analysis, induction and in vitro estrogenicity rudy simons
TRANSCRIPT
Prenylated isoflavonoids from
soya and licorice
Analysis, induction and in vitro estrogenicity
Rudy Simons
Thesis committee
Thesis supervisor: Prof. Dr. Ir. Harry Gruppen
Professor of Food Chemistry
Wageningen University
Thesis co-supervisor: Dr. Ir. Jean-Paul Vincken
Assistent Professor, Laboratory of Food Chemistry
Wageningen University
Other members: Prof. Dr. Renger Witkamp
Wageningen University
Dr. Nigel Veitch
Royal Botanic Gardens, Kew, United Kingdom
Prof. Dr. Robert Verpoorte
Leiden University
Dr. Henk Hilhorst
Wageningen University
This research was conducted under the auspices of the Graduate School VLAG (Voeding,
Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid)
Prenylated isoflavonoids from
soya and licorice
Analysis, induction and in vitro estrogenicity
Rudy Simons
Thesis
submitted in fulfilment of the requirements of the degree of doctor
at Wageningen University
by the authority of the Rector Magnificus
Prof. Dr. M.J. Kropff
in the presence of the
Thesis Committee appointed by the Academic Board
To be defended in public
on Tuesday 28 June 2011
at 1.30 p.m. in the Aula.
Rudy Simons
Prenylated isoflavonoids from soya and licorice
Analysis, induction and in vitro estrogenicity
Ph.D. thesis, Wageningen University, Wageningen, the Netherlands (2011)
With references, with summaries in Dutch and English
ISBN: 978-90-8585-943-7
ABSTRACT
Prenylated isoflavonoids are found in large amounts in soya bean (Glycine max)
germinated under stress and in licorice (Glycyrrhiza glabra). Prenylation of isoflavonoids
has been associated with modification of their estrogenic activity.
The aims of this thesis were (1) to provide a structural characterisation of
isoflavonoids, in particular the prenylated isoflavonoids occurring in soya and licorice, (2)
to increase the estrogenic activity of soya beans by a malting treatment in the presence of
a food-grade fungus, and (3) to correlate the in vitro agonistic/antagonistic estrogenicity
with the presence of prenylated isoflavonoids.
A reversed-phase UHPLC-DAD-MSn-based screening method was developed that
allowed the tentative identification of all prenylated flavonoids in licorice extracts using
neutral losses diagnostic for prenylation. Moreover, both the chain and pyran ring can be
discriminated. This method was also employed on extracts from soya seedlings, which
were challenged with the food-grade fungus Rhizopus spp. Besides known prenylated
pterocarpans, also novel prenylated isoflavones and coumestans were found in soya.
Subsequently, a licorice root extract was fractionated and the fractions were
screened for estrogenic activity using an in vitro yeast ER bioassay. Several fractions were
considered estrogenically active on one or both ER-subtypes (α and β). The estrogenic
activity of some fractions was associated with the presence of glabrene, a prenylated
isoflavene. The predominant phytoestrogen of licorice root, glabridin, did not show any
agonistic activity, but was found to be a potent ERα antagonist.
Large-scale Rhizopus-challenging of soya seedlings resulted in a 10 to 12 fold
increase of the isoflavonoid content, in a diversification of isoflavonoid composition, and
in an increase of estrogenic activity. The measured estrogenicity of the extracts on both
ERs was lower than the theoretically calculated estrogenicity, indicating the presence of
antagonists. This antagonistic activity was hypothesized to be associated with the
presence of prenylated isoflavonoids.
TABLE OF CONTENTS
Chapter 1 General introduction 1
Chapter 2 A rapid screening method for prenylated flavonoids with
UHPLC-ESI-MS in licorice root extracts
37
Chapter 3 Identification of prenylated pterocarpans and other
isoflavonoids in Rhizopus spp. elicited soya bean
seedlings by electrospray ionisation mass spectrometry
59
Chapter 4 Agonistic and antagonistic estrogens in licorice root
(Glycyrrhiza glabra)
77
Chapter 5 Increasing soya isoflavonoid content and diversity by
simultaneous malting and challenging by a fungus to
modulate estrogenicity
95
Chapter 6 General discussion 117
Appendix 139
Summary 155
Samenvatting 159
Acknowledgements 165
Curriculum vitae 170
List of publications 171
Overview of completed training activities 172
Chapter 1
General introduction
Chapter 1
2
STRUCTURAL CLASSIFICATION AND BIOSYNTHESIS OF FLAVONOIDS
Flavonoids are a group of secondary metabolites that naturally occur throughout the plant
kingdom and are structurally characterised by a C6-C3-C6 carbon framework [1]. Flavonoids
have gained increasing scientific interest due to their diverse functions in plant such as
anti-microbial agents, photoreceptors, visual attractants, and feeding repellents. In
addition, flavonoids are considered to provide health-promoting activity through a myriad
of bioactivities including anti-oxidant, anti-inflammatory, anti-cancer and anti-allergic
activities [2]. Flavonoids belong to the superfamily of phenyl benzopyrans, more specifically
the term flavonoid refers to the 2-phenyl benzopyrans. Often isoflavonoids are also
referred to as flavonoids, but actually this is a misnomer, as they are 3-phenyl
benzopyrans. In this thesis, the term flavonoid will refer to the whole family of phenyl
benzopyrans. The family of phenyl benzopyrans can be divided into 4 classes based on the
position of the linkage of an aromatic ring to the benzopyran moiety (or chromene moiety,
i.e. rings A and C): 2-phenyl benzopyrans, 3-phenyl benzopyrans (isoflavonoids), 4-phenyl
benzopyrans (neoflavonoids) and miscellaneous flavonoids (Figure 1). The miscellaneous
flavonoids contain representatives in which the pyran C-ring (flavan core) is not retained.
In these molecules, the C-ring is either open (e.g. chalcones), or ring-closed into a furan
derivative (e.g. aurones).
The first step in flavonoid biosynthesis is the conversion of phenylalanine via
several steps into hydroxycinnamic acids, characterised by a C6-C3 carbon framework.
With the addition of 3 malonyl-CoA units, 2’,4’,6’,4-tetrahydroxychalcone can be formed
from which all four phenyl benzopyran classes are formed [3]. Each of these four classes
can be subdivided into subclasses based on the oxidation level of the C-ring and variations
in the complexity of the carbon framework as a result of hydroxylation, alk(en)ylation
(including methylation and prenylation), glycosylation and sulfonation. Flavonoids often
occur as O-glycosides in which the sugar moiety is connected to the aromatic ring or the C-
ring via a hydroxyl group with the formation of a glycosidic (acetal) bond. Occassionally,
the glycosyl residue is linked to the aromatic ring by a C-C bond, an example of which is
the isoflavone C-glycoside puerarin [4]. Glycosylation involves a broad range of sugars such
as glucose and glucuronic acid, but also substituted sugars. For example, acetyl-glucosides
and malonyl-glucosides are the predominant isoflavonoid conjugates found in soya.
This thesis deals mainly with isoflavonoids from licorice roots (Glycyrrhiza glabra)
and soya (Glycine max), two species belonging to the family of Leguminosae. Both sources
are particularly rich in prenylated isoflavonoids with estrogenic activity. Therefore,
isoflavonoids, prenylation, and estrogenic activity will be more extensively discussed
below, together with background information on the two species and methods to enhance
estrogenic activity.
Figure 1. Flavonoid subdivision into 4 main classes based on the linkage of the aromatic B-ring to the benzopyran moiety.
Chapter 1
4
ISOFLAVONOIDS
The Leguminosae consist of more than 750 genera comprising 18,000 species, and is
considered to be particularly rich in isoflavonoids [5]. Despite the fact that isoflavonoids are
almost exclusively found in Leguminosae [5, 6], the class of isoflavonoids is characterised by
large structural variations. These variations are due to different substitutions, different
degrees of oxidation and the presence of extra heterocyclic rings. Hence, the isoflavonoids
have been subdivided into 13 subclasses, based on oxidation level and the occurrence of
additional heterocyclic rings [7].
Based on their proposed biosynthesis, α-methyldeoxybenzoins and 2-phenyl-
benzofurans are included in the class of isoflavonoids. However, their deviating C-ring
structures, a five-membered C-ring (2-phenyl-benzofurans) and open C-ring (α-
methyldeoxybenzoins), suggest that these subclasses might also be categorised under the
‘miscellaneous flavonoids’. Six subclasses (excluding the 2-phenyl-benzofurans) have a
typical 3-ring carbon frame with a 6-membered C-ring. In addition, 5 subclasses
(pterocarpans, coumestans, rotenoids, coumaronochromones and coumaronochromenes)
are characterised by an additional D-ring. It is thought that the isoflavones and
pterocarpans are the largest two subclasses comprising ~40% and ~20%, respectively, of
all molecular species of isoflavonoids [8]. The biosynthetic relationships of the isoflavonoid
subclasses (including that of α-methyldeoxybenzoins and 2-phenyl-benzofurans) are
illustrated in Figure 2.
