts have also been used for several years in food supplements without any known problems,

also in combination with other plants such as valerian. The Belgian Health Authorities have set upper-

limits for phyto-oestrogens in dietary supplements. For hop extract, the threshold was set at 400 µg

8-PN/day (for soy isoflavones at 40 mg/day)(Belgian legislation on herbals: MB, 25/02/2005).

The potential side-effects of soy preparations such as the thyroid effect have not been mentioned for

hops, so that every phyto-oestrogen should be considered differently.

II. 4.1.3. Genotoxicity

An overview on the genotoxicity of 8-PN was found in the “Comparative toxicogenomics database”

on the internet : http://ctd.mdibl.org/voc.go?voc=chem&acc=C119737&view=ixn

8-PN has been mentioned therein for interactions with alkaline phosphatase, CYP1A2, ESR1 and

2, GSTA1, IL6, PGR, TFF1 and VWF.

In the Ames mutagenicity test, a hydroethanolic extract of hop strobile showed weakly mutagenic

potential in Salmonella typhimurium strains TA98 and TA100 with or without activation (Göggelmann

et al., 1986). These data were confirmed for TA98 strains only for the hop extract enriched in 8-PN

(Menohop ® ) (Publication in preparation).

The hop extract enriched in 8-PN (Menohop ® ) has been tested in the Salmonella typhimurium reverse

mutation assay with four histidine-requiring strains (TA1535, TA1537, TA100 and TA98) and in the

Escherichia coli reverse mutation asay with a tryptophan requiring strain of Escherichia coli

WP2vvrA. The tests were performed in two independent experiments in the presence and absence of

S9-mix (Aroclor-1254 induced rat liver S9-mix). The concentration range varied from 100 to

5000 µg/plate. The hop extract showed up to 3,6 and 2,8-fold, dose-related increases in the number of

revertant colonies in tester strain TA98 compared to the solvent control, both in the absence and

presence of S9-mix (2 experiments). All other bacterial strains showed negative responses over the

entire dose range. Since hop extracts contain quercetin glycosides (McMurrough 1981; De Cooman et

al., 1998), the slight mutagenic effect in the Salmonella typhimurium TA98 strain which was not

observed in the other strains, might be a false positive result, as it is well-known that quercetin gives

positive results in the Ames test but negative results in other genotoxicity tests. Therefore, it was

decided to carry out a mouse lymphoma test in order to show that the hop extracts are not mutagenic.

Hops extract were tested up to concentrations of 160 and 100 µg/ml in the absence and presence of

8% and 12% (v/v), S9 mix respectively in an in vitro mammalian cell gene mutation test with L5178Y

mouse lymphoma cells. In this test the effects of hop extracts on the induction of forward mutation at

the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells were determined. It was

shown that hop extract was not mutagenic in the TK mutation test system under the experimental

conditions and consequently does not cause a point mutation or chromosomal aberrations. Since the

suppressed mutagenic effect in the Ames test was not confirmed in the same mutation test in

mammalian cells, the overall conclusion can be drawn that hop extract is not mutagenic and that

therefore carcinogenicity studies are not needed (Guideline EMEA/HMPC/32116/2005).

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Assessor’s comments

To date, adequate genotoxity studies have not been carried out for the aqueous or hydroalcoholic

extracts of hops included in the monograph on traditional use.

II. 4.1.4. Carcinogenicity

To date, no data on carcinogenicity studies on hop strobiles or its preparations are available.

II. 4.1.5. Reproduction and developmental toxicity

No preclinical data on reproductive and developmental toxicity of hop strobiles or its preparations are

available.

Kumai and Okamoto. (1984) investigated purified fractions of hop strobile extract for reproductive

toxicity in female rats primed with 25 IU of pregnant mare’s serum gonadotrophin.

Fractions F 1 (20 mg/rat) and F 2 (50 mg/rat), administered subcutaneously twice daily for 3 days, did

not induce any change in uterine weights but ovarian weights decreased significantly (p<0,05) by

25,7% and 24,9% respectively. Under the same conditions, two further fractions (4 mg/rat) purified

from F 1 produced significant decreases (p<0,01) of 42,0% and 33,1% in ovarian weight.

