Biotechnology Advances 33 (2015) 10631090

Contents lists available at ScienceDirect

Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Biotransformations and biological activities of hop avonoids

Marcel Karabin, Tereza Hudcova, Lukas Jelinek, Pavel Dostalek
Department of Biotechnology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, Prague, Technick 5, 166 28 Prague 6, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: Female hop cones are used extensively in the brewing industry, but there is now increasing interest in possible
Received 15 September 2014 uses of hops for non-brewing purposes, especially in the pharmaceutical industry. Among pharmaceutically
Received in revised form 13 February 2015 important compounds from hops are avonoids, having proven anticarcinogenic, antioxidant, antimicrobial,
Accepted 16 February 2015
anti-inammatory and estrogenic effects. In this review we aim to present current knowledge on the
Available online 21 February 2015
biotransformation of avonoids from hop cones with respect to products, catalysis and conversion. A list of
Keywords:
microbial enzymatic reactions associated with gastrointestinal microbiota is presented. A comparative analysis
Hop of the biological activities of hop avonoids and their biotransformation products is described, indicating
Flavonoid where further research has potential for applications in the pharmaceutical industry.
Biotransformation 2015 Elsevier Inc. All rights reserved.
Prenylation
Methylation
Demethylation
Glycosylation
Deglycosylation
Hydroxylation
Acylation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064
2. Structure and properties of hop avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065
2.1. Structure of hop avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065
2.1.1. Flavan-3-ols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065
2.1.2. Proanthocyanidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065
2.1.3. Flavonols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065
2.1.4. Flavonol glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
2.1.5. Flavanones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
2.1.6. Prenylavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067
2.1.7. The contents of avonoids in hop cones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067
2.2. Health benets of hop avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067
2.2.1. Cardiovascular effects of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067
2.2.2. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067
2.2.3. Anticarcinogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069
2.2.4. Anti-inammatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070
2.2.5. Estrogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070
2.2.6. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071
2.2.7. Antidiabetic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071
2.2.8. Other health promoting effects of hop avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1071
2.3. Toxicity of hop avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
3. Biotransformations of hop avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
3.1. Human metabolism of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
3.2. Bioavailability of hop avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1073

Corresponding author. Tel.: +420 220444037; fax: +420 224311082.

E-mail address: Pavel.Dostalek@vscht.cz (P. Dostalek).

http://dx.doi.org/10.1016/j.biotechadv.2015.02.009
0734-9750/ 2015 Elsevier Inc. All rights reserved.
1064 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090

3.3. Prenylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074

3.3.1. Impact of prenylation on bioavailability of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074
3.3.2. Enzymes involved in prenylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
3.3.3. Prenylation in biotechnological industries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
3.4. Methylation and demethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
3.4.1. Enzymes involved in methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076
3.4.2. Impact of methylation on the biological activities of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
3.4.3. O-methylation in biotechnological industries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
3.4.4. Demethylation in the digestive tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
3.4.5. Demethylation in biotechnological industries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1078
3.5. Glycosylation and deglycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079
3.5.1. Impact of glycosylation on the biological activities of avonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079
3.5.2. Glycosylation and deglycosylation in biotechnological industries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080
3.6. Hydroxylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
3.7. Acylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
3.8. Biotransformation of the avonoid carbon skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
3.8.1. Prenylavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
3.8.2. Flavan-3-ols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083
3.8.3. Flavanones and avonols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1084
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1084
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1084

1. Introduction
Hops are used in the manufacturing of beer and female infertile
plants are cultivated on high trolleys especially for brewing. Compounds
important for brewing are concentrated inside hop cones, in lupulin
glands that contain hop resins, bitter acids, essential oils and prenylated
avonoids. Other polyphenols are also present in the green part of hop
cones. The main role for hops in brewing is the conservation and
bittering of beer by hop resin isomerization products, avouring of
beer by hop essential oils and their transformation products, and in-
creased levels of polyphenols in wort and beer (Almaguer et al., 2014;
Karabin et al., 2014; Steenackers et al., 2015). The brewing industry
uses either directly pressed and dried hop cones/pellets, or ethanol
and CO2 extracts. Hop avonoids are therefore important for brewing
and beer quality. Flavonoids have antioxidant properties that protect
against oxidation of hop resins, and due to their reactivity, promote a
range of activities including formation of a hot break during wort boil-
ing, beer clarication and sludge elimination reactions with proteins
during wort cooling and beer fermentation (Magalhaes et al., 2011b).
Flavonoids improve the redox properties of wort and beer and are
natural antioxidants that support sensory reactions (De Clippeleer
et al., 2010) and colloidal stability (Jelinek et al., 2014). Flavonoids can
be oxidized, creating Maillard reaction products, or isomerized during
brewhouse operations (Briggs et al., 2004).
The origin of the hop (Humulus lupulus L., Cannabaceae) dates back
to ancient Mesopotamia, but can be also traced to the lowlands of
southern Caucasus and Siberia. Historically, hops were also used in tra-
ditional medicine, and hop was a popular herb, mainly in teas and baths,
or as an ingredient in food (Wilson, 1975). In ancient times, hop was
known as an important natural medicine that was used by healers
against leprosy, foot odour, constipation and for blood purication.
In traditional Chinese medicine, hops were used for treatment of
anxiety and insomnia, as well as to stimulate appetite and combat
dyspepsia. Favourable results were also recorded using alcoholic
extracts of hops for the treatment of diseases such as pulmonary
tuberculosis, silicosis, asbestosis or leprosy (Chang and But, 1986;
Khan and Abourashed, 2009; Zanoli and Zavatti, 2008). Phytotherapy
has focussed on hops as a remedy for the past two centuries, and
in the early 20th century, the rst drugs were based on the hop. The
Committee on Herbal Medicinal Products (HMPC) of the European
Medicine Agency (EMEA) issued a statement in 2007 on the traditional
use of hops (Humulus lupulus L.) for the treatment of mild symptoms
of stress and insomnia. For these purposes, a hop extract is combined
with an extract of valerian (Valeriana ofcinalis, Valerianaceae). Until re-
cently, this preparation represented the most frequently administered
form of plant-based sleeping agents and sedatives (Bent et al., 2006;
Dimpfel and Suter, 2008). These drops are better tolerated and have
no side effects compared with drugs containing synthetic substances.
Hop was identied as a medicinal plant by the European Foundation
for Phytotherapy and European Society for Cognitive Psychology
(ESCOP), which approved hops as a drug for use in the treatment
of mental state disorders such as anxiety, restlessness, and sleep distur-
bances (Zanoli and Zavatti, 2008). During this period, research was
focused on identifying biologically active hop substances. One of these
substances, with apparent miracle effects was the prenylated chalcone,
xanthohumol. Almost 10 year ago, the international journal, Molecular
Nutrition and Food Research, (Vol.49, No.9, 2005) published a special
edition dedicated to xanthohumol (Gerhauser and Frank, 2005). This
substance is now the subject of active research and papers and patents
on this topic have increased steadily. Nowadays, considerable atten-
tion is being paid to 8-prenylnaringenin due to its reported strong
phytoestrogenic activity, stimulating estrogen receptors in the body.
Phytoestrogens can therefore substitute for human steroidal hormones
(e.g. 17--estradiol) and suppress menopausal symptoms in women,
or reduce the risk of cancers associated with changes in the hormonal
system. 8-prenylnaringenin can stimulate certain types of estrogen
receptors up to one hundred times more effectively than genistein,
a phytoestrogen isolated from soybean (Schaefer et al., 2003). A very
important metabolic conversion of isoxanthohumol (isomerization
product of xanthohumol) to phytoestrogenic 8-prenylnaringenin
using selected intestinal bacteria has been reported. This conversion
involves O-demethylation of isoxanthohumol, therefore the nal con-
centration of 8-prenylnaringenin in blood plasma is not only dependent
on total intake of 8-prenylnaringenin, but also on the presence of its
precursor isoxanthohumol, in combination with appropriate intestinal
microora that carry out the demethylation reaction (Possemiers
et al., 2008). This biotransformation can be used in the pharmaceutical
industry for the biotechnological production of 8-prenylnaringenin.
Because of its phytoestrogenic properties, this substance is useful in
hormone replacement therapy (HRT) for the prevention and treatment
of osteoporosis or in the control of climacteric symptoms (Karabin et al.,
2012). There are many other avonoid substances in hops with
biological effects and many transformation reactions that can change
these substances, generating others with different biological effects.
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090 1065

This fascinating topic is the subject of this review. Since only a few
studies have been focused specically on hop avonoids, we include
in review also several studies about pure avonoids which are present
in hop, too.

2. Structure and properties of hop avonoids

Flavonoids are phenolic substances that represent a wide group of

plant secondary metabolites, occurring in practically all parts of the
plant and are widely distributed in food and beverages from plant
(Crozier et al., 2006; Di Carlo et al., 1999). They have a variety of
functions in plant physiology, acting as antioxidants (protecting plants
against oxidative stress), antimicrobials (defence of plants against mi-
croorganisms), photoreceptors that include colouring characteristics
(plant pigments), UV protectors, and also play a signicant role in nitro-
gen xation and plant growth and development (Havsteen, 2002;
Pietta, 2000; Winkel-Shirley, 2001). Flavonoids have been described
as health-promoting agents with proven biological effects in vitro and
in vivo(Das and Rosazza, 2006), such as free-radical scavenging
(Pietta, 2000), modulation of enzymatic activity (Fraga et al., 2010),
and inhibition of cellular proliferation (Miranda et al., 1999), as well
as their potential uses as antibiotic (Gerhauser, 2005b), anticarcinogen-
ic (Ramos, 2007), anti-inammatory(Ferrandiz and Alcaraz, 1991) or
estrogenic agents (Chadwick et al., 2006).
The basic structure of avonoids consists of a avan molecule (Fig. 1)
derived from the heterocyclic structure, chromane, by substitution of its
C ring in position 2 or 3 with a phenyl group (ring B) (Croft, 1998).
Depending on the C ring substitution, avonoids are classied as
avones (phenyl group in position 2) or isoavones (phenyl group in
position 3) (Hodek et al., 2002).
Flavonoids consist of two aromatic rings (A and B) linked through a
three carbon chain, usually organized as an oxygenated heterocyclic
pyran (C) (Pietta, 2000). These three rings are substituted by hydroxy
groups occurring most frequently at carbons 3, 4', 5, 5 and 7 (Croft,
1998; Crozier et al., 2006) followed by methyl, prenyl, isoprenyl and
methoxy groups (Das and Rosazza, 2006). With increasing numbers
of hydroxy groups and glycosidic bonds, the aqueous solubility of
avonoids increases, whereas with increasing number of methyl, prenyl
and/or isoprenyl groups, the hydrophobicity increases (Crozier et al.,
2006).
2.1. Structure of hop avonoids
More than 4000 varieties of avonoids have been identied and can
be classied according to their structure based on the degree of substi-
tution and oxidation (Nijveldt et al., 2001). The predominant avonoids
are most often divided into six subclasses: avones, avonols, avan-3-
ols, isoavones, avanones and anthocyanidins (Ross and Kasum,
2002). In this review, we will only discuss avonoids derived from
hops, consisting of six main groups: avan-3-ols, proanthocyanidins,
avonols, avanones, avonol glycosides and prenylavonoids.
Fig. 2. Structures of most signicant hop avan-3-ols.
2.1.1. Flavan-3-ols
Flavan-3-ols (catechins) are colourless avan monomers containing
two chiral centers and therefore existing as four possible isomers
(Fig. 2). Flavan-3-ols occur usually as aglycone monomers, oligomers
or esteried with gallic acid to form gallocatechin and epigallocatechin.
(+)-Catechins, (+)-gallocatechin, ()-catechin and ()-gallocatechin
have hydrogens at carbons C2 and C3, in an (E)-conguration.
Corresponding epicatechin and epigallocatechin have the same
hydrogen atoms in a (Z)-conguration. In nature, only (+)-catechin
and (+)-gallocatechin occur, i.e., isomers ()-epicatechin and ()-
epigallocatechin (Crozier et al., 2006).
Interestingly, a low level of the avan-3-ol, (epi)afzelechin, was
found in hops but was identied only as a dimer incorporated into a
proanthocyanidin and corresponds to propelargonidin B or its isomer
(Olsovska et al., 2013).
2.1.2. Proanthocyanidins
Oligomeric and polymeric forms of avan-3-ols, are known as
proanthocyanidins or condensed tannins (Fig. 3) (Fraga et al., 2010).
They can differ in their degree of polymerization, subunit composition
and type of binding between subunits, where catechin is the main
terminal unit and epicatechin is the main extension unit (Li and
Deinzer, 2009). Proanthocyanidins consisting of catechin and epicatechin
units only are called procyanidins (Crozier et al., 2006).
Most of the dimeric fraction consists of procyanidin B1 and
B4 (consisting of a molecule of catechin bound to epicatechin,
epicatechin-(4 8)-catechin and catechin-(4 8)-epicatechin)
and procyanidin B3 (consisting of two molecules of catechin,
catechin-(4 8)-catechin). The main trimer is proanthocyanidin
C2 (three units of catechin, catechin-(4 8)-catechin-(4 8)-
catechin). Additionally, small quantities of prodelphinidin B3 (polymeric
tannins composed of gallocatechin, galloepicatechin-(4 8)-catechin)
and propelargonidin B (condensed tannins formed from epiafzelechin)
are also present in hop cones (Li and Deinzer, 2009). Li and Deinzer
(2006) isolated other proanthocyanidin oligomers from hops, including
gallocatechin-(4 6)-catechin, catechin-(4 8)-gallocatechin,
catechin-(4 6)-gallocatechin, afzelechin-(4 8)-catechin,
catechin-(4 8)-catechin-(4 8)-catechin, epicatechin-(4 8)-
epicatechin-(4 8)-catechin, catechin-(4 8)-gallocatechin-
(4 8)-catechin, and gallocatechin-(4 8)-gallocatechin-
(4 8)-catechin (Li and Deinzer, 2006).
Upon acid-mediated oxidative depolymerization, proanthocyanidins
yield anthocyanidins (Li and Deinzer, 2009).

2.1.3. Flavonols
Flavonols are comprised of avone, including a double bond
Fig. 1. Structure of avan. between C2 and C3, conjugated with hydroxyl groups (Fig. 4).
1066 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090

Fig. 3. Structures of most signicant hop proanthocyanidins.

Among the most signicant hop avonols are quercetin (3,5,7,3',4'-
pentahydroxyavone), kaempferol (3,5,7,4'-tetrahydroxyavone)
and myricetin (3,5,7,3',4',5'-hexahydroxyavone) (Crozier et al.,
2006). In hops, less common avonols such as morin, which does
not have a hydroxyl group in the C5 position, also occur (Jelinek
et al., 2010).
Flavonol glycosides have been characterized in hops, including
quercetin (quercetin-5-O-glucoside-3-O-rutinoside, quercetin-3-O-
rutinoside (rutin), quercetin-3-O-(6-O-malonylglucoside), quercetin-
3-O-glucoside and kaempferol (kaempferol-5-O-glucoside-3-O-
rutinoside, kaempferol-3-O-rutinoside, kaempferol-3-O-(6-O-
oxalylglucoside), kaempferol-3-O-glucoside, kaempferol-3-O-(6-
O-malonylglucoside) (Aron et al., 2012).