The isoflavonoid subclasses share the same biosynthetic origin. They are derived
from the chalcones chalconaringenin (2’,4’,6’,4-tetrahydroxychalcone) and isoliquiritigenin
(2’,4’,4-trihydroxychalcone) that are converted into their corresponding flavanones. The
subsequent migration of the aromatic ring from the 2-position to the 3-position of the
flavanone, the so-called aryl rearrangement performed by isoflavone synthase, and water
loss lead to the formation of the isoflavones genistein and daidzein, respectively (Figure 3) [3, 7]. Daidzein and its precursor liquiritigenin differ from genistein and naringenin by the
absence of the 5-hydroxyl group. Isoflavonoids lacking the 5-hydroxyl group are also called
5-deoxyisoflavonoids. From this perspective, the isoflavone biosynthesis consists of 2
parallel routes involving the formation of isoflavones and 5-deoxyisoflavones. All
isoflavonoids are thought to originate from the main isoflavones daidzein and genistein,
which are therefore considered key elements in the structural diversification of
isoflavonoids [9].
Figure 2. Biosynthetic relationships among several representative isoflavonoid subclasses, adapted from Dewick et al. [7].
The subclasses indicated in grey might also be classified under the Miscellaneous flavonoids.
Chapter 1
6
Soya beans are very rich in isoflavones, with a content ranging from 0.2 to 2 mg/g
DW [10-13]. Another isoflavonoid-rich legume species is Glycyrrhiza glabra, or licorice. The
isoflavonoid content of Glycyrrhiza glabra roots is similar or higher than that of soya beans [13]. The root content of the principal licorice isoflavonoid, glabridin, ranges from 0.5-5.7
mg/g DW [14-16]. Generally, also flavanones, chalcones, isoflavones, isoflavenes, and other
isoflavans are abundantly present in the roots of Glycyrrhiza glabra [16].
Figure 3. Biosynthetic conversion of (2S)-flavanones to isoflavones. 2HIS: 2-hydroxyisoflavanone synthase; 2HID:
2-hydroxyisoflavanone dehydratase; adapted from Veitch et al. [17].
Prenylation of isoflavonoids
Prenylation
Prenylation refers to substitution with a C5-isoprenoid (or prenyl) group. In a more broad
sense, prenylation also refers to the substitution with other groups, including C10-
isoprenoid (geranyl) or C15-isoprenoid (farnesyl) (Figure 4) [18]. Prenylation increases the
lipophilicity of flavonoids, which results in an increased affinity to biological membranes
and an improved interaction with target proteins [19]. Furthermore, it has been suggested
that prenylation plays a crucial role in modulating the bioactivity of flavonoids [19, 20].
Representative examples of prenylated flavonoids are 8-prenyl naringenin, a C5-prenyl
substituted flavanone isolated from Humulus lupulus, and kurarinone, a C10-prenyl
substituted flavanone isolated from Sophora flavescens. Both prenylated flavonoids
exhibit strong bioactivities such as estrogenic activity [20-23].
General introduction
7
Figure 4. Representative overview of common prenyl-substituents in flavonoids. 1 7,8-(2,2-dimethylpyran) or pyran ring.
Chapter 1
8
Prenylated flavonoids are predominantly C-prenylated, whereas only a few
studies reported O-prenylation [18]. The prenyl chain generally refers to the 3,3-
dimethylallyl substituent (3,3-DMA), but also includes variations of this structure based on
oxidation and hydroxylation. Hydroxylated prenyl groups are also called prenyl hydrates. A
prenyl chain may undergo cyclisation with an ortho-phenolic hydroxyl group leading to 6-
membered pyran derivatives. The common 2,2-dimethylchromeno substituent is often
termed a pyran ring. Cyclisation of a prenyl chain may also result in the 5-membered furan
derivative. C-prenylation at positions 6 and/or 8, as well as 3’ and / or 5’, appear to be
generally preferred [1]. In this thesis, the term ‘prenyl’ will generally refer to C5-isoprenoid
group unless stated otherwise.
Whereas most prenylated 2-phenyl benzopyrans appear to be constitutively synthesised
compounds in plant tissue, most prenylated isoflavonoids are considered inducible
metabolites [1, 18]. Prenylated isoflavonoids are often produced as part of the plant’s
defensive strategy (see Germination under stressed conditions). The majority of the
prenylated isoflavonoids (~90 %) are restricted to the Leguminosae subfamily
Papilionoideae [19, 20].
Prenyltransferases
Prenyltransferases (PTs) are enzymes that catalyse the transfer of the dimethylallyl moiety
from dimethylallyl pyrophosphate (DMAPP) to an acceptor molecule. PTs are distributed
throughout all living organisms. Aromatic PTs (APTs) are enzymes that catalyse the
substitution of an aromatic ring with a prenyl group. The APTs can be divided into three
subgroups based on their respective substrates, being: quinones, flavonoids, and phenolic
acids, of which the distribution of the flavonoid APTs is limited to plants [20]. Flavonoid
APTs can be subdivided into soluble and membrane-bound enzymes, referring to whether
the APT occurs in the cytosol, or attached to the membrane of e.g. the endoplasmatic
reticulum and plastids [24]. Flavonoid APTs have been isolated from plants belonging to the
families of Leguminosae, Moraceae, Cannabaceae, Euphorbiaceae, Umbelliferae,
Guttiferae, Rutaceae and Compositae [20]. Within the Leguminosae, APTs have been
characterised in Glycine max, Sophora flavescens, Phaseolus vulgaris and Lupinus albus,
and all are membrane-bound [25-31]. The isoprenoid biosynthesis includes the mevalonate
pathway localised in the cytosol and the methyl erythriol phosphate pathway (MEP)
localised in the plastids. Regardless of the location of the APT (soluble or membrane-
bound) or the type of aromatic molecule, it is assumed that the origin of the prenyl-
moiety originates from the MEP pathway [20].
General introduction
9
IDENTIFICATION OF FLAVONOIDS BY MASS SPECTROMETRY
Mass spectrometry is based on the generation of charged molecular species. These ions
are expressed in mass-to-charge (m/z) ratio and are plotted against their (relative)
abundance, resulting in a mass spectrum. Ionisation of compounds can be established by a
variety of different techniques. Ionisation techniques are generally classified as ‘hard’ or
‘soft’ ionisation [32, 33]. Flavonoids are generally studied with soft ionisation techniques
such as electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI),
but ESI is generally preferred over APCI due to its higher sensitivity [34, 35]. Soft ionisation
involves either the protonation or deprotonation of the analyte. Protonation of an analyte
is referred to as positive ion (PI) mode and results in a positively charged compound,
generally indicated by [M+H]+. In the so-called negative ion (NI) mode, the analyte is
deprotonated resulting in [M-H]-. NI mode is considered more sensitive and selective in
flavonoid analysis [35-37].
Another method that provides structural information is the application of
collision-induced dissociation (CID), resulting in the fragmentation of the charged analyte.
The fragment-ions that are produced upon fragmentation of flavonoids can provide
important structural information. Fragmentation of flavonoids is characterised by the
retro-Diels Alder (RDA) reaction in which the C-ring of the flavonoid is cleaved. Cleavage of
the C-ring results in A-ring and B-ring fragment ions that provide information on the
number and type of substituents of these rings [36 , 37, 38]. The nomenclature used for C-ring
fragment ions is based on the systematic nomenclature of carbohydrate fragmentation [38,
39]. A-ring and B-ring fragments are indicated by either i,jA+ and i,jB+ for PI mode
fragmentation, or by i,jA- and i,jB- for NI mode fragmentation. The superscripts i,j represent
the bonds that are cleaved in the C-ring, as indicated in Figure 5. In this figure, the most
common fragmentation pathways resulting from C-ring cleavages are presented for
different subclasses of 2-phenyl benzopyrans, isoflavonoids and miscellaneous flavonoids.
In addition to RDA fragments, neutral losses are also observed upon
fragmentation. Neutral losses involve the loss of small molecules including H2O (18), CO
(28), CO2 (44), but also sugar moieties such as glucosyl (162). Furthermore, the loss of 15
from the parent ion in both PI and NI mode corresponds to the loss of a methyl radical,
indicating the presence of one or more O-methyl or methoxyl groups [40]. Both RDA
fragments as well as neutral losses provide structural information of flavonoids [37].