Caujolle et al. (1969) showed that an alcoholic extract of hop strobile (1 g of dried drug in 10 ml of

70% ethanol) inhibited concentrations of rat uterus with an ED 50 equivalent to 31x10 -6 of hop strobile

per ml.

In view of this in vitro antispasmodic effect on the uterus and lack of toxicity data, the use of hops and

its preparations should be avoided during pregnancy and lactation. It is clear that the extracts enriched

in 8-PN are intended for menopausal complaints and consequently should not be used by fertile

women. This is also the case for the indications of hop preparations as sedative.

II. 4.1.6. Local tolerance

Data on local tolerance for hop strobiles and its preparations are not available. Nevertheless, allergic

reactions have been reported for hops, although only following external contact with the herb and the

oil.

II. 4.2.

Side effects

Based on traditional use and available studies, there have been no serious side effects reported with

hops. Drowsiness or sedation may occur. Use caution if you are driving or operating heavy machinery.

In animal studies, hops have increased stomach acid, but there is no available research in humans in

that area. Based upon animal studies, hops may increase blood sugar levels in diabetic patients, but

may lower blood sugar in nondiabetic patients. Consequently, the effects of hops on blood sugar levels

are unclear (Anonymous, 2003a). It should be noted that oral intake of isohumulones, present in hops

and its preparations, is reported to improve insulin sensitivity in patients suffering from diabetes type

2. Consequently, it might be expected that the blood sugar level in such patients would decrease after

oral intake of hops.

Allergic reactions from hops have been reported, particularly in hop harvesters. The contact dermatitis

that has occurred in hop-pickers is attributed to myrcene, present in fresh oil but readily oxidized

(Mitchell and Rook, 1979). Additionally, a mechanical dermatosis has been attributed to the rough

hairs on the stem and secretions of the yellow glandular hairs on hops (Estrada et al., 2002).

Respiratory allergy caused by handling of hop cones have been documented (Newark, 1978), a

subsequent patch test using dried, crushed flowerheads proved negative. Positive patch test reactions

have been documented for fresh hop oil, humulone and lupulone.

Both of these allergic reactions are not likely to occur when using the hop extract, since allergens are

supposed to be removed (Estrada et al., 2002).

Moreover, no clinical cases of allergy or anaphylaxis resulting from the therapeutic use of hops have

been published (Anonymous, 2003a).

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Since hop extracts might contain small amounts of oestrogens, it is not known as yet what the effects

would be of use for viz . more than three months, on conditions such as breast, uterine, cervical or

prostate cancer or endometriosis. However, it should be noted that oestrogens such as 8-PN are only

present in hop preparations in significant amounts when old hops are used as starting material and

special enrichment procedures are employed for the hop preparations.

It should also be noted that hop preparations may contain antiproliferative compounds such as

humulones, xanthohumol and isoxanthohumol.

Finally, some hop preparations contain high levels of alcohol and should be avoided during pregnancy.

II. 4.3.

Contra-indications, warnings

It has been suggested that hops should not be taken by individuals suffering from depressive illness, as

the sedative effect may accentuate symptoms (Anonymous, Harvard Medical School, 2003b). The

sedative action may potentiate the effects of existing sedative therapy and alcohol.

Allergic reactions have been reported for hops, although only following external contact with the herb

and oil.

Concerning pregnancy and lactation, in vitro antispasmodic effect on the uterus has been documented.

In view of this and lack of toxicity data, the use of hops during pregnancy and lactation should be

avoided.

II. 4.4.

Interactions (see also under II.2.3)

Interactions with drugs, dietary supplements and other herbs have not been thoroughly studied. From

the many publications on hops and its constituents, it is theoretically possible that hops may increase

the drowsiness caused by sedative drugs and alcohol.

Hops may also affect blood sugar levels but no clinical data are available to support this effect.

II. 4.5.

Overdoses

Not known.

Assessor’s comments

In view of its long term use and present use in humans hops is considered to be non-toxic and safe

with no significant adverse effects.