2.1.4. Flavonol glycosides 2.1.5. Flavanones

Flavonoids in nature occur most often conjugated with one or
more sugar molecules and are known as avonoid glycosides (Fig. 5),
where the sugar component consists mostly of D-glucopyranose or
rutinose (-L-rhamnosylpyranose-D-glucopyranose). Non-conjugated
avonoids are called aglycones (Crozier et al., 2006).
Flavanones are substances derived from avone, with the double
bond missing between C2 and C3, thereby forming a new chiral center
at carbon C2 (Crozier et al., 2006). The most signicant hop avanone
is naringenin (5,7,4'-trihydroxyavanone) (Fig. 6) (Jelinek et al., 2010).

Fig. 4. Structures of most signicant hop of avonols. Fig. 5. Structures of most signicant hop avonol glycosides.
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090 1067

commercially grown hops, the avonoid content can be up to 5 g/100 g

of hop cone dry matter (Jelinek et al., 2010).
Catechin, epicatechin and their polymers proanthocyanidins
(proanthocyanidin B3 is the most representative followed by
procyanidin C2) are present in hops at the highest levels (Li and
Deinzer, 2009).
The average amount of avan-3-ols in hops, according to Briggs et al.
(2004), is 8172821 mg of (+)-catechin/kg and the related epicatechin
have also been reported. The amount of proanthocyanidin B3 is
4281472 mg/kg and proanthocyanidin C2 is 287875 mg/kg (Briggs
et al., 2004).
Contents of avonols, according to McMurrough, ranged from 520 to
1630 mg/kg for kaempferol and from 320 to 1440 mg/kg for quercetin
(Mcmurrough, 1981).
Fig. 6. Structure of hop avanones.
Prenylavonoids are also signicant components of hop avonoids.
Major representatives are xanthohumol (0.2-1.1 g/100 g of hop cone
dry matter) (Karabin et al., 2012) and desmethylxanthohumol (Diller
et al., 2009), while others prenylavonoids are present in very small
2.1.6. Prenylavonoids
to trace amounts (Stevens and Page, 2004). Interestingly, the
Prenylavonoids are a special group of avonoids that belong to a
prenylavonoid 8-prenylnaringenin, which is present in hops in
subgroup of chalcones or avanones. Unlike other avanones, they are
very low concentrations, is considered to be the most effective
characterized by the presence of prenyl (Pn) or geranyl (Gn) chains
phytoestrogen isolated yet (Milligan, 2009).
(Fig. 7) on the A ring in position 8 (Fig. 1), through which they exhibit
The average content of the most signicant hop avonoids,
non-polar characteristics (Stevens and Page, 2004).
as demonstrated in two independent studies, is shown in Table 1.
Part of the female hop cones are lupulin glands, which produce,
apart from bitter acids, a mixture of prenylated, geranylated, oxidized,
2.2. Health benets of hop avonoids
and/or cyclized chalcones and avanones. The most signicant
hop prenylavonoids are the chalcones xanthohumol ((E)-1-
There is considerable evidence from epidemiological studies in vitro
[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]-3-(4-
and in vivo that long term administration of avonoids can reduce the
hydroxyphenyl)prop-2-en-1-one), and desmethyxanthohumol
risk of some serious diseases. Flavonoids have been suggested as
((E)-1-[2,4-dihydroxy-6-methoxy-3-(3-methylbut-2-enyl)phenyl]-
chemopreventive agents for cardiovascular and liver diseases, as well
3-(4-hydroxyphenyl)prop-2-en-1-one), while other prenylavonoids
as some types of cancer (Hodek et al., 2002).
(at least 10 more prenyl or geranyl chalcones and 9 prenyl or geranyl
The biological activities of avonoids strongly depend on their
avanones have been identied in hops) are present in very small to
chemical structures and relative orientations of various moieties
trace amounts (Figs. 8 and 9) (Stevens et al., 1997, 2000). The presence
on the molecule. Even minor changes in their chemical structure
of a free hydroxyl group in chalcones makes possible isomerisation
(e.g. position of substituted group on avan skeleton) can have a
to avanones, which also occur in hop cones, although at very
major impact on their biological activities. Flavonoid biological activities
low concentrations. The isomerisation product of xanthohumol is
are frequently linked to their antioxidant properties (redox activity
isoxanthohumol (Stevens and Page, 2004), and the isomerisation
contributes signicantly to free radical defence) and their ability to
product of desmethylxanthohumol, due to the presence of two
modulate several specic enzymes, cell receptors and to stimulate
hydroxyl groups, is a mixture of 6- and 8-prenylnaringenin(Diller
some hormones and neurotransmitters (Havsteen, 2002). Flavonoids
et al., 2009).
have been recognized to exhibit a wide range of positive effects
on human health: antimicrobial, anti-inammatory, antiangiogenic,
analgesic, antiallergic, hepatoprotective, anticarcinogenic and estrogenic,
2.1.7. The contents of avonoids in hop cones
as has been demonstrated in many in vitro and in vivo studies (Ross and
Hop avonoids are present in all parts of hop cones, the largest con-
Kasum, 2002).
tent being present in seeds and bracts. Exceptions are prenylavonoids
In recent years, scientic research has focused especially on
that are made in lupulin glands together with hop resins and essential
prenylated avonoids (Magalhaes et al., 2009; Stevens and Page,
oils. Flavonoid content in hop cones is very variable and depends on
2004) and therefore these will be highlighted in this review.
many factors including hop variety, climatic conditions, cultivation
area, storage conditions, etc. It is therefore not surprising that their
2.2.1. Cardiovascular effects of avonoids
average content varies according to the literature. For conventional,
Population studies have shown that avonoid intake in the human
diet is inversely correlated with mortality from cardiovascular diseases
(Hertog et al., 1993). Epidemiological, in vitro and animal studies
indicate that avonoids have benecial impacts on parameters
associated with atherosclerosis, including lipoprotein oxidation,
blood platelet aggregation, and vascular reactivity (Mladenka et al.,
2010). The cardioprotective effect of avonoids can be attributed to
their antioxidant, antithrombogenic, anti-inammatory, and hypo-
lipidemic properties, and higher avonoid intakes are thought to
play a key role in reduced risk of developing cardiovascular diseases
(Velayutham et al., 2008).

2.2.2. Antioxidant activity

Reactive oxygen species (ROS), (for example hydroxyl, superoxide,
Fig. 7. Prenyl (Pn) and geranyl (Gn) chain. hydroperoxide radicals, and even singlet oxygen or peroxide anions),
1068 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090

Fig. 8. Structure of most signicant hop prenylchalcones.

are formed during normal aerobic metabolism in vivo and can cause
oxidative damage to DNA, proteins and lipids (Cooke et al., 2003;
Pietta, 2000; Ross and Kasum, 2002; Salvi et al., 2001); they
are thus thought to be involved in the development of cancers,
atherosclerosis, heart failure, diabetes, or Parkinson's disease (Mladenka
et al., 2010).
Many studies have shown avonoids to be effective antioxidants in a
wide range of chemical oxidation systems including (Mladenka et al.,
2010; Pietta, 2000):
i. scavenging reactive species (usually oxygenated),
ii. inhibiting enzymes (ability to inhibit the activity of lipoxygenases,
xanthine oxidase and NADPH oxidase),
iii. chelating trace elements (metal divalent cations) involved in free
radical production (prevents their catalytic effects).

The antioxidant activities of avonoids are determined by their

chemical structure, in particular the hydroxylation status of their aro-
matic rings. It has been shown that antioxidant activity is related to a-
vonoids containing a phenol B ring. Hydroxyl groups in the C3' and C4'
positions on the avan skeleton are the main structural determinants
of antioxidant activities of avonoids (Lien et al., 1999). Hydroxyl

Table 1
Content of the main avonoid groups in hop cones.

Group of avonoids Content in hop cones (mg/kg)

(de Keukeleire et al., 1999) (Forster and Back, 1999)