Chapter 1
10
Figure 5. Fragment ions (in either ionisation mode, ±) resulting from common C-ring cleavages of 2-phenyl
benzopyrans and isoflavonoids. The left structure represents the fragmentation of chalcones. The middle and
right panel represent the same structures (2-phenyl benzopyrans and isoflavonoids), but different fragmentation routes. The most common cleavages for 2-phenyl benzopyrans and 3-phenyl benzopyrans (isoflavonoids) are 0/2,
0/3, 1/2, 1/3 and 1/4.
Although the substitution pattern of flavonoids may influence the specific
fragmentation pathway in NI and PI mode, some common fragmentation pathways have
been described for several 2-phenyl benzopyrans and isoflavonoid subclasses. In Tables 1
and 2 an overview is given of fragment ions that are formed upon C-ring cleavage of
different 2-phenyl benzopyrans and isoflavonoid subclasses in both PI and NI mode. The
most common cleavage in both PI and NI mode is 1/3, which is observed in almost every
studied subclass. Nevertheless, the fragmentation of flavonoids appears to follow more
specific degradation pathways, characteristic for each subclass. These unique fragment
ions, or combinations thereof, have been used for identification purposes [41].
The 2-phenyl benzopyran subclasses such as the flavones and flavonols have been
most studied regarding their fragmentation pathways. Studies investigating the
fragmentation of isoflavonoids have so far been limited to isoflavones, isoflavanones,
isoflavans and pterocarpans. The fragmentation of these subclasses in PI mode typically
results in 1,3A+, 1,3B+ and 2,3B+ fragment ions. Their fragmentation in NI mode appears less
straightforward, with several fragment ions for isoflavones and isoflavanones. The C-ring
cleavage of isoflavones is often very limited or not observed in NI mode [42, 43].
Furthermore, a number of studies investigated the fragmentation of pterocarpans in NI
mode, and coumestans in both PI and NI modes. To our knowledge, these studies
reported no C-ring fragmentation [44-47].
General introduction
11
In general, it is not possible to discriminate 2-phenyl benzopyrans and
isoflavonoids solely based on MSn fragmentation. Furthermore, the fragmentation of
different 2-phenyl benzopyran and isoflavonoid subclasses in NI mode appears less
distinctive compared to PI mode. In addition, the limited diversity of RDA fragments in PI
mode might suggest the presence of isoflavonoids.
Table 1. Fragment ions in PI mode resulting from C-ring cleavage reported for subclasses within 2-phenyl
benzopyrans, isoflavonoids and miscellaneous flavonoids.
0/2 0/4 1/2 1/3 1/4 0 2 Remarks
2-Phenyl benzopyrans
Flavones B B A,B
Flavonols A,B (B) A B 1,4A + 2H, 1,3B - 2H
Flavanones A,B A 1,3B - 2H, M+H-Bring
Flavanols A A B
Dihydroflavonols A B A A
Isoflavonoids
Isoflavones A,B
Isoflavanones A,B 2,3B
Isoflavans A,B 2,3B, M+H-Bring
Pterocarpans A 2,3B
Miscellaneous flavonoids
Chalcones A,B A,B
Fragments in parenthesis indicate that these were fragments of minor abundance (<10%).
This table has been compiled from data from the following references: [32, 35, 37, 38, 41, 43, 46, 48-53].
Table 2. Fragment ions in NI mode resulting from C-ring cleavage reported for subclasses within 2-
phenyl benzopyrans and isoflavonoids.
0/3 0/4 1/2 1/3 1/4 Remarks
2-phenyl benzopyrans
Flavones A,B A* A A,B 1,4A + 2H Flavonols A A,B A,B** B* Flavanones A A* A A,B A Isoflavonoids
Isoflavones B,(A) B A** A** Isoflavanones A,B Pterocarpans No RDA fragments were
observed Coumestans
Fragments in parenthesis indicate that these were fragments of minor abundance (<10%). * Ambiguous. ** Only observed for methoxylated derivatives.
This table has been compiled from data from the following references: [32, 37, 41-45, 47, 54-58].
Chapter 1
12
INTERACTION OF FLAVONOIDS WITH THE HUMAN ESTROGEN RECEPTOR
Plant-derived compounds are known to induce biological responses by mimicking or
modulating endogenous estrogens [59]. These so-called phytoestrogens usually exhibit
their activity by binding to the estrogen receptor (ER), due to their structural similarity
with the human sex hormone 17β-estradiol (E2). Phytoestrogens possess a phenol ring,
which together with another hydroxyl group at the other end of the molecule, is one of
the most important prerequisites for binding to the ER [60, 61]. The spacing between the
hydroxyl groups on both ends of the phytoestrogen is critical, and should resemble the
distance between the 3-hydroxyl and 17β-hydroxyl of E2 [61]. Many phytoestrogens belong
to the isoflavonoid subclasses of isoflavones and coumestans, although also other
phenolic compounds, such as lignans and stilbenes, are known to display estrogenic
activity [62, 63].
The ER belongs to the superfamily of nuclear receptors. Its main function is the
regulation of gene expression. The ER is activated by estrogens (C18-steroids) that control
growth, differentiation and functions of many target organs. Two ER subtypes have been
described in humans: the ERα and ERβ. The ERα is mainly expressed in the sex organs such
as the endometrium, mammary gland and uterus. The ERβ is mainly distributed in the
bone, cardiovascular system, central nervous system and the brain [59]. The most potent
hormone in the human body is 17β-estradiol [64]. Estrogens like E2 act as ligands binding to
the ligand binding domain (LBD) of the receptor, upon which a dimeric configuration of
the receptor is enabled, which facilitates binding to the estrogen response elements (ERE).
Binding of the dimeric receptor complex to the ERE can initiate or inhibit DNA
transcription, depending on whether the ER-ligand is an agonist or antagonist [60]. An
agonist induces a conformational change that results in dimerisation of ERs initiating DNA
transcription. E2 is a well-known agonist. Antagonists, on the other hand, induce a
conformational change that hampers dimerization, or the recruitment of so-called co-
factors, resulting in inhibition of DNA transcription [60].
Currently, there is a broad range of in vitro bioassays available that allow the
determination of estrogenic activity of a compound. There are different types of bioassays
varying from ligand binding assays to receptor-dependent gene expression assays and cell
proliferation assays. Whereas the ligand binding assay can only determine binding to the
ER, receptor-dependent transactivation assays and proliferation assays can distinguish
between agonists and antagonists [65]. Although in vitro bioassays help in determining the
estrogenic potential of compounds, their physiological relevance needs to be confirmed in
vivo.
The binding affinity of ligands to a receptor is generally expressed as effective
concentration at half maximal binding, or EC50 value. Figure 6 illustrates typical binding
curves of E2 to the ERα and ERβ, in which the EC50 is indicated.
General introduction
13
Figure 6. A typical binding curve of E2 to estrogen receptors α and β.
Most phytoestrogens have a preferential binding to the ERβ. In general, their
binding affinity is approx. 102 to 105 times weaker than that of endogenous estrogens,
resulting in an EC50 in the µM range, depending on the assay type [66-68]. Apart from
binding to the ER, the estrogenic activity of phytoestrogens might also be mediated via
other mechanisms such as the induction of sex hormone-binding globulin (SHBG) [69].
Furthermore, isoflavonoids have been reported to inhibit the activity of enzymes in the
estrogen and androgen biosynthesis, i.e. aromatase, 5α-reductase and 17β-hydroxysteroid
oxidoreductase [70-72].
Dietary intake of phytoestrogens might be beneficial in prevention or the
treatment of hormone-dependent deficiencies including osteoporosis, cardiovascular
diseases, chemo-prevention, (post-)menopausal symptoms [10, 12, 63, 67, 73-76]. As rich sources
of phytoestrogens, legumes are the main contributor of the dietary intake of estrogenic
compounds. The observation that the consumption of legumes was linked to health
beneficial effects led to the development of health ingredients [77], which are nowadays
used in the development of food products (functional foods) or dietary supplements
(nutraceuticals). Both licorice and soya are legumes belonging to the family of the
Leguminosae.
Chapter 1
14
LICORICE
Licorice is one of mankind’s oldest known medicines. Licorice refers to the dried
underground parts (roots and stolons) of Glycyrrhiza ssp. and is also known as liquorice,
radix Glycyrrhizae (Latin), gancao (‘sweet grass’ in Chinese), kanzou (Japanese) and
gamcho (감초, Korean). For centuries, licorice has been applied in traditional herbal
medicine and its origins can be traced back to Babylonia (Codex Hammurabi, ca. 1700 BC),
Assyria (ca. 700 BC) and ancient China (Shénnóng Běn Cǎo Jíng, Shennong's Materia
Medica) [78]. References of the medicinal application of licorice in East-Asia have been
documented in the Shénnóng Běn Cǎo Jíng, the first Chinese dispensatory, where it is used
for its life-enhancing properties, improving health and detoxification [79].