The experimental toxicological data on hop preparations are rather limited and incomplete but as a

whole show low toxicity.

Substantial toxicological experiments have been carried out on a standardized hydro-alcoholic extract,

enriched in 8-PN, and consisting of 0,22% 8-PN, 0,2% 6-PN, 5,5% xanthohumol and

1,7% isoxanthohumol (Menohop ® ). The single-dose toxicity studies as well as the genotoxicity studies

showed the extract to be safe in all concentrations tested i.e. no adverse effects have been seen in

doses up 1000-fold the intended dose for clinical studies.

Investigations on the toxicity of the oestrogenic active ingredient of hop extracts viz . 8-PN were

carried out during 2 weeks and also three months, indicating that 8-PN had very similar but milder

effects than 17β-oestradiol on all tested oestrogenic parameters. Although, today no data are available

on the long-term toxicology of hop extract when used by menopausal women, it should nevertheless,

be stressed that hop extracts also contain xanthohumol, which is considered to possess antiproliferative

effects in several cancer cell lines.

Hop extracts such as Menohop ® have been used for several years in food supplements without any

known adverse effects. The Belgian authorities have set upper-limits for phyto-oestrogens in dietary

supplements. For hop extracts and soy-isoflavones the threshold was set at 400 µg 8-PN/day and

40 mg/day isoflavonoids, expressed as the glycoside of the main isoflavonoid respectively. It can also

be concluded from the many publications that no detrimental effects can be attributed to phyto-

oestrogens upon moderate beer consumption.

In conclusion, the safety assessment of hops and hop preparations is mainly based upon the many

years of experience from the extensive medicinal use in man, which indicate hop preparations to be

safe pharmaceutical agents.

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II.5.

ASSESSOR’S OVERALL CONCLUSION

The sedative effect of hops and preparations thereof has long been recognised empirically, so that a

period of at least 30 years of traditional medicinal use is easily fulfilled for this indication.

Its use has been made plausible by many in vitro and in vivo pharmacological studies. Recently, it has

been suggested that besides the acids and the essential oil other constituents such as xanthohumol may

play an important role in the sedative effect of hop preparations.

No clinical studies have been conducted to date with hop extracts alone as active drug for insomnia,

but at least seven placebo- or reference-controlled clinical studies have been carried out with fixed

combinations of hop extracts and valerian root extracts. Whether hop extracts act as mild sedative

independently, as a synergist, or not at all remains to be determined.

Although several hop-containing cosmetic preparations have been patented since 1961 for the external

treatment of various gynaecological disorders, almost no traditional oestrogenic formulations of hops

have been found in literature until the discovery of the oestrogenic principle in hops in 1988.

Consequently, the traditional medicinal use of hops as a phyto-oestrogenic preparation does not fulfil

the requirement for at least 30 years on the market. Since 8-PN was recognized as the phyto-

oestrogenic principle in hops, many modern hop extracts enriched and standardised upon the active

constituent have been prepared and pharmacologically investigated. It has been shown that 8-PN

behaves like 17β-oestradiol, but has much milder effects on all oestrogenic parameters tested than the

parent compound.

To date, only two small placebo-controlled clinical studies have been conducted with such extracts to

investigate menopausal discomforts. These studies, however, should be considered only as pilot

studies with results, encouraging enough to justify a full-scale trial. The experimental toxicological

data, however, obtained with these extracts, have shown the extracts to be devoid of genotoxicity and

adverse effects in concentrations up 1000-fold the dose used for the clinical studies.

Consequently, the well-established use as phyto-estrogenic drug for these hop extracts can not be

granted as yet.

In conclusion, single hop preparations can be accepted as traditional herbal medicinal products for

their sedative effects. Fixed combination products consisting of dry extracts of hop strobiles with other

sedative plants will be considered in separate monographs.

The data available on the oestrogenic properties of hop preparations are not sufficient to support a

marketing authorization.

III

ANNEXES

III.1

Community herbal monograph

III.2

Literature references

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Herbal Medicines for Human Use

Herbal Medicines
for Human Use