Proanthocyanidins 600-1500 100-600

Flavanols 300-1100 30-200
Glycosides of quercetin 500-2000 50-250
Glycosides of kaempferol 500-700 50-300
Flavonols b100-200 1-10
Prenylavonoids - 100-10000
Fig. 9. Structure of most signicant hop prenylavanones.
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
groups at carbons C3 and C5, together with the carbonyl group in C4
position, occurring in the molecule of quercetin, results in maximum
radical scavenging potential (Resende et al., 2014). However, the most
signicant antioxidant activities are demonstrated by prenylated
chalcones, mainly due to their prenyl group that produces a more
non-polar character than other avonoids. Xanthohumol was proven
to be the most active amongst hop avonoids in tests for total oxygen
radical absorbance capacity (ORAC), singlet oxygen absorbance capacity
(SOAC), ferric reducing antioxidant power (FRAP) and quenching
activity against superoxides (SOD) (Bartmanska et al., 2013). It was
experimentally conrmed that xanthohumol, at a concentration of
1 M, is signicantly more effective in absorbing hydroxyl (8.9-fold)
and peroxyl (2.9-fold) groups than the reference antioxidant
(a derivative of Vitamin E- TROLOX). Similarly, the ability of
xanthohumol to absorb the superoxide radical was conrmed
(Gerhauser et al., 2002).
A variety of enzymes (particularly NADPH oxidase, xanthine oxidase
and lipoxygenases) are involved in the production of ROS(Mladenka
et al., 2010). Catechins and quercetin have been shown to inhibit
xanthine oxidase activity and thus scavenge peroxide and oxygen
radicals that are generated by this enzyme (Chang et al., 1993).
Flavonoids have been also demonstrated to inhibit NADPH oxidase
(Fraga et al., 2010; Steffen et al., 2008), whereas saturated avonoids
(without a double bond between C2 and C3 are more effective in
comparison with unsaturated compounds. Interestingly, avonoids
containing an unsubstituted ring B do not inhibit NADPH oxidase
(Steffen et al., 2008).
For avonoids, especially prenylavonoids, the ability to chelate
bivalent metals, thereby inhibiting the oxidation of low density
lipoproteins (LDL), one of the major causes of atherosclerosis,
was demonstrated. Bivalent cations (Cu2 +) cause the conversion of
polyunsaturated fatty acids in lipoproteins to conjugated dienes
and hydroperoxides (Miranda et al., 2000b). Lipoxygenases are
responsible for dioxygenation of polyunsaturated fatty acids in
lipids. Flavonoids inhibit the activity of lipoxygenases through
iron reduction or chelation (Mladenka et al., 2010). Not only
prenylavonoids, but also other hop avonoids are known for their
iron-chelating and iron-stabilizing properties (Ferrali et al., 1997).
Mladenka et al. (2011) analysed 26 avonoids from different
subclasses for their iron-chelating activity in comparison with the
iron chelator deferoxamine. The hydroxyl group in the C3 position,
keto group in the C4 position together with double bond between
C2 and C3 were associated with a considerable iron-chelation activ-
ity. These structural determinants occur in molecules of quercetin
and myricetin, and these avonoids showed similar iron-
chelatingactivity as deferoxamine under neutral conditions, but dem-
onstrated lower activity at a lower pH, demonstrating that iron-
chelating activity strongly depends on pH (Mladenka et al., 2011).
In addition, xanthohumol, and to a lesser extent, isoxanthohumol
and 8-prenylnaringenin, have been shown to reduce the rate of
lipid peroxidation and the associated formation of thiobarbituric
acid reactive compounds (TBARS), as well as to inhibit oxidation of
tryptophan residues (Miranda et al., 2000b). The rate of inhibition of
Cu2 + dependent lipid peroxidation in LDL was expressed as the per-
centage formation of TBARS and was 26.6% for xanthohumol (25 M),
42.7% for 8-prenylnaringenin(25 M) and 64% for isoxanthohumol
(25 M). On the contrary, naringenin (25 M) acted as pro-oxidant and
caused 160% formation of TBARS. Xanthohumol, desmethylxanthohumol
and quercetin, at concentrations of 25 M, were most effective inhibitors
of the oxidation of tryptophan residues (Miranda et al., 2000b).
Noroozi et al. (1998) tested the effects of several avonoids on
oxygen radical-generatedDNA damage from hydrogen peroxide in
human lymphocytes, in comparison with vitamin C. Pretreatment
with all avonoids led to a reduction in oxidative DNA damage, particu-
larly pretreatment with myricetin caused only 10% DNA damage
compared with quercetin (22%), kaempferol (32%), quercitrin (45%),
1069
quercetin-3-glucoside (62%) and rutin (quercetin-3-beta-D-rutinoside)
(82%). The protective effects of aglycones such as quercetin, myricetin
and kaempferol were signicantly better than the protective effect of
vitamin C against DNA damage (78%). The study therefore showed
that aglycones possess greater antioxidant activity than the correspond-
ing glycosides such as quercetin-3-glucoside(Noroozi et al., 1998).
2.2.3. Anticarcinogenic activity
Studies performed in recent years have proved the existence of
potential anticarcinogenic agents in hops, especially phenolic com-
pounds. Flavonoids are able to inhibit tumor cell proliferation, to stop
tumor growth and to actively participate in inhibiting carcinogenesis
in the initial, promotional, and late phases of cell proliferation.
Anticarcinogenic activities of avonoids have been attributed to
a wide variety of mechanisms, especially inhibition of metabolic
activation of procarcinogens, and the induction of carcinogen detox-
ication enzymes. In the advanced stage of tumor growth avonoids
are able to suppress the process by inhibiting DNA synthesis, inhibiting
angiogenesis and inducing apoptosis of tumor cells (Gerhauser, 2005a;
Ramos, 2008).
Attention was focused especially on prenylavonoids that have
strong and broad-spectrum anticarcinogenic activities (Gerhauser,
2005b). From other avonoids, ones that can be mentioned include
quercetin and myricetin (Araujo et al., 2011). Quercetin particularly, is
a compound with anticarcinogenic properties including cell cycle
regulation, reversal of multidrug resistance and induction of tumor
cell apoptosis (Thangasamy et al., 2008). Kaempferol has been also
reported to inhibit cancer cell growth and angiogenesis and to induce
apoptosis in cancer cells (Chen and Chen, 2013).
Xanthohumol and some other hop prenylated avonoids, such as
isoxanthohumol, are capable of inhibiting enzymes of cytochrome P450
that are able to transform exogenous and endogenous procarcinogens
to their reactive forms carcinogens (Hodek et al., 2002; Moon et al.,
2006). In the study of Henderson et al. (2000) it was demonstrated that
10 M xanthohumol almost completely inhibited cytochrome P450 1A1
and 1B1 enzymes. Cytochrome 1A2 enzymes were 90% inhibited by
10 M 8-prenylnaringenin or isoxanthohumol (Henderson et al., 2000).
These and other results showed that prenylavonoids are potent selective
inhibitors of cytochrome P450 enzymes (Moon et al., 2006).
In another study with human liver cells (HepG2), antimutagenic ef-
fects of xanthohumol were demonstrated, even at a concentration of
0.01 M, on procarcinogens 2-amino-3-methylimidazo [4,5-f] quinoline
(IQ) and tert-butyl hydroperoxide (t-Booh) that are activated by
cytochrome P450 enzymes (Ferk et al., 2010; Plazar et al., 2007).
Resende et al. (2014) investigated antimutagenic activities of
avonoids against mutagens (namely: 4-nitro-o-phenylenediamine,
sodium azide, mitomycin C,benzo-[a]-pyrene, aatoxin B1 and
2-aminouorene) and showed that they differed from one another
only by the hydroxylation status of their aromatic rings (quercetin
and kaempferol), demonstrating that antimutagenic potential strongly
depends on the number and position of hydroxyl groups. Additional
hydoxyl groups caused signicant increases in antimutagenic activities.
Among the monohydroxylated avonoids, changes due to the position
of the hydroxyl group groups were not evident (Resende et al., 2014).
Chemopreventative effects can also occur by detoxifying carcinogens
through the action of specic enzymes. One of these enzymes is
NAD(P)H: quinone reductase that catalyses the reduction of quinone
to hydroquinones, which are more suitable substrates for subse-
quent conjugation. It was found that xanthohumol and at least six
other prenylavonoids, at concentrations above 1 M, increased by
several fold, the activity of quinone reductase in murine liver cells
(Hepa 1c1c7). For each substance, the concentration necessary to
double enzyme activity ranged from 1.8 to 10.1 M, the better
(lower) values being obtained for prenylated chalcones. In contrast,
avonoids without a prenyl or geranyl chain were practically ineffective
(Miranda et al., 2000a).
1070 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
Uda et al. (1997)have determined the structural requirements for a-
vonoids to induce quinone reductase in murine Hepa1c1c7 cells. Their
study showed that the avonols (kaempferol N quercetin N myricetin)
are the most effective inducers of quinone reductase activity in
Hepa1c1c7 cells, where the concentration of kaempferol required
for a 50% increase in quinone reductase activity in the hepa1c1c7
cells was 3 M (Uda et al., 1997).
Progressive stages of carcinogenesis are characterized by uncon-
trolled cellular proliferation that is growth factor independent, and
newly formed cells resist apoptosis. Anticarcinogenic activities of
avonoids in the progressive stages of carcinogenesis are attributed to
their ability to affect DNA synthesis. In vitro tests have conrmed that
xanthohumol is capable of inhibiting human DNA polymerase ,
which is responsible for the initiation of DNA synthesis. This ability
was demonstrated even for isoxanthohumol, but at a much lower
efciency (Gerhauser et al., 2002). Flavonoids such as quercetin and
myricetin effectively inhibit the activities of topoisomerase I and
II,enzymes that regulate the overwinding or underwinding of
DNA(Bensasson et al., 2011).
Flavonoids can also affect cancer cells by triggering apoptotic
processes (Araujo et al., 2011) through the modulation of a number of
key elements involved in cellular signal transduction pathways linked
to apoptosis (caspases and bcl-2 genes, tumor necrosis factor alpha
TN) (Deeb et al., 2010; Ramos, 2007). Recent studies have shown
that quercetin can induce apoptosis in numerous types of cancer cell
lines such as human leukemia Jurkat T cell lymphoblasts (the half
maximal inhibitory concentration - IC50 was 107 M) (Baran et al.,
2014; Chen et al., 2005), MCF-7 breast cancer cell line (IC50 was
30.8 M) (Oh et al., 2010; Zhang et al., 2012), various ovarian and
endometrial tumor cell lines in a concentration range of 0.01-10 M
(Scambia et al., 1992), A549 pulmonary cancer cell line (Nguyen et al.,
2004), HSC-3 oral cancer cell line (IC50 was 20 M) (Huang et al.,
2013), EPG85-257P and EPG85-257RDB gastric cell lines (IC50
was 12 M) (Borska et al., 2012) and LoVo colonic cancer cell line
(IC50 was 40.2 M) (van Erk et al., 2005; Zhang et al., 2012).
Several studies have demonstrated the activity of avonoids in effec-
tively inhibiting the proliferation of tumor cells (Hudcova et al., 2014a).
Kuntz et al. (1999) tested over 30 avonoids for their effects on cell
proliferation and potential cytotoxicity on two human colon cancerous
cell lines. All avonoids tested, including avonols and avanones, dem-
onstrated antiproliferative activities (IC50 was within a concentration
range of 15.5 -39.7 M) without cell cytotoxicity (Kuntz et al., 1999).
Moreover, the main prenylavonoid of hops, xanthohumol, can in-
hibit growth or induce apoptosis in a wide range of types of cancers
in vitro and in vivo, in a dose-dependent manner. Xanthohumol affected
cell proliferation in the following cell lines: the ovarian cell line, SKOV3
(at a concentration range of 2030 M), OVCAR3 (at a concentration
range of 1020 M) (Drenzek et al., 2011), breast cancer cell line
MCF7 (at a concentration range of 1100 M) (Monteiro et al., 2008),
colon cancer cell line HT29 (IC50 was 1.2 M) and SW620 (IC50 was
2.5 M) (Hudcova et al., 2014b; Miranda et al., 1999; Pan et al., 2005),
prostate cancer cell lines (at a concentration range of 2040 M)
(Deeb et al., 2010; Delmulle et al., 2006), hepatic cancer cell lines
HepG2 and Huh7 (at a concentration of 25 M) (Dorn et al., 2010),
leukemic cell line B-ALL L1210 (IC50 was 4 M) (Benelli et al., 2012).
2.2.4. Anti-inammatory activity
It is estimated that approximately 18% of all cancers are associated
with chronic inammations (Gerhauser, 2009).
Excessive production of tissue activators, especially prostaglandins
(a group of hormone-like lipid compounds) and nitric oxide, initiates
a general inammatory response and avonoids have been shown to
inhibit key enzymes involved in the biosynthesis of these tissue activa-
tors (Gerhauser et al., 2002). An intermediate in the biosynthesis
of prostaglandins is arachidonic acid, and therefore the release of
arachidonic acid is a essential in the development of inammation
(Ferrandiz and Alcaraz, 1991). Flavonoids as anti-inammatory agents,
have been shown to be effective inhibitors of arachidonic acid metabo-
lism through inhibition of gene expression of cyclooxygenase 1 enzymes
(COX-1, IC50 for xanthohumol is 16.6 M) and cyclooxygenase 2 (COX-2,
IC50 xanthohumol is 41.5 M) (Gerhauser, 2005a). Isoxanthohumol did
not show such an activity when tested under the same conditions
(Gerhauser et al., 2002) whereas 8-prenylnaringenin inhibited prosta-
glandin synthesis by COX-2 in RAW 264.7 murine macrophages in an
in vitro model of inammation (Paoletti et al., 2009).
Several studies have demonstrated that avonoids, especially
prenylavonoids such as xanthohumol, can inhibit the release of
NO by direct inhibition of inducible nitric oxide synthase (iNOS).
Paoletti et al. (2009) examined the effect of 8-prenylnaringenin on
iNOS expression and thus the production of nitric oxide. 30 M of
8-prenylnaringenin considerably increased iNOS gene expression in
LPS-stimulatedRAW 264.7 cells (Paoletti et al., 2009). Karin (2006)
demonstrated that xanthohumol, isoxanthohumol, kaempferol, and
quercetin inhibit iNos induction in murine macrophages with IC50
values ranging from 18.7 to 40.6 M (Gerhauser, 2009). This inhibits ex-
pansion of the vascular system and thus the formation and maintenance
of a tumor (Nozawa et al., 2009). Flavonoids with a higher number of
hydroxyl groups are only weakly active but methylation or acetylation
of an hydroxyl group improves their activities (Mladenka et al., 2010).
NF-B (nuclear transcription factor kappa beta) regulates the
expression of genes encoding proteins involved in the formation
proinammatory factors. NF-B allows transcription of genes for
cyclooxygenase enzymes (especially inducible forms of COX-2), catalyz-
ing the formation of prostaglandins by oxidation and cyclization of ara-
chidonic acid (Araujo et al., 2011; Ramos, 2008) Inhibition of activation
of NF-B being the key chemopreventative target (Monteiro et al.,
2008). It was also found a in vitro study that xanthohumol and
other prenylavonoids such as 8-prenylnaringenin strongly inhibited
activation of NF-B. Colgate et al. (2007) investigated the effect of
xanthohumol on the inhibition of NF-B in BPH-1 cells. Xanthohumol,
at a concentration of 20 M, caused a 42% decrease in NF-B activity
(Colgate et al., 2007; Dorn et al., 2010; Paoletti et al., 2009). Not only
prenylavonoids but also catechin, epicatechin and procyanidin dimers
B1, B2 and B5, can inhibit NF-B at different positions in the activation
pathway. Treatment with 100 nM of procyanidin dimers B1 or B2
resulted in the reduction of binding activity of NF-kB to DNA (by 83
and 90%, respectively) (Mackenzie et al., 2009). Flavonol monomers
and procyanidins were shown to supress production of other pro-
inammatory agents that participate in the regulation of immune
responses and inammatory reactions, namely the cytokines
interleukin (IL)-1 and IL-6 in the cell line RAW 264.7. Treatment
with quercetin blocked (by more than 80%) the release of tumor
necrosis factor (TNF-), another signicant contributor to chronic
inammatory responses (Garcia-Lafuente et al., 2009).
2.2.5. Estrogenic activity
Another important effect of some avonoids (mainly prenylavonoids)
is their estrogenic activities that mimic human steroids (17--estradiol),
and thus suppress critical symptoms or reduce the risk of hormone-
associated cancers (Chadwick et al., 2006). Structural similarities be-
tween estrogens and avonoids provide these molecules with the
ability to act as estrogen agonists or antagonists. The prenylavonoid,
8-prenylnaringenin, was identied as the most potent phytoestrogen
known to date. Subsequent investigations in vitro show that
8-prenylnaringenin can generally mimic the action of 17--estradiol,
although with a lesser (1020,000-fold) potency (Chadwick et al.,
2006). The concentration required to achieve 50% of its maximum
estrogenic effect (EC50) was determined for 8-prenylnaringenin to
be 4.4 nM, while the EC 50 for 17--estradiol is 0.82 nM (Milligan
et al., 2002).
Estrogenic properties of avonoids can be expressed in two ways.
The rst is their binding to specic estrogen receptors. The mechanism
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
of action consists of the activation of estradiol receptors (ER), which
exist in two subtypes: ER and ER. Flavonoids possess estrogenic
activity based on direct interactions with ERs (Milligan et al., 2000).
8-prenylnaringenin exhibits approximately a 2 fold greater afnity for
ER where it is about 70-fold less active than 17--estradiol(Schaefer
et al., 2003). The main hop prenylavonoid, xanthohumol, showed no
afnity for either ERs (Milligan et al., 2000).
The second way is via the ability of avonoids to act as enzyme
effectors. Flavonoids can interfere with the enzyme aromatase, which
regulates the level of blood estradiol by converting androgens to
estrogens in the breast (from androstenedione and testosterone)
(Monteiro et al., 2007). Monteiro et al. (2007) have shown that
all prenylavonoids tested (8-prenylnaringenin, xanthohumol and
isoxanthohumol) inhibited aromatase activity (IC50 = 0.08 M,
IC50 = 3.2 M, IC50 = 25.4 M, respectively) and thus play a crucial
role in the development of aromatase-expressing breast cancer
(SK-Br-3), where breast tumors have been demonstrated to express
abnormally high levels of aromatase, in comparison with normal
tissues (Monteiro et al., 2007; van Duursen et al., 2013). In addition,
8-prenylnaringenin stimulates the production of alkaline phosphatase
in endometrial derived adenocarcinoma cell line Ishikawa Var
Iin vitro.Milligan et al. (1999) determined the EC 50 values for
8-prenylnaringenin and 6-prenylnaringenin as 4 and 500 nM,
respectively, compared with the EC50 value for 17--estradiol
(0.8 nM) (Milligan et al., 1999, 2002), and stimulates estrogen-
responsiveMCF-7 cell proliferation breast cancer cells (Effenberger
and Westendorf, 2009).
It is assumed that the intake of avonoids with estrogenic potential
is positively associated with the prevention of osteoporosis and protec-
tion of bone health, and thus avonoids exert protective effects against
post-menopausal bone loss. Osteoporosis is a progressive decrease in
bone mass and density, whereas the main cause of this disease is an
imbalance between bone formation and bone resorption, especially
affecting postmenopausal women.
The mechanism of the action of prenylavonoids is still not
completely understood. It is assumed that prenylavonoids can modu-
late gene expression in bone cells, enhance expression in osteoblasts
(cells responsible for the bone formation) and suppress the expression
in osteoclasts (bone cell that resorb bone tissue). It was demonstrated
that 8-prenylnaringenin and naringenin, at a concentration of 1 M,
signicantly enhanced differentiation of osteoblasts, and thus minerali-
zation, in rat bone marrow stromal cells in vitro(Ming et al., 2012). Jeong
et al. (2011) investigated the ability of xanthohumol to increase the
expression and transcription of RUNX2, a major gene of osteoblast dif-
ferentiation (Jeong et al., 2011). Suh et al. (2013) demonstrated the abil-
ity of xanthohumol to suppress receptor activator of NF-B ligand
(RANKL) signaling in RAW264.7 cells, which plays a major role in
osteoclastogenesis and thus bone resorption (Suh et al., 2013).
Humpel et al. (2005) tested 8-prenylnaringenin in an adult ovariec-
tomized rat model in vivo, showing that 8-prenylnaringenin has the
ability to protect against ovariectomy-induced bone loss through com-
pensation for loss of bone mineral density in an animal model in vivo,
with minimal effects on uterus weight and the endometrium (Humpel
et al., 2005).
2.2.6. Antimicrobial activity
Flavonoids have wide antimicrobial activities against a variety
of microorganisms, including bacteria, viruses, fungi and protozoa
(Gerhauser, 2005b).
It was found that xanthohumol inhibits the growth of the Gram-
positive bacteria Staphylococcus aureus (with a minimal inhibitory
concentration (MIC) of 17.7 M) and Streptococcus mutans (with a
MIC of 35.3 M) (Gerhauser, 2005b). Myricetin has been shown to
inhibit the growth of the multidrug resistant bacterium Burkholderia
cepacia, vancomycin-resistant enterococci and other medically impor-
tant microorganisms such as Klebsiella pneumoniae and Staphylococcus
1071
epidermidis.MIC values of myricetin were in a concentration range of
64 to 256 mg/mL (Xu and Lee, 2001).
Antiviral activity of xanthohumol, in combination with interferon
-2b, was demonstrated against the virus that causes bovine diarrhea
(bovine viral diarrhea virus -BVDV E2), which shows considerable
similarities with the human hepatitis C virus, and against herpes virus
(herpes simplex virus -HSV 1 and HSV 2), cytomegalovirus (CMV) and
rhinovirus (Buckwold et al., 2004; Zhang et al., 2010). The concentra-
tions required for antiviral activity (IC50) were in a range of 4.2-7.6 M
xanthohumol. The discovery and development of avonoids as anti-
HIV agents has expanded (Wang et al., 2004), where most studies
have focused on avonoid inhibition of reverse transcriptase, or RNA-
directedDNA polymerase (Ono et al., 1990). Wang et al. (2004) investi-
gated the activity of xanthohumol to suppress several crucial steps in
the replication of HIV-1 such as interfering with HIV-1co-
receptors(Wang et al., 2004). Anti-HIV activities against HIV1-
protease and reverse transcriptase by quercetin and its derivatives
have been reviewed (Xu et al., 2000).
2.2.7. Antidiabetic effects
Flavonoids exhibit antidiabetic activities through inhibition of
-glucosidase(Liu et al., 2014a), an enzyme that controls the postpran-
dial blood glucose level, thus preventing excess glucose absorption in
the small intestine, and easing hyperglycemia. They have potential for
use in the treatment of type 2 diabetes (Havsteen, 2002).
Diabetes mellitus is connected with another enzyme, aldose reduc-
tase, which reduces excess D-glucose to D-sorbitol. Aldose reductase
is a key enzyme in the polyol pathway (sorbitol-aldose reductase
pathway) that plays an important role in the development of degener-
ative complications of diabetes, especially in microvascular damage to
the retina, kidney and nerves. Mok and Lee investigated (2013) the
inhibitory activity of the hop avonols kaempferol, quercetin and
myricetin against aldose reductase from rat lens (Mok and Lee, 2013).
Quercetin was also demonstrated to be an inhibitor of glycogen
phosphorylase, a key enzyme in the regulation of glycogen metabolism.
Inhibition of this enzyme is considered to be benecial in treating type II
diabetes (Kantsadi et al., 2014).
Liu et al. (2014a) investigated inhibitory effects of xanthohumol
on -glucosidase, an enzyme that controls blood glucose levels,
and thus xanthohumol provides glycemic control over hyperglyce-
mia in diabetes mellitus type 2, particularly with regard to postpran-
dial hyperglycemia. It is assumed that the mechanism of inhibition
consists of binding xanthohumol to -glucosidase and induction of
changes in enzyme conformation. Additionally, xanthohumol reduces
the hydrophobicity of -glucosidase(Liu et al., 2014a).
Legette et al. (2013) determined the effect of oral administration
of xanthohumol on biomarkers of a metabolic syndrome connected
with obesity, including atherogenic dyslipidemia, insulin resistance,
impaired glucose tolerance, hypertension, and the pro-inammatory
and prothrombotic states in obese male rats. It was demonstrated that
administration of xanthohumol, at a concentration of 16.9 M/kg b.w.
(body weight) had a positive effect on glucose metabolism (Legette
et al., 2013, 2014).
2.2.8. Other health promoting effects of hop avonoids
In addition to the described biological activities, avonoids
exhibit other health benets, for instance avonoids have antiallergic
(Makino et al., 2013), anticoagulant, antiplatelet (Guerrero et al.,
2005), and anticholinesterase activities (therapeutic strategy in
Alzheimers disease) (Ding et al., 2013). Moreover avonoids modulate
hepatitic expression of genes involved in thyroid hormone distribution
and metabolism (Radovic et al., 2010), preventing disuse muscle
atrophy (Mukai et al., 2012) or can have neuroprotective activities
in vitro(Oberbauer et al., 2013).
In addition, some avonoids (kaempferol, quercetin, xanthohumol,
isoxanthohumol and 8-prenylnaringenin) have potential for use in
1072 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
dermatology, treating skin diseases including atopic eczema, contact
dermatitis or pigmentary disorders (Chen et al., 2014a).
2.3. Toxicity of hop avonoids
When the positive effects of hop avonoids on human health are
discussed, it is also necessary to mention the potential toxicity of
these substances. Extensive toxicological studies in vitro, and in vivo
using animals and humans, have investigated their safety in pharma-
ceutical products for the prevention of certain pathological conditions.
Safety studies in vitro and in vivo(Nagasako-Akazome et al., 2007),
and clinical trials (Tagashira, 2007), have been carried out on high
level and long-term intakes of hop bract polyphenols including
procyanidins, avan-3-ols and avonols (Nagasako-Akazome, 2014).
Nagasako-Akazome et al. (2007) used 14-day, 28-day and 90-
dayin vivo toxicity tests using SpragueDawley (SD) rats (group of 10
males and 10 females) to evaluate the safety and possible harmful
effects of hop bract polyphenols, including avonoids. SD rats were
fed an extract of hop polyphenols at doses of 0, 500, 1000 or
2000 mg/kg body weight (b.w.)/day and all animals were observed
daily for 14 days; toxicological parameters including body weight,
changes in faecal condition, and pathological gastrointestinal tract
abnormalities were measured (Nagasako-Akazome et al., 2007).
No changes in faecal condition and no pathologic abnormalities in
the gastrointestinal tract were observed in any of the groups of rats.
Even very high doses of polyphenol extract (N2000 mg/kg b.w.) caused
no mortality and did not represent a risk to any of the groups of rats.
Clinical studies on 12 healthy adults showed no clinical changes
in hematological and biochemical tests, before and after intake of
avonoids (2500 mg of hop bracts polyphenols/day) for 28 days
(Tagashira, 2007).
Despite all studies documenting the positive effects of quercetin on
health, the safety of quercetin has long been a highly controversial
issue because of its potential carcinogenic properties (Dunnick and
Hailey, 1992; Pamukcu et al., 1980) and in 1998 it was even declared
a carcinogen by the International Agency for Research on Cancer
(Harwood et al., 2007). However, the carcinogenic properties of querce-
tin have repeatedly been investigated but carcinogenicity could not be
conrmed by other investigators (Ito, 1992). Harwood et al. (2007)
summarized the safety results of quercetin in mutagenicity tests, and
short- and long-term animal and human studies in vivo. From the
numerous toxicological studies (Garcia-Saura et al., 2005; Nakamura
et al., 2000), it was concluded that quercetin, at an estimated dietary
exposure level, posed no safety risk for human or animal health
(Harwood et al., 2007). Ruiz et al. (2006) evaluated possible adverse
effects of high doses of quercetin in Swiss mice, where test animals
were administered with quercetin in a 28-day test at doses of 0, 30,
300, and 3000 mg/kg b.w. There were no animal mortalities at high
doses of quercetin and no changes or abnormalities were observed
(Ruiz et al., 2006).
For catechins, no health risks have been reported from trials in vitro
and in vivo, nor from clinical studies that were almost always performed
on catechins isolated from green tea (Morita et al., 2009; Wang et al.,
2011; Yoneda et al., 2009). The toxicity of prenylavonoids such as
xanthohumol were investigated in numerous studies in vitro(Dorn
et al., 2010; Hudcova et al., 2014a) and in vivo(Dorn et al., 2010;
Vanhoecke et al., 2005) but with negative results. Dorn et al. (2010)-
reported no adverse effects after daily administration of xanthohumol
(1000 mg/kg b.w.) to BALB/c mice for 3 weeks compared with control
mice without dietary xanthohumol. Histopathological evaluation of
major organs (liver, kidney, colon, lung, heart, spleen and thymus),
as well as biochemical serum analysis, conrmed that xanthohumol
does not pose any risk and no signs of xanthohumol-toxicity were
observed (Dorn et al., 2010).
Particular research emphasis has been placed on the prenylavonoid
8-prenylnaringenin, which is already part of dietary supplements
available on the market. Milligan et al. (2002) reported negative effects
of 8-prenylnaringeninin vivo(15.9 mg/kg b.w. - a concentration that
exceeds the normal concentration of 100 g/pill once per day in food
supplements) on uterine function, inuencing the development of
vaginal mitoses (Milligan et al., 2002). Therefore, despite the num-
ber of in vitro(Effenberger and Westendorf, 2009; Tokalov et al.,
2004), in vivo and clinical studies (van Breemen et al., 2014), more
epidemiological and clinical studies are needed for the safety of
8-prenylnaringenin to be fully elucidated.
3. Biotransformations of hop avonoids
3.1. Human metabolism of avonoids
Polyphenols from hops are transferred during processing into one
of the most popular beverages worldwide beer and thereby
become an integral part of the human diet. The most important
biotransformation processes then involve metabolism of these com-
pounds in the human digestive tract. A diagram describing individual
stages of the metabolism of avonoids, and their locations in the
body, is shown in Fig. 10.
Many products of human avonoid metabolism exhibit interesting
biological effects and these physico-chemical processes can be the rst
step in future industrial applications of avonoids biotransformation.
Crucial steps in the human metabolism of avonoids are processes
taking place in the liver. Flavonoids, either unmodied or partly
transformed (hydrolysed, demethylated, deglycosylated, conjugated)
by the acidic environment in the stomach, in epithelial cells of the
small intestine, and/or by colonic microbiota (Spencer et al., 1999),
are bound to albumin (Kuhnle et al., 2000; Manach et al., 1996) and
transported through the hepatic portal vein to the liver. The most
common reactions in the liver are conjugations that form avonoid
glucuronates, sulphates and O-methylated derivatives, and oxidative
transformations catalysed by cytochrome P450 enzymes (Breinholt
et al., 2002; Hodek et al., 2002). The role of microsomal enzymes and
UDP-glucuronyl transferase in glucuronidation processes has been
demonstrated (Oliveira and Watson, 2000; Otake et al., 2002; Singh
et al., 2011; Vaidyanathan and Walle, 2002; Yilmazer et al., 2001a)
and the production of glucuronides has also been carried out using
recombinant forms of these enzyme (Chen et al., 2008). Human
sulphotransferases capable of conjugating a wide range of dietary avo-
noids to sulphonates (Huang et al., 2009) were also isolated and tested.
The resulting conjugates and unconverted avonoids are distributed
by the circulatory system to individual tissues and further transformed
by intracellular metabolism (Spencer et al., 2004). However, not all
avonoids are capable of uptake into all types of tissues. For example,
methylated forms of quercetin are capable of entering cells, while
their glucuronides are not (Spencer et al., 2003), and the most impor-
tant group of hop polyphenols, avan-3-ols, are able to enter several
types of cells, such as dermal broblasts (Spencer et al., 2001b) and
cortical neurons (Spencer et al., 2001a), although in only a relatively
small amounts. Moreover, conjugation of avonoids with glutathione
also occurs in tissues. This has been shown for quercetin, where the
regiospecicity of conjugation was strongly pH-dependent(Awad
et al., 2002).
Little is known about the ability of avonoids to overcome the
bloodbrain barrier (BBB), a highly selective lter that separates the
circulating blood from the brain extracellular uid, and controls
the entry of xenobiotics into the brain (Bicker et al., 2014). After oral
administration of epicatechin (Ferruzzi et al., 2009), avanones (Peng
et al., 1998), avonols (Rangel-Ordonez et al., 2010) and anthocyanidins
(Talavera et al., 2005), their concentrations in brain cells increased.
It is assumed that the ability to cross the BBB is based on polarity
(Youdim et al., 2003), so lipophilic compounds are capable of greater
brain uptake than hydrophilic sulphates and glucuronides.
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
Fig. 10. Human metabolism and distribution of avonoids (Corona et al., 2014).
3.2. Bioavailability of hop avonoids
Although the use of hop avonoids in therapeutic drugs is not the
primary topic of this article, it is appropriate to at least partially describe
the bioavailability and pharmacokinetic properties of these substances.
It is evident that questions of absorption of bioactive avonoids and
their levels in tissues and blood serum will be an integral part of future
studies examining the use of polyphenolic biotransformation products
for therapeutic purposes. This chapter summarizes basic information
regarding the bioavailability of avonoid compounds found in hops.
Since only a few studies have been carried out with hops or hop
extracts, results of research dealing with the bioavailability of pure
standards, as well as avonoids from other foods, will be described.
Differences in the bioavailability caused by structural variations in
avonoids (glycosylation, prenylation, methylation etc.) will be
addressed in the respective chapters.
The United States Food and Drug Administration (FDA) dene
bioavailability as "the rate and extent to which the active ingredient
or active moiety is absorbed from a drug product and becomes available
at the site of action" (FDA, 2014).
Given the complex character of human metabolism of prenylavonoids,
it is clear that in addition to exogenous factors (chemical structure, the
presence of carbohydrate or prenyl moieties, the route of administration),
human bioavailability of avonoids is affected by a number of endogenous
factors such as age and sex (Guo and Bruno, 2014), and in particular, by the
individual-specic composition of intestinal microora (Guarner and
Malagelada, 2003).
Considerable attention has been paid to the bioavailability of hop
prenylavonoids, particularly with regard to their metabolic conversion
into the estrogenically active 8-prenylnaringenin(Possemiers et al.,
2009b). The intracellular uptake and distribution of xanthohumol
has been studied in vitro in hepatocellular carcinoma cells, hepatic
1073
stellate cells, cultured primary hepatocytes and colorectal adenocarci-
noma cells (Wolff et al., 2011). Incubation of cells in medium containing
10 M of xanthohumol, after 4070 minutes, resulted in a 60-
foldaccumulation of xanthohumol within the cells, compared to the
original concentration in the medium. A study, carried out on
SpragueDawley rats (Legette et al., 2012) conrmed that orally
dosed xanthohumol is absorbed by intestinal cells and subsequently
transported in the blood, like other avonoids, and the highest con-
centrations of xanthohumol in plasma occurred after approximately
4 hours. Changes in concentration of xanthohumol and other
prenylavonoids in plasma were studied after injection (1.86 mg/kg
b.w.) and oral (three different doses - 1.86, 5.64 and 16.9 mg/kg b.w.)
administration of xanthohumol (Legette et al., 2012). Using the proce-
dure recommended by the FDA,dose-dependent bioavailability of
xanthohumol was determined. Values obtained were inversely propor-
tional to the dose, and amounted to 33.1%, 13.4% and 10.8% respectively.
In another study (Hanske et al., 2010) rats were fed a diet enriched in
xanthohumol to a level of 50 mol/kg b.w. Maximum concentrations
(Cmax) of xanthohumol in the blood were achieved after 4 hours
(Cmax = 0.65 M), and isoxanthohumol and its conjugates after 8
to 10 hours (C max = 1.04 and 4.87 M). During a randomized,
Table 2
Concentrations of xanthohumol, isoxanthohumol and 8-prenylnaringenin aglycone
equivalents in serum and breast tissue upon prenylavonoid supplementation (expressed
as mean + standard error of mean).
Compound Serum (nmol/L) Breast tissue (pmol/g)
Adipose Glandular
Xanthohumol 4.99 1.79 0.69 0.17 2.58 0.45
Isoxanthohumol 14.86 2.71 3.68 1.27 12.02 7.20
8-Prenylnaringenin 2.20 1.26 1.44 0.34 2.50 0.46
1074 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090