The Glycyrrhiza genus belongs to the legume family (Leguminosae) and contains 5
different species: G. glabra, G. uralensis, G. korshinskyi, G. inflata, and G. aspera [80]. G.
glabra is considered to consist of 3 main varieties. G. glabra L. var. typica is commonly
referred to as Spanish or Italian licorice. G. glabra L. var. violacea is known as Persian or
Turkish licorice. G. glabra L. var. glandulifera is mainly known as Russian licorice [78, 81].
Confusion often occurs as the term licorice is applied for all species and varieties within
the genus of Glycyrrhiza [82]. Several species (G. glabra and G. uralensis) have been
reported to be distributed in the same geographical regions in Central Asia and Western
China, and are able to form hybrids [14, 83]. In general, materials originating from G. glabra
are used in the food and pharmaceutical industry.
Nowadays, licorice is mainly applied in the tobacco, confectionary and
pharmaceutical industries as a flavouring agent [16, 84]. One of the principal components of
licorice root is glycyrrhizin (Figure 7), a triterpenoid glycoside, which is considered 50-170
times sweeter than sucrose and primarily responsible for the licorice flavour and
sweetness [16, 85]. Glycyrrhizin has long been considered as the bioactive constituent of
licorice. However, it has been shown that many biological activities of licorice, including
estrogenic, anti-cancer, anti-microbial, skin whitening and metabolic syndrome
preventive, can be ascribed to its isoflavonoid constituents [15, 86-99]. In the past decades,
extensive investigations have resulted in the identification of over 300 different flavonoids
in Glycyrrhiza ssp., of which half were isolated from the underground parts [16].
General introduction
15
Licorice isoflavonoids
G. glabra is predominantly rich in isoflavans, isoflavenes and isoflavones. In addition,
other flavonoids are abundantly present such as chalcones (miscellaneous flavonoids) and
flavanones (2-phenyl benzopyrans). The chalcones and flavanones in licorice roots are
represented by isoliquiritigenin and liquiritigenin, respectively, which occur as free
aglycone form and as glycoside-conjugate. Furthermore, licorice is known to be a rich
source of prenylated isoflavonoids. So far, studies have reported the isolation of ~75
prenylated isoflavonoids from the roots of G. glabra [16, 100-108]. Glabridin is the main
prenylated isoflavonoid of licorice (Figure 7), and is considered a species-specific marker
compound for G. glabra [14, 82]. Glabridin is a prenylated isoflavan with a pyran-substitution
on the A-ring. Its content in the dried roots varies from 0.08-0.35% (w/w) [109]. In addition
to glabridin, several different glabridin-derivatives have been isolated from licorice,
including hydroxylated, methoxylated and other prenylated derivatives [16].
In the past two decades, licorice has been investigated for its estrogenic activity.
The estrogenic activity of the licorice extracts has been reported in different estrogenicity
assays, implicating the presence of phytoestrogens [110-113]. The main estrogenic
compounds that have been isolated from licorice root are glabridin and the isoflavene
glabrene (Figure 7) [114-118]. Glabrene is considered the most estrogenic compound with an
EC50 of 1 µM, followed by glabridin with an EC50 of 5 µM. Further investigations have
shown that glabridin-derivatives were less estrogenic: hispaglabridin A and B, both B-ring
prenyl-substituted glabridin-derivatives, were also estrogenic, but with an EC50 of ~10
times higher than glabridin. Methylation of the hydroxyl groups on the B-ring of glabridin
was shown to reduce the estrogenic properties of glabridin. The estrogenicity of glabrene
and glabridin were determined using a mammalian cell based proliferation assay.
However, their specific estrogenic potency on the ERα and ERβ, as well as possible
antagonistic activities on both ER-subtypes, have not yet been established.
Figure 7. Molecular structures of glycyrrhizin, the main licorice triterpenoid, glabridin and glabrene, the main
prenylated isoflavonoids of licorice. GlcA, glucuronic acid.
Chapter 1
16
SOYA
Soya often refers to all species within the genus Glycine and belongs to the family of
Leguminosae. The genus Glycine is divided into two subgenera, Glycine and Soja (Moench)
F. J. Herm. The cultivated soya bean, Glycine max (L.) Merr., is derived from its wild
ancestor Glycine soja. In this thesis the term soya refers to Glycine max unless stated
otherwise. The soya bean is a complex matrix of proteins, oils, carbohydrates and
bioactive compounds such as isoflavones, saponins, and phytosterols [119]. In Asia, soya
forms an integral and important part of the diet. Soya beans are often processed resulting
in a broad variety of food products. Common soya products are tofu (originating from
China), tempeh (originating from Indonesia) and natto (originating from Japan).
Soya beans are one of the richest sources of isoflavones. The isoflavone
composition of soya is characterised by 3 main isoflavones, genistein, daidzein and
glycitein, and their respective glucosides, acetyl-glucosides and malonyl-glucosides [120].
The isoflavone content can be affected by variety (cultivar), cultivation year, geographical
location, temperature, storage conditions, and germination period [120-122]. The isoflavone
content is highly variable and ranges between 0.2 and 2.0 mg/g dry weight, with
occasional extremes up to 9.5 mg/g dry weight [123, 124]. With its high isoflavone content,
soya is considered one of the richest dietary sources of phytoestrogens, and is associated
with health-beneficial effects towards hormone-related conditions such as cancer-
prevention, menopausal complaints and osteoporosis [10, 12, 63, 67, 73-76].
METHODS TO INCREASE ESTROGENIC BIOACTIVITY OF SOYA
Hydrolysis of isoflavone glycosides / Increasing bioavailability
The isoflavones in soya beans and soya foods are predominantly present in their glycosidic
forms. Although isoflavone glycosides are more water soluble than the aglycones, in their
conjugated form isoflavones are poorly absorbed in the intestinal tract [125]. Hydrolysis of
the isoflavone glycoside is, therefore, essential in the bioavailability of isoflavones. Upon
ingestion, isoflavone glycosides are hydrolysed by β-glycosidase of the brush border cells
in the small intestine and of the intestinal microflora such as Lactobacillus spp.,
Bacteroides spp. and Bifidobacterium spp. [126]. Due to their stability, isoflavone glycosides
remain intact during soya bean processing and, therefore, predominate in many soya
foods [127]. Fermented soya foods such as miso, natto and tempeh, on the other hand,
contain relatively high levels of isoflavone aglycones, due to bioconversion by microbial
cultures used in fermentation [128-131]. Rhizopus spp., used in the production of tempeh, is
for example mainly responsible for the conversion of isoflavone glycosides into their
aglycones [132-134]. In relation to the enhanced bioavailability of isoflavone aglycones, the
General introduction
17
consumption of soya foods enriched with isoflavone aglycones is associated with reduced
blood cholesterol and improves markers of cardiovascular risk [135].
Identification of different bacterial cultures capable of the hydrolysis of
isoflavone glycosides stimulated the development of fermented functional foods that are
enriched in isolavone aglycones. A common example is the enrichment of soymilk with
isoflavone aglycones by fermentation with Bifidobacteria [136-138]. Several strains of the
probiotic Bifidobacterium were capable of converting up to 90% of daidzin into its
aglycone daidzein.
Enrichment of equol
For years, daidzein and genistein were considered the only estrogenic compounds that
were responsible for the health benefits related to soya consumption. Studies on the
microbial metabolism of soya isoflavones led to the discovery that daidzein can be
converted into equol by intestinal microflora [139]. ER binding studies showed that equol is
a more potent estrogen than daidzein, with a preferential binding affinity to the ERβ [140].
The intestinal conversion of equol was found to occur only in 25-30% of Western adults [141-143]. The prevalence of these so-called equol-producers is approximately twice as high
among adults from Japan, Korea or China [144-147]. It has been hypothesised that the health
benefits from soya consumption may be more pronounced in equol producers than in
equol non-producers [139].
The additional health benefits for equol-producers led to the large-scale
production of equol by chemical synthesis. However, this resulted in a racemic mixture of
S-(-)equol and R-(+)equol as equol has a chiral carbon atom. S-(-)equol is the natural form
that is produced by intestinal conversion, and is considered the most active form of both
enantiomers [148, 149]. A few years ago, a specific bacterial strain (Lactococcus garvieae) was
isolated from dairy products. This bacterium is capable of converting daidzein into S-(-)
equol from daidzein-rich soya hypocotyls under controlled conditions [150]. This provided
an alternative way to large-scale production of the natural S-(-)equol, and led to the
development of new health ingredients and functional foods based on the enrichment of
equol [151-153]. These equol-enriched health ingredients offer both equol-producers and
non-producers the potential health benefits of equol.
Chapter 1
18
Figure 8. The microbial conversion of daidzein to (S)-equol.
Germination
Germination or sprouting is another common process that is applied to different legumes,
such as mung beans and soya beans, to enhance digestibility and the nutrient’s profile [154-
156]. Soya bean sprouts are a common vegetable mainly found in the diet of Asian counties,
particularly in Korea (kongnamul; 콩나물), where soya bean sprouts are a common
ingredient in soups, salads and side-dishes. Studies have shown that germination improves
the nutritional quality by decreasing anti-nutritional factors such as trypsin inhibitors,
phytates and undesirable (beany) flavour and odour caused by lipoxygenase [157-160], and
increasing the levels of phytonutrients such as vitamins, phytosterols, saponins and
tocopherols [160-164]. Germination is also known to induce the biosynthesis of bioactive
phenolics and other phytonutrients as illustrated in e.g. broccoli and rye seedlings [155, 165,
166]. Their relatively high content of bioactive phytonutrients makes seedlings an
interesting source for phytonutrient-rich extracts serving as a basis for developing new
functional food ingredients.
A summary of studies that have investigated the effects of soaking and
germination on the isoflavone content of soya beans is shown in Table 3. Soaking or
imbibition of soya beans refers to the uptake of water during which the moisture content
increases from 5-10% to 55-65% (w/w) [167]. Studies on the effect of soaking on the
isoflavone content show contradictory results. In general, if soaking affected the
isoflavone content, it resulted in either a moderate increase or decrease [159, 168-170].
Soaking generally leads to a decrease in isoflavone glycoside forms, which are hydrolysed
by β-glucosidase [165, 169, 170]. Some studies suggested that the observed decrease in
General introduction
19
glycoside forms could also be due to leaching, i.e. the extraction of water-soluble
isoflavone glycosides by the soaking water [169, 170].
The changes in isoflavone profile and content are more pronounced upon
germination. Short-term germination (12-48 h) generally leads to a 9-57% increase of the
total isoflavone content. This increase is usually followed by a decrease after prolonged
germination (Table 3). The changes in isoflavone profile are characterised by a decrease in
glycoside forms (with the exception of malonyl-glucoside) and an increase in free
aglycones. The malonyl-glucoside forms appear to dominate throughout in non-soaked,
soaked and germinated beans, and often show an absolute increase. The absolute and
relative content of aglycones appears to continuously increase during short and long-term
germination.
In general, short-term germination of soya beans is an effective method to
increase the absolute isoflavone content by 10 to 60%. Moreover, both short and long-
term germination lead to an increase of isoflavone aglycone content, thereby increasing
the bioavailability of the soya isoflavones.
Table 3. Overview of the effects of soaking, short-term and long-term germination on isoflavone content and
profile of soya beans. The changes in isoflavone content (%) are compared to the non-soaked beans (100%).
Soaking Short-term
germination (12-48 h)
Long-term
germination (>48 h)
Range of
conditions
4-20 h, 15-50 °C 12 h to 8 days; 18-30°C; 100% relative humidity
Bean/sprout
characteristics
Increase moisture content from ca. 10% to max. 60%
Development of main root and side-roots after 24 h; Loss in dry weight ranging from 10% up to 40% after1-6 days
Isoflavone
content
Slight increases to slight decreases
Increases ranging from 9 to 57%
Stabilisation or decreases up to 40%
Isoflavone
profile
Relative increase of aglycone content due to hydrolysis of glucoside forms
Rel. increase of aglycone and decrease of glycoside forms except malonyl-form; Malonyl-forms predominate and continue to increase
Increase of aglycones and decrease of other forms
This table has been compiled from data of the following references: [119, 158-160, 165, 168-176].
Germination under stressed conditions
The exposure of plants and seedlings to pathogens can result in the mobilisation of
defensive chemicals. This class of inducible secondary metabolites is called phytoalexins
and are defined as low molecular weight, anti-microbial compounds that are both
synthesized de novo by, and accumulated in, plants after exposure to micro-organisms [177]. Phytoalexin biosynthesis and accumulation are not only induced by micro-organisms
(biotic elicitation), but also by other factors (abiotic elicitation) such as heavy metal salts,
pesticides and UV irradiation, and by components of microbial origin, such as cell wall
polysaccharides, proteins and fatty acid [178].
Chapter 1
20
One of the most studied legume species regarding phytoalexins is Glycine max.
The main phytoalexins induced in soya are coumestrol (coumestans) and glyceollins
(pterocarpans). The classification of coumestrol as a phytoalexin is rather ambiguous as
coumestrol has also been reported in non-germinated soya beans [179, 180]. Glyceollins are a
mixture of 6 different forms of which glyceollin I, II and III are predominantly produced in
Glycine max (Figure 9) [181]. Glyceollin I and II are characterised by a pyran ring-substitution
of the A-ring, whereas glyceollin III is substituted with an isopropenyl dihydrofuran on the
A-ring. Only trace amounts of glyceollin IV have once been isolated from CuCl2-induced
cotyledons of Glycine max[182]. Whereas glyceollins V (canescacarpin) and VI
(clandestacarpin) have only been isolated from other members of the genus Glycine such
as G. canescens, G. clandestina and G. tomentella, a recent study reported the isolation of
trace amounts of glyceollin V (2 mg/kg DW) from the leaves of field grown plants of G.
max [183].
The isoflavone daidzein is the main precursor of the glyceollin biosynthesis.
Daidzein is converted into glycinol, which is subsequently substituted with a prenyl chain
(originating from the MEP pathway) on either the 4 or 2 position, resulting in glyceollidin I
and II, respectively [25]. Cyclisation of the prenyl chain of glyceollidin I leads to glyceollins I
and VI, whereas cyclisation of glyceollidin II results in glyceollins II, III and glyceofuran. The
formation of glyceollin IV involves methylation of the 3-hydroxyl group of glyceollidin II
instead of cyclisation (Figure 9).
Both glyceollins and glycinol possess a broad range of anti-bacterial and
antifungal activities against pathogenic micro-organisms, including the well-known soya
pathogen Phytophthora megasperma f. sp. glycinea [184-186]. In the past decade, studies
have shown that glyceollins also possess health-promoting activities such as anti-oxidant [187, 188], anti-cancer [189-191], anti-diabetic and anti-obesity activity [192]. However, particular
interest goes out to the estrogenic activity of glyceollins. Due to their estrogenic activity,
glycollins have been investigated in a number of in vitro and in vivo studies.
The estrogenic activity of the glyceollin mixture was first reported a decade ago
by means of a ligand binding assay [193]. Since its affinity for the ER was established, a
number of studies demonstrated the in vitro and in vivo estrogenicity of the glyceollin
mixture. Mixtures of glyceollins have been suggested to contain potent antagonists, which
was demonstrated by the inhibitory effects of ER-mediated gene expression and
proliferation of breast and ovarian tissue [189-191, 194, 195]. Moreover, it has been reported
that glyceollins also show ER-mediated inhibition in the prostate cancer proliferation
assay, indicating antagonistic activity towards prostate cancer [196]. Recently, glyceollin I
has been identified as the active component of the glyceollin I-III mixture with an
antagonistic activity that is somewhat different from the known ERα antagonists 4-
hydroxytamoxifen and ICI 182.780 [194]. Similar to equol, glyceollin I can be chemically
synthesised leading to a racemic mixture of (-)-glyceollin I and (+)-glyceollin I. Contrary to
the natural form, (-)-glyceollin I, that possesses antagonistic activity, (+)-glyceollin I
General introduction
21
behaves as an agonist on the ERα [195]. Recently, the estrogenic activity of the non-
prenylated glycinol has been reported [197]. Glycinol showed potent agonistic activity
towards both ER-subtypes comparable to another potent phytoestrogen found in soya,
coumestrol.
The type of elicitation is of influence on the isoflavonoid content as well as the
isoflavonoid profile. The differences between abiotic and biotic elicitation are summarised
in Table 4. In general, abiotic elicitation results in glyceollin levels ranging from trace
amounts to 1528 µg/g FW. On average, biotic elicitation appears to induce higher levels of
glyceollins ranging from 65 to 4327 µg/g FW. This may indicate that biotic elicitation is a
more effective way of inducing glyceollins compared to non-biotic elicitation. Within the
class of biotic elicitors, fungal elicitors appear to be the most effective inducers of high
levels of glyceollins. In glyceollin research, the response of soya to the pathogenic fungus
Phytophthora megasperma f. sp. glycinea is studied most, although the application of food
grade fungi such as Rhizopus spp. and Aspergillus spp. have become popular more
recently in view the development of novel food applications such as fermented soya bean
yoghurt [198].