double-blind, placebo-controlled study in postmenopausal women
(Rad et al., 2006) receiving 8-prenylnaringenin at doses of 50, 250
and 750 mg, maximum serum concentrations of this substance
occurred after 8 to 10 hours. These concentrations were dose-
dependent from 235 ng/mL and decreased to zero 48 hours after
administration.
The bioavailability of hop prenylavonoids in human breast tissue
was the subject of a study performed on 21 women (Bolca et al.,
2010). Following daily administration for 5 days of a food supplement
made of hops and containing 6.12 mg of xanthohumol, 3.6 mg of
isoxanthohumol, and 0.3 mg of 8-prenylnaringenin, the concentrations
of these compounds in blood serum, adipose and mammary glandular
tissues were monitored. The average values are given in Table 2.
It should be noted that the presence of 8-prenylnaringenin in the
serum and breast tissue was shown for only approximately half of the
subjects, which conrms the theory that only a proportion of the popu-
lation possesses intestinal microora that are capable of signicant con-
version of isoxanthohumol into 8-prenylnaringenin(Possemiers et al.,
2009b).
A very extensive study using 48 human subjects (24 male and
24 female) on the pharmacokinetic parameters of hop prenylavonoids
was published in 2014 (Legette et al., 2014). After a single-dose oral
application of 20, 60 or 180 mg of xanthohumol, the pharmacokinetic
prole was measured, based on concentrations of xanthohumol
and isoxanthohumol conjugates in the plasma, where rst peak
concentrations occurred after 1 hour and the second after 45 hours.
Dose-dependent maximum concentrations were 33 7 g /L, 48
11 g/L and 120 24 g/L respectively.
Information regarding bioavailability of other polyphenols obtained
from hops and/or beer is, so far, limited to phenolic acids. For avonoids,
data are almost exclusively available only from analyses using other
sources of polyphenols such as tea, wine, cocoa, various fruits and
vegetables, or pure isolated substances. Publications summarizing the
available information on this topic were published in 2005 (Manach
et al., 2005) and in the form of a monograph in 2012 (Crozier et al.,
2012). Taking into account that food properties signicantly affect the
bioavailability of individual components (Guarner and Malagelada,
2003; Guo et al., 2013; Urpi-Sarda et al., 2012), it would be more appro-
priate to describe studies using pure compounds. The results of selected
studies are summarized in Table 3.
Regardless of the source of avonoids, it is generally agreed that the
bioavailability of avonoids in the human organism is quite low and the
rst works dealing with possibilities of increasing this have just been
published (Song et al., 2014).
3.3. Prenylation
Prenylation plays an important role in the diversication of plant
polyphenolic compounds. The substitution site, chain length and
structure of prenyl side chains can signicantly affect the properties
of the polyphenol (Yazaki et al., 2009). Moreover, the presence of a
nonpolar isoprene chain structure can also signicantly affect metabolic
transformations of these compounds and their bioavailability (Terao
and Mukai, 2014). While other polyphenols are synthesized in non-
specic tissues of the hop plant, prenylated avonoids, together with
- and -bitter acids, are formed through parallel biosynthetic path-
ways, predominantly in lupulin glands; glandular trichomes present
on hop cones and leaves (Stevens and Page, 2004). The similarity of
these pathways and structures of both groups of compounds is clearly
apparent from Fig. 11(Tsurumaru et al., 2012). Biosynthesis is initiated
by the condensation of thioesters, obtained by degradation of the
corresponding amino acid (leucine, isoleucine and valine for bitter
acid analogs and phenylalanine for xanthohumol), with three
malonylcoenzyme A molecules and this step is unique to hop. The
arising conjugates are then prenylated and from this point, the pathways
diverge. While the precursors of bitter acids are prenylated again,
desmethylxanthohumol is methylated, giving rise to the formation of
xanthohumol. O-methyltransferase, which catalyses this methylation in
lupulin glands, was identied in 2008 (Nagel et al., 2008). As a result
of the potential use of prenylated avonoids in the pharmaceutical
industry, close attention has been focussed on regiospecic biosynthesis
of these compounds (Chen et al., 2013), isolation and characterization of
relevant enzymes (Yu and Li, 2011), and identication and verication of
possible health-promoting properties of prenylavonoid metabolites
(Possemiers et al., 2006; Tronina et al., 2013a, 2014; Zierau et al., 2004).
3.3.1. Impact of prenylation on bioavailability of avonoids
The question of bioavailability of prenylated avonoids and their
bioaccumulation in tissues has not yet been fully resolved. It is assumed
that the presence of the prenyl group decreases the polarity of
prenylavonoids (Jelinek et al., 2013), which would increase their
uptake through the semi-permeable enterocyte cell membrane,
increasing the rate of absorption and thus their bioavailability.
It is therefore surprising that results of studies focused on bioavail-
ability in vitro of other hop-derived substances containing a prenyl
group (- and -bitter acids and prenylated fenolic acids) suggests
that, on the contrary, prenylation decreases bioavailability (Cattoor
et al., 2010; Konishi, 2005). This discrepancy can be explained by the
fact that molecules of prenylated compounds can easily pass into
epithelial cells, however efux of their conjugates (glucuronide and/or
sulphate formed by IIphase-enzymes) to the basolateral side of
the cell is signicantly lower when compared with nonprenylated
analogues. This difference could be caused by a low afnity of conju-
gates for transport proteins such as adenosine triphosphate-binding
cassette transporters (Terao and Mukai, 2014), although work focused
on this specic issue is not yet available.
The presence of a prenyl group also confers resistance to degrada-
tion of xanthohumol by intestinal microbiota. Analyses of extracts