Table 4. Induction and accumulation of glyceollins and other phytoalexins in soya bean seedlings or selected tissues categorised by the type and subtype of elicitation.
Type of eliciation Total glyceollins I, II and
III (µg/g FW)
Other compounds (µg/g
FW)
References
Biotic elicitation Bacteria
1 270 Glycinol: 60-100
Glyceollidins: 10 Glyceofuran: <10
[181, 185, 199, 200]
Fungus- Phytophthora
megasperma f. sp. glycinea
65-4327 n.d. [201-204]
Fungus – other fungi2 159-3000
(1000-68003) Coumestrol: 20 [188, 189, 192, 205-209]
Fungal cell wall
polysaccharides
500 Glycinol: 34 [185, 210]
Abiotic elicitation
UV radiation 4.3 Glycinol: 150 [185, 211] Heavy metal salts
4 11-42 Glyceollin IV [181, 182, 185, 211-213]
Herbicides5 79-338 Glyceofuran: 16 [214, 215]
Miscellaneous chemicals6 Traces - 1528 [181, 211, 216]
1 Include Erwinia carotovota, Pseudomonas fluorescens and Curtobacterium spp. 2 Include Fusarium spp., Rhizopus spp., Aspergillus spp, white rice yeast and Mucor ramosissimus. 3 1000-6800 µg/g DW reported for black soya bean cultivar [207]. 4 Heavy metals salts include CuCl2, AgNO3 and HgCl2. 5 Diphenyl ether herbicides acifluorfen and lactofen. 6 Include hydroperoxides, sulfhydryl reagent (e.g. iodoacetate) and TX-100 detergent.
Figure 9. Biosynthesis of glyceollins, involving the pterocarpan biosynthesis and MEP pathway. Glyceollin V* refers to the structure indicated as glyceollin V in Yuk et al. [217]
General introduction
23
Furthermore, a number of studies investigated the influence of tissue and type of
elicitation on ratio of glyceollins I, II and III. In addition, other phytoalexins have been
reported to accumulate, such as glycinol, glyceollidins I/II, glyceollin IV and glyceofuran [185, 199]. A summary on the effects of both tissue and elicitation types on the ratio of
glyceollins I, II and III is presented in Table 5. In the late 1980s, the effects of tissue and
elicitor on the glyceollin I-III ratio were reviewed by Keen et al. [181]. It was concluded that
not the elicitor but the soya tissue mostly determined the glyceollin ratio. Although some
variation was observed, tissue-specific ratios could be observed. In leaf tissue, the ratio
was approximately 2:1:3 to 1:7:11, indicating the predominance of glyceollin III. In the
other tissues the ratio was characterised by more or less equal amounts of glyceollin II and
III, and a 2 to 50 fold higher amount of glyceollin I relative to glyceollin II.
Table 5. The ratio of glyceollins I, II and III in different soya tissues from Glycine max induced with different
types of elicitors.
Biotic Abiotic
Tissue Fungi Cell wall poly-
saccharides
Misc.
Heavy
metal salts
Misc. Ref.
Seedling
11:1:1 to
51:1:2
2:1:2 [202, 212]
Hypocotyl 3:1:1 to
15:2:1
[202, 218]
Cotyledon 6:2:1 to
1:2:2
2:1:3 2:1:2 to
4:1:3
3:1:21 to
6:1:12
[181, 186, 202,
206, 219]
Leaves 1:1:3 to
2:1:3
1:2:33 to
1:5:113
1:3:51 to
1:7:114
[181, 199, 202,
215]
Roots 6:1:25 [181]
Callus 17:1:2 [181]
Pods 5:3:1 [212]
Misc. – miscellaneous. 1 1 mM sodium iodoacetate. 2 0.2% sodium dodecyl sulfate. 3 Pseudomonas syringae pv pisi (bacterium). 4 Acifluorfen (herbicide). 5 Meloidogyne incognita (nematode).
The accumulating evidence that phytoalexins can promote health-beneficial
effect in humans makes the induction process a potentially interesting method for
enhancing the bioactivity of soya. With respect to functional food ingredients,
phytoalexin-enriched soya seedlings could provide an alternative source of
phytoestrogens in the development of new health-beneficial food ingredients.
Chapter 1
24
AIM AND OUTLINE OF THIS THESIS
In the field of functional foods and nutraceuticals, there is a constant need for the
development of new and innovative health ingredients. Although phytoestrogen-rich
extracts are one of the main health ingredients in this area, most of these ingredients are
derived from a limited number of sources. The increasing knowledge on phytoestrogens
and their structure-activity relationships provide an important basis for the diversification
of phytoestrogen-rich extracts. Although prenylation has been suggested to play an
important role in the modification of the structure-activity relationship of phytoestrogens,
still relatively little is known regarding its effect on the estrogenic properties of
isoflavonoids.
In this thesis we aim (1) to provide a better structural characterisation of
isoflavonoids, in particular the prenylated isoflavonoids occurring in soya and licorice, (2)
to increase the estrogenic activity of soya beans by the application of malting in the
presence of a food-grade fungus, (3) to correlate the agonistic/antagonistic estrogenicity
of extracts, and fractions thereof, with the presence of prenylated isoflavonoids.
Chapter 1 presents an overview of prenyl-substituted flavonoid (sub-)classes in
relation to their analysis by mass spectrometry and their estrogenic activity. Special
attention is paid to methods that enhance the estrogenic potential of soya. In Chapter 2,
the development of an LC-MS screening method is described that allows the screening of
prenylated flavonoids in complex mixtures such as plant extracts. An ethyl acetate extract
from licorice root extract was used as a source of flavonoids. In Chapter 3, the prenyl
screening developed is used as a tool to analyse for novel prenylated isoflavonoids that
were induced in Rhizopus-challenged soya seedlings grown on lab-scale. Furthermore, the
MS fragmentation behaviour in NI mode of particularly pterocarpans and coumestans is
described. In Chapter 4, the licorice root extract was fractionated by centrifugal
partitioning chromatography. The estrogenic potential of the fractions was determined
towards both ERα and ERβ, and some of the fractions were analysed for antagonistic
activity. The induction of soya seedlings with Rhizopus microsporus performed on a larger
scale with malting technology used in the brewing industry is described in Chapter 5. The
isoflavonoid content and composition, as well as the estrogenic potential towards both ER
subtypes, were monitored over a 10 day period. The results of these chapters are
discussed in Chapter 6, including the implications for further development of novel
phytoestrogen-rich extracts.
General introduction
25
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Chapter 2
A rapid screening method for prenylated
flavonoids with UHPLC-ESI-MS in licorice root
extracts
Based on: Rudy Simons, Jean-Paul Vincken, Edwin J. Bakx, Marian A. Verbruggen, Harry
Gruppen, A rapid screening method for prenylated flavonoids with UHPLC-ESI-MS in
licorice root extracts, Rapid Communications in Mass Spectrometry 2009, 23, 3083-3093 Reprinted with permission
Chapter 2
38
ABSTRACT
Due to their substitution with an isoprenoid group, prenylated flavonoids have an
increased affinity for biological membranes and target proteins, enhancing their potential
bioactivity. Although many prenylated flavonoids have been described, there are no
methods that specifically screen for their presence in complex mixtures, prior to
purification. We describe a method based on ultra-high performance liquid
chromatography (UHPLC) with electrospray ionisation mass spectrometry (ESI-MS) that
allows rapid screening for prenylated flavonoids in multi-component plant extracts.
Identification of the prenylated flavonoids is based on screening for neutral losses of 42
and 56 in the positive-ion mode MS2 and MS3 spectra within the MS chromatograms. In
addition, this method discriminates between prenyl chain and ring-closed prenyl (pyran
ring), based on the ratio of the relative abundances of the ions that lose 42 and 56 (42:56).
The application of this screening method on a 70% aq. ethanol, ethanol and ethyl acetate
extract of the roots of Glycyrrhiza glabra indicated the presence of 70 mono and di-
prenylated flavonoids. In addition, of each prenylated flavonoid the type of prenylation,
chain or pyran ring, was determined.