Table 3
Selected bioavailability studies of pure avonoids or avonoid-containing foods.

Compound No. of subjects Source Dose Tmaxh Cmax in plasma mol/L References

Rutin 9 pure 100 mg (quercetin eq) 9.3 0.3 (Hollman et al., 1997)
9 pure 190 mg 6 0.18 (Hollman and Katan, 1999)
3 pure 500 mg 4-7 0.13-0.73 (Boyle et al., 2000)
16 pure 8/20/50 mg (quercetin eq) 6.5/7.4/7.5 0.08/0.16/0.30 (Erlund et al., 2000)
Quercetin 16 pure 8/20/50 mg 2.0/2.7/4.9 0.14/0.22/0.29 (Erlund et al., 2000)
12 pure 0.14 mg/kg b.w. 0.5 0.15-0.42 (Goldberg et al., 2003)
35 pure 50/100/150 mg/day for 2 weeks 2/3/6 0.19/0.30/0.43 (Egert et al., 2008)
Quercetin-3-glucoside 9 pure 156 mg 0.6 5 (Olthof et al., 2000)
Catechin 12 pure 0.36 mg/kg b.w. 0.5 0.14-0.49 (Goldberg et al., 2003)
9 red wine 35 mg 1.44 0.077 (Bell et al., 2000)
Flavan-3-ols 6 cocoa 115.25 mg/serving 0.9-2.3 0.024-0.042 (Neilson et al., 2009)
EGCG/EGC/ECG 39 green tea extract 414/362/51 mg/per day 1-2.5 0.55/0.50/0.35 (Hodgson et al., 2014)

EGCG/EGC/ECG - Epigallocatechin gallate/Epigallocatechin/Epicatechin gallate;

quercetin eq - quercetin equivalent
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090 1075

Fig. 11. Biosynthesis of xanthohumol and bitter acids in glandular trichomes of hop (Tsurumaru et al., 2012).