Keywords: Prenylation, isoflavonoids, licorice, mass spectrometry, screening
LC-MS based screening method for prenylated flavonoids in licorice
39
INTRODUCTION
Flavonoids are a class of naturally occurring compounds ubiquitous in plants, with a broad
variety of bioactivities [1]. Flavonoids can be decorated with one or more C5 isoprenoid
substituents, and these are referred to as prenylated flavonoids [2-4]. Prenyl groups can be
attached to a flavonoid in the form of a prenyl chain (3-methyl-2-butenyl or dimethylallyl)
or in the ring-closed form of a pyran ring (2,2-dimethylpyran). In general, most flavonoids
are C-prenylated, whereas O-prenylation is less common [5]. Prenylation increases the
lipophilicity of flavonoids leading to an increased affinity to biological membranes and to
an improved interaction with target proteins. This results in enhanced biological activities
such as anti-microbial and estrogenic activities [5, 6]. Examples of prenylated flavonoids
with estrogenic activities are 8-prenylnaringenin and xanthohumol from hop (Humulus
lupulus) [7, 8]. Prenylated compounds seem to offer interesting possibilities for developing
new food supplements, e.g. for the alleviation of osteoporosis and menopausal
complaints, which are both under hormonal control.
In the past two decades, many prenylated flavonoids were reported, mainly in
plants belonging to the Leguminosae and Moraceae [2]. Most of these flavonoids have
been structurally elucidated by means of NMR spectroscopy and mass spectrometry upon
their isolation and purification. A screening method to directly detect prenylated
flavonoids in multicomponent samples is unavailable until now, but might offer
opportunities in facilitating the discovery of potential bioactivities.
Flavonoids have been extensively investigated with electrospray ionisation (ESI)
and atmospheric pressure chemical ionisation (APCI), both in negative-ion (NI) and in
positive-ion (PI) mode, each with their own advantages. In general, NI mode is considered
more sensitive and selective for the analysis of flavonoids [9]. An essential part of the
identification of flavonoids by MS is the fragmentation of the deprotonated molecules in
MSn, in which the flavonoid is degraded into fragment ions. So far, there are no clear
indications for the degradation of the prenyl group of prenylated flavonoids in the MS2
spectra in NI mode. This supports the hypothesis that due to the nucleophilic nature of the
prenyl-group degradation is highly unfavourable [10].
Licorice refers to the roots from Glycyrrhiza glabra and has been traditionally
used in herbal medicine for over 4000 years [11, 12]. In the past decades, extensive chemical
studies resulted in the identification of many flavonoids in the roots of Glycyrrhiza glabra ,
including about 75 prenylated flavonoids [13-22].
The aim of the present study was to develop an UHPLC-MS method that allows
rapid screening of prenylated flavonoids in multi-component plant extracts. Licorice roots
were used as a source because of the presence of many different prenylated and non-
prenylated flavonoids.
Chapter 2
40
EXPERIMENTAL
Materials
The roots of Glycyrrhiza glabra, collected in Afghanistan, were provided by Frutarom US
(North Bergen, NJ, USA). Glabridin and glycyrrhizinic acid were purchased from Wako
Chemicals GmbH (Neuss, Germany). UHPLC/MS grade acetonitrile (ACN) was purchased
from Biosolve BV (Valkenswaard, The Netherlands). Water was prepared using a Milli-Q
water purification system (Millipore, Billerica, MA, USA). All other chemicals were
purchased from Merck (Darmstadt, Germany).
Sample preparation
The roots were milled with a Retsch Ultra Centrifugal Mill ZM 200 (Haan, Germany) to
yield a root powder with a particle size of 1 mm. The root powder (0.01 g powdered root/
ml solvent) was extracted with either 70% (v/v) aq. ethanol (EtOH), EtOH or ethyl acetate
(EA). The compounds were extracted by a two-step sequential extraction with each
solvent for 30 min in a sonication bath at 30°C. The extracts were centrifuged at 2500 g for
15 min. The dried extracts were obtained after evaporation (EtOH and EA) or after
subsequent lyophilization (70% (v/v) aq. EtOH). The extracts was resolubilised in methanol
(MeOH) and stored at -20°C. All samples were thawed and centrifuged before analysis.
RP-UHPLC analysis
Samples were analysed on a Thermo Accela UHPLC system (Thermo Scientific, San Jose,
CA, USA) equipped with pump, autosampler and PDA detector. Samples (1 µl) were
injected on an Acquity UPLC BEH C18 column (2.1 x 150 mm, 1.7 µm particle size) with an
Acquity UPLC BEH C18 Vanguard pre-column (2.1 x 5 mm, 1.7 µm particle size; Waters,
Milford, MA, USA). Water acidified with 0.1% (v/v) acetic acid, eluent A, and acetonitrile
(ACN) acidified with 0.1% (v/v) acetic acid, eluent B, were used as eluents. The flow rate
was 300 µl/min, and the PDA detector was set to measure at a range of 205-400 nm. The
following elution profile was used: 0-18 min, linear gradient from 10%-100% (v/v) B; 18-22
min, isocratic on 100% B; 22-23 min, linear gradient from 100%-10% B 23-25 min, isocratic
on 10% B.
Electrospray ionization mass spectrometry (ESI-MS)
Mass spectrometric data were obtained by analysing samples on a Thermo Scientific LTQ-
XL (San Jose, CA, USA) equipped with an ESI-MS probe coupled to the reversed-phase (RP)-
LC-MS based screening method for prenylated flavonoids in licorice
41
UHPLC, 150 µl/min of the flow from the RP-UHPLC was directed to the MS. Helium was
used as sheath gas and nitrogen as auxiliary gas. Data were collected over an m/z-range of
150-1500. Data dependent MSn analysis was performed with a normalised collision energy
of 35%. The MS3 fragmentation was always performed on the most intense daughter ion
in the MS2 spectrum. Most settings were optimised using “tune plus” (Xcalibur 2.07,
Thermo Scientific) via automatic tuning.
The system was tuned with glabridin in both positive and negative ionisation
mode. In the NI mode, the ion transfer tube temperature was 350°C and the source
voltage 4.5 kV. In the PI mode, the ion transfer tube temperature was 350°C and the
source voltage 5.0 kV. Data acquisition and reprocessing were done with Xcalibur 2.07
(Thermo Scientific). Mass spectral data interpretation and peak determination were
performed with Mass Frontier 4.0 (Highchem, Bratislava, Slovakia).
RESULTS AND DISCUSSION
Chromatographic analysis of root extracts from Glycyrrhiza glabra
The extraction of the licorice roots resulted in extraction yields of 23.7, 6.3 and 4.7 g/100g
for 70% aq. EtOH (v/v), EtOH and EA, respectively. Each of the UHPLC-UV-profiles of the
three licorice root extracts indicated a complex mixture of phenolic compounds (Figure 1).
Preliminary chemical analysis of the roots confirmed the presence of glabridin, which is a
marker compound for G. glabra roots [23]. The 70% aq. EtOH (v/v) extract contained
compounds eluting in the first 10 min of the chromatogram. Extracting with EtOH resulted
in an extract with relatively less polar compounds and relative more apolar compounds,
eluting in the second half of the chromatogram. With EA, mainly apolar compounds were
extracted. A total of 18 peaks could be tentatively assigned in the UHPLC profiles by
means of UV spectra, the m/z values of the parent ion, and Collision Induced Dissociation
(CID) fragmentation of these parent ions in PI and NI mode (Figure 2 and Table 1).
Peaks 1-9
Peaks 1-6 represent the flavonoid glycosides in the 70% (v/v) aq. EtOH and EtOH extracts,
i.e. the three glycosidic forms of both liquiritigenin and isoliquiritigenin. The
fragmentation of liquiritigenin, isoliquiritigenin and their corresponding glycosides have
been described previously in PI and NI modes [24, 25]. Our observations are in line with
these results (Table 1).
Table 1. Compounds tentatively assigned in roots Glycyrrhiza glabra by UHPLC-ESI-MS.
No Rt.