from the faeces of laboratory rats fed with a hop extract containing high
levels of xanthohumol showed that approximately 89 rel.% of the
avonoid fraction consisted of unconverted xanthohumol (Nookandeh
et al., 2004). The rest consisted of a wide range of cyclization,
hydroxylation, O-methylation, O-acetylation, isomerization and
glucuronidation products (Bartmanska et al., 2013; Herath et al.,
2003b; Nikolic et al., 2005; Ruefer et al., 2005). An important biotrans-
formation reaction occurring in the digestive tract is demethylation of
the isomer of xanthohumol that produces 8-prenylnaringenin, one of
the most active phytoestrogens known (Milligan et al., 1999, 2000).
Given the importance of this reaction, a separate chapter dedicated
to it has been added.
1076 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
3.3.2. Enzymes involved in prenylation
Prenylation of hydroxylated aromatic structures is the outcome of
two important biosynthetic pathways. The aromatic acyphloroglucinol
structure is produced by the shikimate or polyketide pathways and
the prenyl chain (mostly as dimethylallyl pyrophosphate) is the product
of isoprenoid synthesis from the mevalonate and methyl erythritol
(MEP) pathways (Yazaki et al., 2009). Prenylation itself is essentially a
Friedel-Crafts alkylation reaction catalysed by prenyltransferases.
These enzymes catalyse the transfer of isoprenoid units, such as
dimethylallyl, geranyl or farnesyl groups, to a variety of acceptors
(Liang et al., 2002). In addition to the biosynthesis of prenylavonoids,
these enzymes form part of biosynthetic pathways leading to the
formation of lipoquinones, ubiquinones, terpenoids, cholesterol, and
many other plant secondary metabolites (Heide, 2009; Liang et al.,
2002). Plant prenyltransferases are typically divided into two groups,
membrane-bound in plastids, and soluble enzymes. (Yazaki et al.,
2009). This simple division however, fails to address the state of our
knowledge and therefore divisions based on the types of reactions
catalysed were created (Heide, 2009). Unlike other prenyl-
transferingenzymes, isolation and characterization of specic avo-
noid prenyltransferases represents a signicant challenge. So far,
only a few of these enzymes, namely from Sophora avescens(Sasaki
et al., 2008), soybean (Akashi et al., 2009) and Lupinus albus(Shen
et al., 2012) have been isolated.
In hops the rst candidate gene for membrane-bound prenyltransferase
HlPT-1(Humulus lupulus prenyltransferase 1) was identied in 2012
(Tsurumaru et al., 2012). The enzyme contains aspartate-rich motifs
(NQxxDxxxD and KDxxDxxGD), which are common in other known
plant magnesium-dependent aromatic prenyltransferases (Stec and
Li, 2012). HlPT-1, in contrast to similar plant enzymes, exhibited
relative substrate promiscuity and utilized precursors of bitter acids,
as well as naringenin chalcone (Fig. 11), which is a precursor of
xanthohumol, as acceptors of the prenyl moiety (Tsurumaru et al., 2012).
3.3.3. Prenylation in biotechnological industries
The rst attempts to prepare hop prenylated compounds were car-
ried out in the last decade of twentieth century (Zuurbier et al., 1998).
Although not all methods for the biotechnological prenylation of
polyphenols have been developed for the commercial preparation of
hop prenylavonoids, due to their health-promoting properties, it is
likely that this will occur in the near future. Preparation of biologically
active prenylavonoids by genetic and metabolomic engineering,
using plant and microbial enzymes, has received considerable attention.
Using the expression of genes encoding the formation of
prenyltransferase HlPT-1 in baculovirus infected cells of Autographa
californica, a fraction with strong prenyltransferase activity was
obtained and used for the production of precursors of prenylated
components of hops - the bitter acids and xanthohumol (Tsurumaru
et al., 2012). Enzyme activity measured using naringenin chalcone
as a substrate was 68 rel.% of that using phlorisovalerophenone.
Furthermore, it was found that the enzyme exclusively required
the presence of Mg2 + ions and had a relatively sharp pH optimum
around 7. However, this enzyme was not able to catalyse direct
prenylation of naringenin and 6-O-methylchalcone, which means that
the glandular trichomes must contain other prenyltransferases in
order to create prenylnaringenins and conrms the view that the
nal step in the biosynthesis of xanthohumol is O-methylation of
desmethylxanthohumol. Enzymes catalyzing this type of methylation
reaction have already been identied (Nagel et al., 2008). Altogether,
three different fractions (OMT 13) were isolated, but only one
of them (OMT 1) was able to catalyse direct methylation of
desmethylxanthohumol to xanthohumol. OMT 2 is involved in
the methylation of desmethylxanthohumol, chalconaringenin,
and xanthohumol to other products, only one of which (4-O-
methylxanthohumol) has been identied and reactions catalysed
by OMT 3 have not been identied at all.
A study of the hop transgenic metabolome and perspectives for
its application is included in the work by authors from the Czech
Republic. They are currently seeking to understand the complex regula-
tion of the hop prenylavonoid biosynthetic pathway, in particular
through analysis of lupulin gland transcription factors (Matousek
et al., 2012). In future, this knowledge should allow the induction of
metabolically active transgenic tissue cultures of hop.
To examine the possibility of using prenyltransferase from the plant
Sophora avescens, the avonoid prenyltransferase gene (SfFPT) was
cloned and successfully expressed in the yeast, Saccharomyces cerevisiae
YPH499 (Chen et al., 2013). The enzyme was able to catalyse the
prenylation of a broad spectrum of substrates, among others naringenin,
to form 8-prenylnaringenin. Regiospecicity could become an advan-
tage of this enzyme, because 8-prenylnaringenin is a substance with
signicantly stronger estrogenic activity than 6-prenylnaringenin.
The use of some recombinant enzymes from microbial sources
to prepare prenylavonoids is also possible. The CloQ/NphB class of
prenyltransferases from Streptomyces sp. (NovQ) over-expressed in
E. coli was tested for the prenylation of a wide range of polyphenolic
compounds (Ozaki et al., 2009) and it has been shown that these
enzymes are capable of C-andO-prenylation on the B ring of avonoids,
such as naringenin. Flavonoids can be also geranylated on 7-OH or 6-C
part of the A ring by the use of NphB soluble prenyltransferase
from Streptomyces coelicolor(Kumano et al., 2008). In 2011, the work
summarizing possible prenylations using microbial and plant genes,
was published (Sugiyama et al., 2011). The authors tested over-
expression of three Streptomyces genes (NphB, SCO7190 and NovQ)
and two plant genes from Sophora avescens (N8DT and G6DT) in
the model legume plant Lotus japonicus. The formation of prenylated
derivatives of naringenin and genistein by these transgenic plants
required supplementation of the avonoid substrate, and the resulting
concentrations in the plant tissues were not high (maximum 86 g/g
dry weight). Despite this, the study is an important step for future
production of these interesting secondary metabolites.
3.4. Methylation and demethylation
3.4.1. Enzymes involved in methylation
O-methylation of avonoid substrates is a common enzymatic
modication, catalysed by O-methyltransferases (OMT) that transfer
the methyl group from S-adenosylL-methionine (AdoMet) to the
hydroxyl group of methyl-acceptor molecule (Cho et al., 2012). OMTs
have been isolated from several plants and have been functionally
characterized. Most of these enzymes have high substrate specicities
(Kim et al., 2006c). OMTs involved in avonoid modications have
been studied extensively, showing that their molecular weights range
from 38 to 43 kDa and they contain a conserved AdoMet binding site
(Kim et al., 2006a). O-methylation affects the physicochemical
Fig. 12. Structures of methylated metabolites of quercetin.
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
Fig. 13. Structures of methylated metabolites of catechin and epicatechin.
properties of substrates and the chemical reactivity of phenolic
hydroxyl groups (Kim et al., 2006b). O-methyltransferases are
expressed mostly in the liver, but also in some other human tissues
such as kidney, intestine, lung, brain, erythrocytes and platelets
(Chen et al., 2014b).
Methylated metabolites can also be reverse O-demethylatedin vivo
by cytochrome P450 enzymes CYPs (Walle, 2009).
Flavonoids with hydroxyl groups including avon-3-ols (catechins)
and avonols (quercetin), are metabolized by catechol-O-
methyltransferase (COMT), a phase II enzyme that transfers a methyl
group from a donor molecule (S-adenosylmethionine, SAM) to form
methylated metabolites (Chen et al., 2014b). A variety of bacterial and
fungal catechol-O-methyltransferases are included in biosynthesis of
various compounds. Dhar and Rosazza (2000) described an assay for
the purication and characterization of catechol-O-methyltransferase
from Streptomyces griseus, a bacteria known to mimic catechin metabo-
lism (Das and Rosazza, 2006; Dhar and Rosazza, 2000).
Quercetin is O-methylated to isorhamnetin (3-O-methylquercetin)
or tamarixetin (4-O-methylquercetin) (Fig. 12). Similarly, avan-3-ols
such as catechin and epicatechin are transformed into O-methylated de-
rivatives such as 3-O-methyl-(epi)catechin, 4-O-methyl-(epi)catechin
and 3,4-O-dimethyl-(epi)catechin (Fig. 13) (Das and Rosazza, 2006;
Duenas et al., 2010). 3-O-methylcatechin is the main (+)-catechin me-
tabolite in human urine (Hosny et al., 2001).
Methylation mediated by COMT is regioselective. In the case of
quercetin, there are two hydroxyl groups (meta/para corresponding to
the 3/4positions) on the B ring that can be methylated. Although
both regioisomers (4-O-methyl and 3-O-methyl metabolites) occur,
quercetin and (epi)catechins prefer 3-O-methylation, with the 4-O-
methylated metabolite being a minor product (Cho et al., 2012; Kim
et al., 2006b).
Zhu et al. (2000)have demonstrated that ()-epicatechin, (+)-
epicatechin, and ()-epigallocatechin are very rapidly
O-methylated by the human cytosolic placental COMT (150500
pmol of methylated avonoid/mg of protein/min) in comparison with
()-epicatechin gallate and ()-epigallocatechin gallate (b50 pmol
of methylated avonoid/mg of protein/min) (Zhu et al., 2000).
3.4.2. Impact of methylation on the biological activities of avonoids
Free hydroxyl groups that can serve as acceptors for rapid conjuga-
tion by glucuronidation and sulphation are the main reason for low
oral bioavailability of avonoids, mainly due to extensive intestinal
and hepatic metabolism. Very low oral bioavailability of quercetin is
especially due to glucuronidation (Walle, 2009).
Methylation of free hydroxyl groups of avonoids produces deriva-
tives that are less sensitive to glucuronidation and sulphation, resulting
in increased metabolic stability and a signicant improvement in
oral bioavailability (Wen and Walle, 2006b). Walle suggested that
1077
methylation is the main reaction to protect free hydroxyl groups in
avonoids and thus increase their oral bioavailability (Walle, 2009).
Wen and Walle investigated differences in hepatic stability (using a
pooled human liver S9 fraction as a model) and intestinal absorption
(using human colon adenocarcinoma Caco-2 cells of methylated and
unmethylated avonoids. It was demonstrated that cells were 5- to
8-fold more permeable to methylated avonoids than to unmethylated
avonoids (Wen and Walle, 2006a).
Methylated metabolites are more lipophilic in comparison to
glucuronidated or sulphated metabolites due to the methyl group and
readily accumulate in vivo due to improved transport and intestinal
absorption through biological membranes (Chen et al., 2014b).
It was also demonstrated that methylation often results in deriva-
tives with improved and/or altered biological effects on human health,
in particular increased anticarcinogenic and antimicrobial activities
(Chen et al., 2014b). Methylation preserves anti-inammatory activities
in comparison to glucuronidation or sulphation, which are suggested to
abolish positive benets on human health (Lotito et al., 2011).
Isorhamnetin is a biologically active compound that is used to treat
cardiovascular diseases, and exhibits anticarcinogenic activities toward
several cancer cell lines, for instance human hepatocellular carcinoma
cells (BEL-7402) with an IC50 of 74.4 g/mL (Teng et al., 2006),
or human oesophageal squamous carcinoma cells (Eca-109) with an
IC50 of 40 g/mL (Ma et al., 2007). In addition, biological effects depend
on the methylation position; 3-O-methylquercetin exhibits antiviral
activities, whereas 7-O-methylquercetin, known as rhamnetin, inhibits
the formation of -amyloid(Sung et al., 2011b), a protein that is the
main component of the amyloid plaques involved in Alzheimer's
disease (Sung et al., 2011a).
Methylated metabolites of naringenin, known as sakuranetin (7-O-
methylnaringenin) and ponciretin (4-O-methylnaringenin), exhibit
antifungal activities against pathogens of rice (Pyricularia oryzae or
Magnaporthe grisea) and antibacterial activity against Helicobacter
pyroli(Cho et al., 2012; Sung et al., 2011b). In addition, methylation re-
duces the possibility of toxic side-effects, an essential feature for poten-
tial therapeutic utility (Walle, 2009).
O-methylation of the catechol groups of quercetin and catechins
has a signicant effect on their antioxidant activities and scavenging
properties (Duenas et al., 2010; Lotito et al., 2011). Duenas et al.
(2010) characterised antioxidant activities of methylated quercetins.
Antioxidant activity was measured by the ferric reducing power
(FRAP) assay and two methods based on the ability to scavenge the
ABTS + radical cations at different pH values were used. The O-
methylation of the hydroxyl groups resulted in a decrease in antioxidant
activities in comparison with the corresponding demethylated com-
pounds. Quercetin and its O-methylderivatives demonstrated greater
activity than (epi)catechins and their O-methyl derivatives. The antiox-
idant activities of the avonoids were clearly dependent on pH, showing
the greatest radical scavenging activities at pH 7.4 (Duenas et al., 2010).
3.4.3. O-methylation in biotechnological industries
In recent years, there has been growing interest in biotechnological
approaches using transgenic microorganisms expressing genes in-
volved in modifying natural compounds such as avonoids. Structural
modications of avonoids can lead to products with improved proper-
ties (such as increased metabolic stability) or health promoting activi-
ties such as antimicrobial activities (Cho et al., 2012). Cloning of genes
for O-methyltransferases (OMT) could be used to create O-methylated
avonoids, some of which are difcult to isolate from plant sources or
to synthesize chemically, and to develop and produce novel drugs that
may have potential for application in the pharmaceutical industry
(Kim et al., 2006a).
Cho et al. (2012) isolated OMT (specically SlOMT 3) from the
miniature tomato cultivar Micro-Tom, and expressed this enzyme in
E. coli, which was subsequently used to modify avonoids. Transgenic
1078 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
E. coli expressing OMT was used, for example, to convert quercetin to
isorhamnetin (Cho et al., 2012).
Kim et al. (2006a) isolated 7-O-methyltransferasePOMT 7 from pop-
lar (Populus deltoids) that has a regiospecic methylation activity for
transforming 7-hydroxyl groups of avonoids to 7-methyl groups. For
example, biotransformation kinetics of quercetin indicated that the en-
zyme converted more than 80% of quercetin (which was added at con-
centration of 70 M) to the corresponding 7-methoxy compound,
within 24 h (Kim et al., 2006a). Rhamnetin, the reaction product of
7-O-methylation of quercetin, has been shown to inhibit the formation
of -amyloid and thus has potential for future use in treatments for
Alzheimer's disease (Sung et al., 2011b).
Kim et al. (2005) investigated the conversion of naringenin,
catalysed by a 4-O-methyltransferaseSOMT 2 isolated from soya
(Glycine max.), and the gene for this enzyme was expressed in E. coli.
SOMT 2 is an enzyme with the ability to transfer a methyl group to
avonoids containing a hydroxyl group in the C4 position on
the B ring. Naringenin (added at a concentration of 150 M) was
O-methylated by SOMT 2 to form ponciretin (4-methoxy-5,7-
dihydroxyavanone) and the ponciretin reached a maximum concen-
tration of 150 M after 15 h of incubation (Kim et al., 2005).
3.4.4. Demethylation in the digestive tract
The enzymatic demethylation of avonoids, consisting of the
removal of a methyl group from the substrate, occurs partly in the
liver by the action of cytochrome P450 enzymes and also in the gut
where avonoids are demethylated by intestinal microora.
Many signicant biotransformation reactions occur in the liver,
a tissue rich in phase I and II enzymes (Hodek et al., 2002). Specic
human liver cytochromes P450 (CYP1A2) enzymes are responsible
for O-demethylation of isoxanthohumol to form 8-prenylnaringenin(Guo
et al., 2006; Nikolic et al., 2005).
Low levels of 8-prenylnaringenin (concentrations of 8-
prenylnaringenin corresponding to up to 5% conversion) were
detected in urinary samples after oral intake of isoxanthohumol
by two healthy subjects, and they attributed this activation to oxidative
demethylation by the liver (Possemiers et al., 2005, 2009a; Schaefer
et al., 2005).
Interestingly, although O-demethylation of chalcones and non-
prenylated analogs of 8-prenylnaringenin has been demonstrated,
demethylation of xanthohumol into desmethylxanthohumol was not
observed (Nikolic et al., 2005).
In this section we will emphasise demethylation transformation of
prenylavonoids. Among the prenylavonoids, 8-prenylnaringenin
has been identied as one of the most potent phytoestrogens identied
so far, and its estrogenic properties have been conrmed in numerous
in vitro and in vivo studies (Milligan et al., 2000). Isoxanthohumol has
much lower estrogenic activity, however it can be metabolically trans-
formed by intestinal bacteria into 8-prenylnaringenin. Isoxanthohumol
is therefore referred to as a proestrogen (Possemiers et al., 2009a).
A variety of anaerobic bacteria are known to carry out O-
demethylation reactions. These bacteria can use methyl esters for
respiratory growth, and some of them use fumarate as an electron
acceptor (Desultobacterium) or are homoacetogens and convert
carbon dioxide into acetate, while others are able to produce buty-
rate or ethanol as minor products (Eubacterium) (Chang et al.,
1999; Possemiers et al., 2005). Incubation of faecal samples with
isoxanthohumol showed that intestinal bacteria are capable of
O-demethylation of isoxanthohumol into 8-prenylnaringenin and
bacterial strains capable of this biotransformation were isolated
from human faeces. This metabolic conversion can be carried out
by the strictly anaerobic homoacetogenic Gram positive bacterium,
Eubacterium limosum (DSM 20543), and the efciency of this
conversion was almost 90% (Possemiers et al., 2006, 2005, 2008;
Possemiers and Verstraete, 2009a,b; Possemiers et al., 2009a).
The extent of conversion of isoxanthohumol by intestinal Eubacterium
limosum, in vivo, is very variable and depends on the composition of the
microbial ora in the individual (between individuals or within the
same individuals at different times). Possemiers et al. (2005) incubated
faecal samples from 12 healthy subjects with isoxanthohumol, at a
concentration of 25 mg/L, for a period of 8 days and demonstrated the
importance of inter-individual variability in the intestinal microbiota,
where biotransformation of isoxanthohumol into 8-prenylnaringenin
was observed in only one-third of the samples (Possemiers et al., 2005).
The biotransformation capacity clearly separated individuals into
high (16% unconverted), moderate (22% unconverted) and low
(63% unconverted) producers of 8-prenylnaringenin(Bolca et al., 2007).
According to the literature, some fungi, such as Aspergillus sp.,
Mortierella sp., Cunninghamella echinulata and Eupenicillium sp. were
found to have demethylation enzymes. Fu et al. (2011) searched
for suitable fungal strains to produce 8-prenylnaringenin from
isoxanthohumol and isolated three fungal species capable of
demethylating isoxanthohumol: Eupenicillium javanicum, Cunninghamella
blakesleana and Ceriporiopsis subvermispora, however the transformation
yields were very low (4%) in comparison with intestinal Eubacterium
limosum(Bartmanska et al., 2013; Fu et al., 2011).
The analogous demethylation reaction by intestinal bacteria was
not observed for xanthohumol. Bacterial strains of E. limosum have
been observed to be involved in the demethylation of avonoids
from various plant sources. It is also known that E. limosum is
involved in the biotransformation of biochanin A(Hur and Rai,
2000), formononetin and glycitein into genistein, daidzein and
6,7,4-trihydroxyisoavone, respectively (Zhang et al., 2014). A related
eubacterial strain was identied as a producer of lignan enterodiol
(Possemiers et al., 2009a).
3.4.5. Demethylation in biotechnological industries
Insufcient production of estradiol during menopause is, in medical
practice, often compensated by hormone replacement therapy (HRT),
which uses relatively high doses of estrogenic hormones. This treatment
effectively eliminates the negative symptoms of menopause, such as hot
ashes, anxiety, insomnia or osteoporosis, and reduces the risk of
certain cardiovascular diseases. Despite these benets, HRT can have
negative effects, especially in increased risk of breast and uterine cancer
and an increased risk of thrombosis due to blood clotting (Cos et al.,
2003; Effenberger and Westendorf, 2009; Wuttke et al., 2002).
Despite the signicantly weaker (three to four orders of magnitude
in comparison to estrogens) estrogenic properties of phytoestrogens,
these substances could become an important supplement to conven-
tional treatment of menopausal complaints and the prevention of
osteoporosis (Zanoli and Zavatti, 2008).
The estrogenic activities of 8-prenylnaringenin have been conrmed
in several animal studies in vivo.Heyerick et al. (2006) demonstrated
that administration of low dosages (100 g) of 8-prenylnaringenin to
postmenopausal women over 6 or 12 weeks reduced menopausal
discomforts (Heyerick et al., 2006).
Although 8-prenylnaringenin can be easily produced by chemical
synthesis from isoxanthohumol or naringenin, scientists are searching
for new ways to produce 8-prenylnaringenin especially, due to the
high cost of chemical synthesis. Biological approaches using microbial
biotransformation to produce biologically active compounds have
attracted considerable interest because of factors such as high
stereo-and regioselectivity, as well as milder reaction conditions, in
comparison with chemical synthesis. Moreover, biotransformation
is environmentally safe (Fu et al., 2011).
Fu et al. (2011) used the fungus Eupenicillium javanicum, for produc-
tion of 8-prenylnaringenin. However, in comparison with the intestinal
bacterium Eubacterium limosum, biotransformation yields were low
(Fu et al., 2011).
Due to the high efciency of Eubacterium limosum, being capa-
ble of 90% conversion of isoxanthohumol, it is expected that
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
biotransformation reactions such as this may, in future, be used for the
production of the phytoestrogen 8-prenylnaringenin(Possemiers et al.,
2006, 2005, 2008; Possemiers and Verstraete, 2009a,b; Possemiers
et al., 2009a).
Possemiers et al. (2009a) designed a Simulator of the Human
Intestinal Microbial Ecosystem (SHIME) for localising the part of the
gastrointestinal tract where transformation of isoxanthohumol into
8-prenylnaringenin occurs. The SHIME consists of a succession of ve
reactors that represent the different parts of the human gastrointestinal
tract. Demethylation of isoxanthohumol, with up to 80% yield of
8-prenylnaringenin, occurred in the distal part of the descending
colon. It was observed that 8-prenylnaringenin was also produced
to a lesser extent in the transverse colon. Rates of transformation in
other parts of the gastrointestinal tract were insignicant (Possemiers
et al., 2009a).
3.5. Glycosylation and deglycosylation
In recent years, isolation and identication of plant avonoids
and their glycosides has become the subject of considerable scientic
interest and the number of identied avonoid glycosides has reached
several hundred (Xiao et al., 2014b). Many avonoids exhibit biological
activities and are in demand for their pharmaceutical or clinical effects.
However, the presence of the sugar moiety signicantly affects
bioavailability, pharmacokinetic properties and most of the positive
health effects of avonoids, including antioxidant, anticancer, anti-
inammatory and antibacterial actions. Although, as previously stated,
the majority of avonoids are present in hops as aglycones, their
biotransformation through glycosylation and deglycosylation reactions
could be important factors that can greatly alter their potential as
biological agents.
Primary deglycosylation of avonoid glycosides occurs in the oral
cavity during the rst few minutes after consumption (Walle et al.,
2005), as a result of the collective action of salivary enzymes and
bacteria, particularly Streptococci, that are commonly found in the oral
cavity (Laires et al., 1989). Flavonoid glucosides are transformed
relatively easily, whereas other glycosides, such as rhamnosides and
rutinosides, show signicant resistance to hydrolysis (Macdonald
et al., 1983; Walle et al., 2005). Subsequent decomposition of some
polyphenolic compounds, such as oligomeric procyanidins, continues
in the acidic environment of the stomach (Stoupi et al., 2010) whereas,
under normal conditions (pH 2; for up to 4 h) monomeric avonoid gly-
cosides are fairly stable and do not undergo signicant non-enzymatical
deglycosylation (Gee et al., 1998).
Important contributions to the metabolism of polyphenols are
processes that occur in the small intestine, where a relatively small
proportion (up to 20 rel.%) is absorbed by epithelial cells (Steidle,
1932). In contrast however, the permeability of nonpolar enterocyte
membranes for the relatively polar avonoid glycosides is quite low,
so absorption requires cleavage of glycosidic bonds (Hollman, 2004).
This is achieved by a broad spectrum of hydrolytic enzymes from epi-
thelial cells and gut microbiota, especially -glucosidases(Viskupicva
et al., 2008). Lactase (lactase-phlorizin hydrolase) is one enzyme that
is able to decompose avonoid monoglucosides (Day et al., 2003;
Sesink et al., 2003) and diglucosides (Nemeth et al., 2003), but does
not hydrolyse quercetin-3-O-rutinoside(Day et al., 2000). There are
also indirect indications that the transport of avonoid glycosides
in an unchanged form can occur via a sodium-dependent glucose
transporter (SGLT1). This fact was conrmed by experiments
monitoring the absorption of polyphenols in the gastrointestinal
tract of patients with ileostomy (surgical construction of an articial
excretory opening into the terminal part of the small intestine). The
result showed a high rate of absorption of quercetin-3-O-glucoside,
which was even higher than absorption of the aglycone itself
(Hollman et al., 1995; Olthof et al., 2001a,b).
1079
Flavonoid glycosides that are not absorbed in the small intestine
can be deglycosylated by enzymes of the gut microbiota, namely
-glucosidases and -rhamnosidases(Bokkenheuser et al., 1987;
Kim et al., 2008; Schneider et al., 1999). Some bacteria from the gut
microora, e.g., Bacteroides uniformis, B. ovatus(Bokkenheuser et al.,
1987) and Enterococcus casseliavus(Schneider et al., 1999) are capable
of hydrolysing quercetin-3-O-glucoside and quercetin-3-rutinoside.
Products resulting from these reactions are further metabolized through
demethylation, dehydroxylation, hydrolytic and ring-cleavage reactions
(Aura, 2008; Selma et al., 2009), followed by binding to albumin,
transportation to the liver via the portal vein (Manach et al., 1996)
and transformation into glucuronates and sulphates (Rice-Evans,
2001; Zhang et al., 2007).
3.5.1. Impact of glycosylation on the biological activities of avonoids
3.5.1.1. Antioxidant properties. In most cases, health related properties
of avonoid glycosides and aglycones differ signicantly. Detailed
knowledge of these differences is an essential prerequisite for the
use of biotransformation in the production of modied natural
substances that have potential for the prevention or treatment of
many serious diseases.
Over the last 20 years, most attention has been focused on compar-
ing the antioxidant effects of avonoid glycosides and their correspond-
ing aglycones. Antioxidant activities that neutralize negative effects of
reactive oxygen species (ROS), chelate redox-active metal ions and
inhibit the activity of lipoxygenases were investigated and it was
shown that substitution on the avonoid molecule with a sugar
moiety signicantly affected these properties. Generally speaking,
O-glycosylation decreases antioxidant properties. Choi et al. (Choi
et al., 2012) conrmed that quercetin and kaempferol have higher
scavenging activities for the peroxynitrite radical than their glycosides
that occur naturally in hops (quercetin-3-O-rutinoside, quercetin-3-O-
glucoside and kaempferol-3-O-glucoside). Burda and Oleszek reached
similar conclusions by studying antioxidant properties of these com-
pounds during heat-induced oxidation in a beta-carotene-linoleic acid
model system (Burda and Oleszek, 2001). In another study (Omololu
et al., 2011), data obtained by 3 different methods for the determination
of antioxidant properties (decolourisation of DPPH, ability to chelate Fe
(II) ions and preventing degradation of deoxyribose by hydroxyl radi-
cal) were compared. It was found that quercetin shows higher radical
scavenging potential, but rutin is a better chelator of Fe ions, suggesting
that both substances, which commonly occur together in nature, can act
synergistically. A reduction in the ability of kaempferol-3-O-glucoside
to absorb the DPPH radical and superoxide ion, compared to the
aglycone, was also described (Masuoka et al., 2012).
The impact of O-glycosylation on the antioxidant properties of
prenylated avonoids is very signicant. Investigation of the antioxi-
dant properties of xanthohumol and its glycosides by means of DPPH
radical scavenging revealed that xanthohumol-4-O--D-glucoside
exhibits higher radical scavenging potential than xanthohumol
(Tronina et al., 2013b).
The question of C-glycosylation is not signicant in terms of its effect
on the antioxidant properties of hop avonoids because this type
of compound is virtually absent from the hop plant. Studies of plant
C-glycosides and their aglycones have shown that antioxidant
properties of C-glycosides of quercetin (Huber et al., 2009), apigenin
and luteolin (Cai et al., 2006; Omar et al., 2011) are greater than
their aglycones.
3.5.1.2. Anticarcinogenic properties. The xanthohumol glycosides,
xanthohumol 4-O--D-(4-O-methyl)glucoside and 4-O--D-
glucoside showed higher antiproliferative activity against the colon
carcinoma cell line HT-29 than xanthohumol itself (Tronina et al.,
2013b). For quercetin glycosides the ndings are much more interest-
ing. A product of partial hydrolysis of rutin containing a high level of
1080 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
quercetin-3-O-glucoside showed signicantly higher antiproliferative
activity against nine cell lines of various types of cancer (glioma,
breast adenocarcinoma, kidney adenocarcinoma, chronic myeloid
leukemia etc.) than pure rutin or pure quercetin (de Araujo et al.,
2013). The fact that substitution with rutinose signicantly reduced
the anticancer effects of quercetin was conrmed in the study
of colorectal carcinoma in rats (Dihal et al., 2006) and in in vitro-
studies of the apoptosis-inducing effect of prenylavonoids on
promyeloleukemic Human HL-60 Cells (Shen et al., 2003) and
Caco-2 colon cancer cells (Lea et al., 2010).
3.5.1.3. Antiviral. Although the rst indications of possible anti-HIV activ-
ity of quercetin and kaempferol derivatives were acquired as early as
the last decade of the twentieth century (Mahmood et al., 1996), this
issue has not been denitively resolved. Studies of Seal and colleagues
(Seal et al., 2011), based on the evaluation of the binding energy of a
ligand, yielded efciency scores that indicated that kaempferol 3-O-
glucoside may be an effective inhibitor of HIV-1 reverse transcriptase,
whereas rutin did not exhibit this activity (Kashiwada et al., 2005).
In another study (Suedee et al., 2013) kaempferol-3-O-rhamnoside
and quercetin 3-O-rhamnoside did not inhibit HIV-1 integrase. On the
other hand, authors of the study examining anti-HIV properties of a
broad set of avonoid glycosides (Tewtrakul et al., 2002) suggested
that the anti-HIV activity of quercetin glycosides was slightly higher
than that of kaempferol glycosides. Yarmolinsky at al. (Yarmolinsky
et al., 2012) investigated the activities of kaempferol and quercetin
diglycosides against herpes simplex virus 1 and 2 (HSV 1/2). Both
3-O-rutinosides showed higher anti-HSV activity than corresponding
aglycones. Also rutin showed higher activity against rotavirus infection
than its aglycone, quercetin (Bae et al., 2000).
3.5.1.4. Other health promoting effects. Differences between hop
aglycones and glycosides of avonoids are also apparent in terms
of their ability to stimulate key enzymes responsible for certainly
diseases that have been discussed previously.
Glycosylation reduces -glucosidase stimulating activity by
avonoids. It was demonstrated that quercetin is more effective
than rutin as an -glucosidase inhibitor, and glycosylation of quercetin
clearly weakened this inhibitory effect (Li et al., 2009; Liu et al., 2014a).
Moreover, it was also shown that glycosylation of quercetin signi-
cantly reduced its inhibitory action against glycogen phosphorylase.
Glycosylation also suppresses some of the potential positive effects of
avonoids on human health such as inactivation of their antitubercular
potential. Moreover the 3-, 7- or 8-O-glycosylation of kaempferol, and
quercetin signicantly reduced their ability to inhibit neuraminidase
(Xiao et al., 2014a).
Among the positive effects of avonoid glycosylation are enhanced
effects of myricetin 3-O-glycosides on inhibition of pro-inammatory
cytokines (TNF-, IL-1, and IL-6) and NO production (Xiao et al.,
2014a).
An interesting nding is the fact that even the position of sugar
moiety can play an important role in the biological effects of avonoids.
Mok and Lee (2013) investigated the ability of the hop avonols
kaempferol, quercetin and myricetin to inhibit aldose reductase from
rat lens, showing that glycosylation at the C3 position of kaempferol,
quercetin, and myricetin increased their inhibitory activities. In con-
trast, glycosylation on the C7 and C4' positions of avonoid molecules
reduced the inhibition (Mok and Lee, 2013).
3.5.2. Glycosylation and deglycosylation in biotechnological industries
The inuence of carbohydrate substituents on most biological
activities of avonoid compounds has promoted interest in the
biosynthetic preparation of various avonoid glycosides. This in-
cludes the production of new substances, virtually absent in nature,
and the production of known glycosidic structures, in amounts
sufcient for clinical applications.
There is considerable interest in the glycosylation of prenylated
avonoids, found in signicant quantities in hops, or in nished beer,
especially dark beer (Magalhaes et al., 2008, 2011a) due to isomeriza-
tion during wort-boiling(Karabin et al., 2013). Glycosylation of the
most important hop prenylavonoid, xanthohumol, by Penicilium
chrysogenumKCTC 6933, led to the formation of the corresponding
4-O--glucoside and 4,4-O--diglucoside, while the fungal strain
Cunninghamella elegans var. ellegansKCTC 6992 was able to carry out
glycosylation at the C7 position with simultaneous isomerization,
resulting in the formation of isoxanthohumol 7-O--glucoside.
Biotransformation yields, however, were low and ranged from
112 rel.% (Kim and Lee, 2006). A similar O-glucosylation at the C4'
position was also performed using the fungi, Absidia glaucaAM 133
(Huszcza et al., 2008). High glycosylation yields of up to 49 rel.%
were achieved by Tronina et al. using other fungi, including Rhizopus
nigricans UPF701, A. coeruela AM93, Beauveria bassiana AM446
and Mortierella mutabilis AM404 (Tronina et al., 2013b). In addi-
tion to these O-glucosides, 4-O--D-(4-O-methyl)glucoside was
obtained using B. bassiana AM446. As part of a larger study,
A. glauca, A. coreuela and B. bassiana were also used for glycosylation
of other prenylavonoids. The reaction yield for biotransformation of
isoxanthohumol ranged from 50 to 60 rel.% and, using a combination
of LC/MS and NMR, the two main products were identied as
isoxanthohumol 7-O--D-glucoside and isoxanthohumol 7-O--D-
4-methoxyglucoside. Estrogenic active 8-prenylnaringenin was
glucosylated with a yield between 30 and 40 rel.% (Bartmanska et al.,
2012). In addition, 8-prenylnaringenin-7-O--D-glucoside was
obtained using Mucor hiemalis KCTC6165, and 8-prenylnaringenin-
6-O-hydroxypropionyl glucoside using Rhizopus oryzae KCTC6399
(Kim et al., 2008).
Simple glycosylation of other avonoids found in hops has
not been intensively studied so far. Non-enzymatic synthesis was
used to prepare catechin and epicatechin C-glycosides containing
glucose, maltose and maltotriose (Stark et al., 2007), as well as
galactose and rhamnose (Hasslauer et al., 2010) units. One of the
few microorganisms tested was Cunninghamella elegansATCC 9245,
which glycosylated quercetin and kaempferol to 3-O--D-glucosides
(Zi et al., 2011).
Another avenue for the glycosylation of avonoid compounds is
through genetic engineering. A recombinant enzyme capable of produc-
ing (+)-catechin-3'-O--D-glucoside, and (+)-catechin-3'-O--D-
maltoside was synthesized by expressing the Deinococcus geothermalis
gene for production of amylosucrase in Escherichia coli(Cho et al.,
2011). Escherichia coli engineered for the overexpression of glycosyl-
transferase genes from Arabidopsis thaliana produced rhamnosides of
quercetin and kaempferol (Kim et al., 2012; Simkhada et al., 2010).
Expression of the oleandomycin glycosyltransferase gene from
Streptomyces antibioticus in E. coli led to production of an enzyme
that is able to glycosylate various avonoids, including naringenin,
kaempferol and quercetin (Choi et al., 2012). Another procedure,
often used for biotechnological deglycosylation of avonoids, is cul-
tivation with intestinal bacteria. For example, deglycosylation of
rutin was investigated using bacterial species of the genera Bacillus,
Bacterioides and Veillonella, and analysis of metabolites demonstrat-
ed that this is a two-stage process, where quercetin-3-O-glucoside is
initially formed, followed by leukocyanidin (Yang et al., 2012).
Studies based on cultivation of avonoid glycosides with the probi-
otic bacterium Bidobacterium animalis ssp. Lactis(de Lacey et al.,
2014) Lactobacillus plantarum(Landete et al., 2014) and by enzymatic
hydrolysis using enzymes from Penicillium sp. (de Araujo et al., 2013)
showed an increase in antioxidant properties of the medium within
72 hours. This is a prerequisite for the production of functional foods
with elevated levels of polyphenols.
Some avonoids glycosides were also deglycosylated by fungal strains.
The yeast Saccharomyces cerevisiae was able to hydrolyse certain gluco-
sides of avonols, avanones and anthocyanins (Schmidt et al., 2011)
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
and fermentation by Aspergillus awamori led to the deglycosylation of
rutinosides of kaempferol and quercetin (Lin et al., 2014).
3.6. Hydroxylation
The number and position of hydroxyl groups in avonoids
determines their colour, stability, and antioxidant capacity. The hy-
droxylation of the avonoid B ring is catalysed mostly by avonoid
hydroxylases (Leonard et al., 2006; Liu et al., 2014b) and hydroxyl
groups are frequently found ortho to the generally present hydroxyl
group at position 4. Enzymes responsible for hydroxylation at the 3- or
3,5-positions are avonoid 3 (F3H) and avonoid 3,5 (F3,5H)
hydroxylases, members of the multifunctional cytochrome P450
enzyme family (Schlangen et al., 2009).
Nikolic and van Breemen (2004) investigated the metabolism of the
hop avanone naringenin by rat liver microsomes and identied
eriodicytol as the major metabolite (Nikolic and van Breemen, 2004).
The production of 2,3-dihydroxy derivatives of avonoids is of
interest because hydroxyl groups on the B ring are essential for their
effective antioxidant activities.
Other enzymes catalyse biotransformation to form hydroxylated
avonoids, from which the best-studied in terms of biochemical and
genetic characterization, are the biphenyl dioxygenases. Eukaryotic
dioxygenases are known to transform avonoids into hydroxylated
avonoids with the formation of an epoxide intermediate. In contrast,
prokaryotic dioxygenases generally catalyse conversion of aromatic
compounds into their corresponding dihydrodiols without the epoxide
intermediate. However, Han et al. (2005) described a biphenyl
dioxygenase from Pseudomonas pseudoalcaligenes that transformed
avanones into their corresponding epoxide structures between carbon
C2and C3on the avanone B ring (Han et al., 2005). Chun et al. (2003)
prepared 2,3-dihydroxyderivatives of avones and avanones by
using Streptomyces lividans cells expressing the gene for a modied
biphenyl dioxygenase. Flavone was converted into two products: 2,3-
dihydroxyavanone and 3-hydroxyavanone. Flavanone was convert-
ed into three products: 2,3-dihydroxyavanone, 2-hydroxyavanone
and 3-hydroxyavanone (Chun et al., 2003).
Matsuda et al. (2008) isolated the bacterium Burkholderia sp. from
tropical peat soil, and demonstrated that it could 4-hydroxylate
(+)-catechin, initially to leukocyanidin, and subsequently transform
this to taxifolin (Matsuda et al., 2008).
Barkova et al. (2011) investigated regioselective hydroxylation of
quercetin by a fungal aromatic peroxygenase isolated from Agrocybe
aegerita, and described an enzyme involved in the H2O2-
dependenthydroxylation or epoxidation of aromatic rings. Quercetin
yielded only one monohydroxylated avonoid with a hydroxyl group
in the C6 position, quercetagin (Barkova et al., 2011).
Quercetagin was also prepared with high efciency and specicity
by a cytochrome P450 dependent momooxygenase, 6-hydroxylase
(F6H), isolated from petals of Tagetes patula and Tagetes erecta. An addi-
tional hydroxyl group in the C6 position of quercetin causes the yellow
colour of this avonol (Halbwirth et al., 2004).
Zierau et al. (2004) investigated 12 metabolites of 8-prenylnaringenin
after incubation with human liver microsomes: one of the main products
was (E)-8-(4-hydroxyisopentenyl)naringenin. This compound exhibited
estrogenic activity in the yeast estrogen receptor assay in vitro, although it
was less potent than the parental compound 8-prenylnaringenin(Zierau
et al., 2004).
More recently, production of hydroxylated avonoids has been
carried out using transgenic microorganisms expressing hydroxylase
genes. Leonard et al. (2006) demonstrated biosynthesis of avonols in
E. coli by expression of avonoid 3-hydroxylase (F3H) and avonoid
3,5-hydroxlase (F3,5H). Fermentation of the recombinant strains re-
sulted in synthesis of the hydroxylated avonols kaempferol, quercetin
and myricetin from precursor phenylpropanoid acids (p-coumaric acid
and caffeic acid) (Leonard et al., 2006). Additionally, biotransformations
1081
resulting in hydroxylated avonoids such as 4-hydroxyavone and
3,4-dihydroxyavone can be performed by many fungi, including
species of Aspergillus, Cunninghamella, Helicostylum, Penicillium,
Linderina, and Streptomyces(Seo et al., 2011).
3.7. Acylation
In addition to their direct health-promoting effects, antioxidants,
including polyphenols, can also act in the food industry to prevent
lipid oxidation and other oxidative degradation reactions of food
components, thereby prolonging shelf life of food.
Their application however is limited by their relatively high polarity
and thus low solubility in the lipophilic environment of most foods,
although this can be modied by acylation of one or more hydroxyl
groups. This increases their uptake across the semi-permeable cell
membrane and improves health-promoting properties such as
inhibiting oxidation of human low-density lipoprotein (LDL) (Lue
et al., 2010), inducing apoptosis of leukemic cells (Sakao et al., 2009)
and preventing undesirable changes in skin cells due to UV irradiation
(Viskupicova et al., 2010). Although research in the eld of the biosyn-
thesis of avonoid acylesters is in its infancy, future applications in
cosmetics, food technology and a wide range of therapeutic areas
are expected.
Preparation of acylavonoids is limited almost exclusively to
enzymatic biosynthesis. The most commonly used enzyme is
immobilized lipase B isolated from Candida antartica (CALB). The
transesterication reaction of quercetin and vinyl acetate, catalysed
by CALB, led to the formation of two acetyl derivatives, 4'-O-acetyl
quercetin and 3'-O-acetyl quercetin, with reaction yields of 35%
and 10% respectively (Kyriakou et al., 2012). Candida antarcticalipase
was also used to prepare rutin fatty acid esters with up to an 18-
carbon acyl chain (Viskupicova et al., 2010). In another study
(Chebil et al., 2007), methods for preparing di- and tri- acetates of
quercetin and its glucoside, isoquercitrin, using CALB and lipase
isolated from Pseudomonas cepacea (PSL-C), were proposed. In addition
to these two lipases, the possibility of using a carbohydrate esterase
from Trichoderma reesei for the preparation of rutin acetate and
propionate was tested, with reaction yields of 60% and 30% respec-
tively (Biely et al., 2014). The synthesis and testing of rutin esters of
oleic, linoleic and linolenic acids also demonstrated the antiangiogenic
properties of these substances and their potential for tumor prevention
(Xanthakis et al., 2010).
3.8. Biotransformation of the avonoid carbon skeleton
The complete range of avonoid biotransformations is not only
based on the attachment or cleavage of various functional groups or
units. Especially during the metabolic changes in the digestive tract of
humans and mammals, a large group of metabolites arise as a result of
extensive oxidation, cyclization and degradation of the avonoid carbon
skeleton. Most studies have focused on the identication and precise
determination of the structure of these metabolites, and only few have
addressed the question of their biological activities.
3.8.1. Prenylavonoids
Much is known, particularly through the work of Bartmanska et al.
(Bartmanska et al., 2013), about transformations of hop prenylated
avonoids. While studying the pharmacokinetic parameters of
xanthohumol in rats (Legette et al., 2012), it was conrmed that the
most prominent metabolites were isoxanthohumol, 6-prenylnaringenin
and 8-prenylnaringenin, all of which are commonly found in hops.
Two previously unknown metabolites were identied by Yilmazer et al.,
studying the biotransformation of xanthohumol by rat liver microsomes
(Yilmazer et al., 2001b). Subsequently, 22 other metabolites were identi-
ed and characterized by HPLC-NMR and HPLC-MS analyses of the faeces
of xanthohumol-fed rats (Nookandeh et al., 2004). Most of them are
1082 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090

Fig. 14. Biotransformation products of prenylated avonoids (A: xanthohumol, B: isoxanthohumol, C: 8-prenylnaringenin).

products of prenyl chain hydroxylation or cyclization of the prenyl
chain with one hydroxyl group on the A ring (Fig. 14 - part A). In addition,
7 metabolites of isoxanthohumol (Fig. 14- part B) and 12 metabolites of 8-
prenylnaringenin(Fig. 14- part C) were identied in studies of liver
microsomal metabolism of prenylavonoids (Nikolic et al., 2004, 2005).
A signicant amount of research has been focused on the use of fungal
strains for the biotransformation of prenylavonoids. Tronina et al.
(Tronina et al., 2013a) demonstrated the capabilities of certain Fusarium,
Penicillium and Spicaria strains to transform xanthohumol to six different
metabolites (1, 11, 19, 20; Fig. 14- part A and 1, 2; Fig. 15), which were
also screened for their antiproliferative effects. One of the metab-
olites tested, , -dihydroxanthohumol(20; Fig. 14- part A), showed
equivalent activity against MCF-7 human breast carcinoma and PC-3
prostate cancer cell lines as the commonly used therapeutic agent, Cis-
platin. The formation of metabolites 6, 11 and 19(Fig. 14- part A) were
also demonstrated in Pichia membranifaciens(Herath et al., 2003a).
Transformation using Aspergillus ochraceus(Tronina et al., 2014) led to
the cyclization of chalcones to avanones and the simultaneous rear-
rangement of the middle heterocyclic ring to a ve-member ring struc-
ture (Fig. 15). Metabolite 1(Fig. 15) had 8.6-fold greater antioxidant
properties than xanthohumol and 2.3-fold greater than ascorbic acid.
One of the cyclized metabolites and its glycosides were prepared
by the biotransformation of 8-prenylnaringenin, using Rhizopus oryzae
and Cunninghamella elegans(Kim et al., 2008) and the analogous metab-
olite of isoxanthohumol was obtained using Fusarium equiseti(-
Bartmanska et al., 2009).
 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
Fig. 15. A. ochraceus metabolites of xanthohumol (Tronina et al., 2014).
3.8.2. Flavan-3-ols
Flavan-3-ols, and particularly catechins, are susceptible to enzymatic
and non-enzymatic oxidation. Reactions of (+)-catechin, catalysed
by mushroom tyrosinase (van Rensburg et al., 2000) horseradish
peroxidase or laccase (Hosny and Rosazza, 2002), resulted in the
formation of dimeric and oligomeric structures, some of which had
28 times greater antioxidant activity than D,L-R-tocopherol. Oxidation
of the dimeric avan-3-ol, procyanidin B2, by dioxygenase isolated
from Aspergillus fumigatus(Roopesh et al., 2010) led to the formation
of compounds in which one or both catechol rings were oxidized to a
(5-oxo-tetrahydro-furan-2-yl) acetic acid ring (Fig. 16).
Many biotransformations of avan-3-ols using microbial cells and
enzymes also lead to extensive changes in the avonoid structure, and
sometimes even to its disintegration, with the formation of simpler
polyphenolic compounds (Das et al., 2011).
By studying the metabolism of catechin, epicatechin, gallocatechin
and related compounds in the pig cecum model (van't Slot and
Humpf, 2009) it was found that intestinal microora, including
strains of Bidobacterium, Enterococcus, Bacterioides, Eubacterium and
Clostridium metabolize avan-3-ols within 48 hours (van't Slot et al.,
2012). The major metabolites are phloroglucinol and substituted
hydroxybenzoic acids. 5-(3,5-dihydroxyphenyl)-4-hydroxyvaleric acid
was identied as the predominant metabolite in a similar study,
dealing with the degradation of ()-epigallocatechin gallate by rat
intestinal microora (Takagaki and Nanjo, 2010). Two -
valerolactonederivatives analogous to those shown in Fig. 17 were
Fig. 16. Product of procyanidin B2 oxidation by dioxygenase from A. fumigatus.
1083
Fig. 17. Gamma-valerolactone products of avan-3-ol degradation by intestinal
microora.
also identied in human studies of the metabolism of avan-3-ols
from tea (Li et al., 2000).
Not all bacterial strains are capable of completely cleaving the
avan-3-ol molecule. A strain of the human intestinal bacteria,
EubacteriumSDG-2, transforms cycle C only partially, giving arise to
1,3-diphenylpropan-2-ol derivatives (Wang et al., 2001).
Interesting results were obtained by Das et al. (2011). Nine of 36
microbial strains tested (Aspergillus giganteusUI 10, A. ochraceusATCC
1008 Cylindrocarpon radicicolaATCC 11011, ATCC 35203 Amycolata
autotrophica, Mycobacterium avescensATCC 14474, M. fortuitumUI
53378, Streptomyces rimosusNRRL 2234, S. griseolusATCC 11796,
and S. griseusATCC 13273) were able to transform (+)-catechin
to completely new avonoid metabolites B ring ssion lactones
(Fig. 18). Metabolic pathways leading to their formation were also
proposed and the incorporation of two oxygen atoms into the structure
during transformation was demonstrated (Das et al., 2011).
3.8.3. Flavanones and avonols
Flavanones and avonols in hops are mainly represented by deriva-
tives of naringenin (avanone) and quercetin (avonol), and are subject
to microbial transformations in the heterocyclic C ring.
Quercetin is converted via oxygenolytic ring-cleavage, catalysed
by avonol 2,3-dioxygenase (quercetinase), leading to the formation
of 2-protocatechuoylphloroglucinol carboxylic acid (Fig. 19). This
reaction has been theoretically described in detail by Antonczak et al.
(Antonczak et al., 2009).
Quercetinase activity was found in several fungal strains such as
Streptomyces urythermus(Merkens et al., 2007), Aspergillus niger(Hund
et al., 1999), A. avus(Yadav and Yadav, 2008), Penicillium olsonii(-
Iacazio, 2005; Tranchimand et al., 2008) and P. decumbens(Mamma
et al., 2004). Some quercetinases are also able to catalyse the conversion
of kaempferol, with a relative enzymatic activity equal to 70 rel.%
(Merkens et al., 2007) or 218 rel.% (Tranchimand et al., 2008) of that
with quercetin.
Flavonols are also subject to similar microbial degradation as
avan-3-ols, leading to the formation of phloroglucinol and mono- or
Fig. 18. Structure of B ring ssion lactones.
1084 M. Karabin et al. / Biotechnology Advances 33 (2015) 10631090
Fig. 19. Oxidation of quercetin catalysed by quercetinase.
dihydroxyphenyl acetic acid. This degradation was described for the
bacterium Clostridium orbiscindens(Schoefer et al., 2003).
The rst step in the degradation of naringenin and other ava-
nones involves isomerization and reduction that yield chalcones
and dihydrochalcones. These biotransformations have been described
in the genera Rhodococcus, Gordonia(Stompor et al., 2013), and
Eubacterium ramulus(Herles et al., 2004); the chalcone isomerase
from Eubacterium ramulus was successfully expressed in E. coli(-
Gall et al., 2014). The chalcones were subsequently converted to
phloroglucinol 3-(4-hydroxyphenyl)propionic acid (Zou et al.,
2014).
There are also other possible biotransformations of naringenin.
In 2014 a study was published (Madej et al., 2014) describing the
biotransformation of naringenin to the stronger antioxidants (Miyake
et al., 2003) carthamidin and isocarthamidin by the yeast Rhodotorula
marina. The biotransformation was performed as a one-step process
and the authors describe its industrial potential.
Of interest is also the possibility of producing the estrogenically
active genistein by biotransformation of naringenin. For this purpose,
a procedure using E. coli engineered for the production of an isoavone
synthase-cytochrome P450 reductase fusion protein has been
developed (Kim et al., 2009).
4. Conclusions
Hop (Humulus lupulus L.), from the family Cannabaceae, has been
grown since the beginning of our modern age, especially for beer
production. The wild form of hop has been used since ancient times as
a medicinal plant. The hop elicits positive effects on health due to
its content of biologically active substances, including polyphenolic
compounds especially avonoids, bitter resins and essential oils.
Hop avonoids can be divided to six subgroups: avan-3-ols,
proanthocyanidins, avonols, avanones, avonol glycosides and
prenylavonoids. Hop avonoids can be transformed in the human
body by metabolism or by intestinal microbiota. These transformation
reactions include prenylation, methylation, demethylation, glycosyla-
tion, deglycosylation, hydroxylation and acylation. Metabolites created
from hop avonoids have altered molecular structures that affect
their polarity and solubility, bioavailability and biological activities.
Pharmaceutically important substances are prenylated avonoids.
Prenylavonoids have been described in many studies, and have a
wide variety of positive health properties including anticarcinogenic,
estrogenic, anti-inammatory and antimicrobial effects. An important
biotransformation is the metabolic conversion of isoxanthohumol to
8-prenylnaringenin, involving O-demethylation of isoxanthohumol by
intestinal bacteria. Due to its phytoestrogenic properties, this avonoid
may be valuable in hormone replacement therapy, in the prevention
and treatment of osteoporosis, or in the control of climacteric symp-
toms. Bacterial strains that are capable of this biotransformation have
been isolated from stool samples, and this process may have uses
in the pharmaceutical industry for the biotechnological production of
8-prenylnaringenin. Generally, biotransformation can be a valuable
tool for the generation of biological active substances with potential
applications in pharmacy. Biotransformations moreover, have many ad-
vantages over methods of classical chemical synthesis they are
cheaper, more selective, even more regioselective, and reaction
conditions are milder and do not require protection of other functional
groups on the molecule. Research in the eld of biotransformation
of hop avonoids may lead to the future large scale production of bio-
logically active substances for clinical and pharmaceutical applications.
Acknowledgments
This work was supported by the Ministry of Agriculture of The Czech
Republic(QI111B053). Thanks are also due to John Brooker for editing
and critical proof-reading.
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