(min)
UVmax (nm) Identification Class Molecular
formula
[M-H]- MS
2 product ions
(relative intensity)
[M+H]+ MS
2 product ions
(relative intensity)
1 6.01 216, 271, 316 Liquiritin apioside Flavanone C26H30O13 549 429(84), 417(4), 297(13), 255(100) 551 419(35), 257(100)
2 6.15 214, 277, 312 Liquiritin apioside Flavanone C26H30O13 549 429(8), 417(14), 297(13), 255(100) 551 419(100), 257(84)
3 6.34 215, 275, 310 Liquiritin Flavanone C21H22O9 417 255(100) 419 257(100)
4 7.32 240, 361 Isoliquiritin apioside Chalcone C26H30O13 549 429(10), 417(15), 297(15), 255(100) 551 419(88), 257(100)
5 7.45 240, 369 Isoliquiritin apioside Chalcone C26H30O13 549 429(80), 417(4), 297(12), 255(100) 551 419(100), 257(40)
6 7.80 240, 370 Isoliquiritin Chalcone C21H22O9 417 255(100) 419 257(100)
7 8.17 215, 360 Licochalcone B Chalcone C16H14O5 285 270(100), 253(8), 225(1), 209(1), 191(7), 150(2)
287 245(100), 193(10), 167(7), 147(7), 121(6)
8 10.00 219 Glycyrrhizinic acid Triterpenoid C42H62O16 822 803(15), 759(7), 645(10), 351(100) 823 647(30), 471(16), 453(100)
9 10.70 216, 248, 297 Formononetin Isoflavone C16H12O4 267 252(100), 223(1), 208(1), 191(1) 269 254(100), 237(39), 213(33), 107(10)
10 12.26 217, 284, 324 Glabrene Isoflav-3-ene C20H18O4 321 306(100), 303(27), 293(18), 277(27), 175(18), 145(14)
322 Not determined
11 13.50 222, 250, 302 Glabrone Isoflavone C20H16O5 335 320(18), 307(10), 292(11), 291(100), 213(17)
337 295(41), 283(100), 239(17), 137(22)
12 13.53 230, 281 Glabridin Isoflavan C20H20O4 323 213(37), 201(71), 147(28), 135(100), 121(35)
325 269(14), 215(8), 203(21), 189(100), 123(35)
13 14.09 220, 283 Glabrol Flavanone C25H28O4 392 203(100), 187(24), 159(5) 394 337(100), 205(15), 203(10)
14 14.59 223, 271, 323 3’-hydroxy-4’-O-methyl-glabridin
Isoflavan C21H22O5 353 338(31), 201(100), 175(28), 165(45) 355 215(4), 189(100), 153(73), 147(5)
15 15.35 208, 226, 281 4’-O-methyl-glabridin
Isoflavan C21H22O4 337 322(47), 213(11), 201(100), 175(54), 149(10), 123(11)
339 215(4), 189(100), 147(3), 137(52)
16 15.83 206, 228, 280 Hispaglabridin A Isoflavan C25H28O4 391 215(36), 203(100), 201(49), 189(40), 177(63)
393 337(100), 189(79), 191(79)
17 15.93 215,265, 280,383
3-hydroxy-glabrol Flavanone C25H28O5 407 203(100), 221(3), 159(2) 409 353(100), 335(29), 229(6), 205(21), 177(6)
18 17.06 228, 280 Hispaglabridin B Isoflavan C25H26O4 389 201(100), 187(5), 175(13) 391 189(100), 147(10)
LC-MS based screening method for prenylated flavonoids in licorice
43
The fragmentation pattern of both liquiritigenin (flavanone) and isoliquiritigenin
(chalcone) in ESI were very similar, making it difficult to discriminate between chalcone
and flavanone. This is caused by thermal isomerisation by which the C-ring of the
flavanone is opened to form its corresponding chalcone [26]. Based on the UV spectra
described by Fu et al. [24], the peaks were assigned either liquiritigenin glycoside or
isoliquiritigenin glycoside.
Peaks 7, 8 and 9 were assigned as licochalcone B, glycyrrhizinic acid and
formononetin, respectively. Peaks 7 and 9 were confirmed by UV spectra and MS
fragmentation patterns in NI mode, comparable to those found by Wang et al. [27]. Peak 8
was assigned as the main triterpenoid glycoside in the roots of Glycyrrhiza glabra by
means of the authentic standard.
Figure 1. RP-UHPLC-UV profile of Glycyrrhiza glabra roots extracted with 70% aq. EtOH (A), EtOH (B) and EA (C).
Peak numbers refer to compounds in Table 1.
Chapter 2
44
Figure 2. Structures of compounds assigned in a Glycyrrhiza glabra root extract.
LC-MS based screening method for prenylated flavonoids in licorice
45
Peaks 10-18
Peaks 10-18, eluting in the second half of the chromatogram, were assigned as prenylated
flavonoids. The predominant prenylated flavonoid in the chromatogram was glabridin
(12). There were 4 other prenylated isoflavans assigned with the same skeleton as
glabridin: 3’-hydroxy-4’-O-methyl-glabridin (14), 4’-O-methyl-glabridin (15), hispaglabridin
A (16) and hispaglabridin B (18). The fragmentation patterns of these 5 isoflavans are
shown in Figure 3. The C-ring is cleaved in several places resulting in different retro-Diels
Alder (RDA) fragments. The nomenclature for the RDA fragmentation was adapted from
Ma et al. [28] and is shown in Figure 4. The RDA fragments dominate the MS2 spectra of the
isoflavans (12, 14-16 and 18). In addition, a number of small neutral losses were observed,
such as 28 (CO) and 44 (CO2). This indicated that the C-ring of the isoflavans studied is less
stable, and more prone to degradation, than that of, for example, glabrene (isoflav-3-ene)
and glabrone (isoflavone) (see Appendix I and II, respectively).
Figure 3. Proposed fragmentation pathway of glabridin (12), 3’-hydroxy-4’-O-methyl-glabridin (14), 4’-O-methyl-
glabridin (15), hispaglabridin A (16) and B (18) in NI mode. The numbers inside the parenthesis refer to the
compounds in Table 1.
Chapter 2
46
The fragmentation patterns of 12, 14-16 and 18 were basically the same in NI
mode. Fragments 1,3B- and 2,3A- were the most abundant ions in all spectra. The other RDA
fragments were present in all MS2 spectra of these isoflavans, with the exception of
fragment ions 1,4B- and 2,3B-. These ions were only formed in the MS2 of 12 and 16. This
might be due to the conversion of the para hydroxyl group of the B-ring into a carbonyl,
which is only possible if the para hydroxyl group is unsubstituted.
Both fragmentation patterns of glabrol (13) and 3-hydroxy-glabrol (17) in NI
mode were characterised by a predominant 1,3A- ion and a much smaller 1,3B- ion (as given
in see Appendix III). Furthermore, the 1,3A- ion underwent a further CO2 loss yielding a [1,3A
- CO2]- fragment ion, a mechanism that has been described for flavonoids in NI mode [9].
The fragmentation of glabrene (10) is characterised by the losses of a methyl
radical CH3•, a CO and a CO2. Furthermore, the fragment ions [Bring-H]- and [M-H-Bring]
-
were observed, as well as four RDA fragments (see Appendix I).
The main fragment ions in the MS2 spectrum of the isoflavone glabrone (11) were
due to small neutral losses such as a methyl radical (CH3•) and CO2. The most prominent
RDA fragment observed was [1,4B - H2O]- (see Appendix II).
This is the first report on the fragmentation of prenylated flavonoids 10-18 in ESI-
MS. The fragmentation patterns indicate that prenylated flavonoids follow the same
degradation pattern, like the formation of RDA fragments and small neutral losses like CO
and CO2. Furthermore, it appeared that the prenyl-groups are not directly involved in the
MS2 fragmentation in negative mode.
Figure 4. Nomenclature adopted for the different retrocyclization cleavages on a flavanone and an isoflavan.
LC-MS based screening method for prenylated flavonoids in licorice
47
Neutral losses 42 and 56 in PI mode MSn spectra of prenylated flavonoids
The MS2 spectra of prenylated flavonoids 13, 16 and 17 in PI mode were characterised by
the dominant fragment ion [M+H-56]+. Furthermore, the fragmentation of the isoflavans
12, 14, 15 and 18 all had similar degradation patterns in PI mode characterised by the
presence of RDA fragments (see Appendix IV). The dominant RDA fragment ion 1,3A+ is
substituted with a pyran ring. Upon fragmentation of 1,3A+, a predominant loss of 42 was
observed in the MS3 spectra.
The MS2 spectrum of glabrone (11) in PI mode was characterised by neutral losses
of 42, 54 and 56, next to RDA fragment ions 1,3A+ and 1,3B+.
Table 2 summarises the prenylated flavonoids, assigned by us in the licorice root
extract, for which neutral losses 42 and 56 were observed. The [M+H-56]+ ion is the most
abundant ion in the MS2 spectra of compounds 13, 16 and 17, which have in common that
they are substituted with at least one prenyl chain. Furthermore, in the MS3 spectra of 12,
14-16 and 18 a dominant neutral loss of 42 was observed. These 5 prenylated flavonoids
have in common that they are substituted with at least one pyran ring.
Table 2. The presence of neutral losses 42 [C3H6] and 56 [C4H8] originating from the degradation of the prenyl
group in PI mode.
MS2 MS3
No1 Name 42 56 42 56
10 Glabrene n.d. n.d. n.d. n.d.
11 Glabrone ++ + n.d. n.d.
12 Glabridin - ± ++ ± 13 Glabrol - ++ + +
14 3’-hydroxy-4’-O-methyl-Glabridin - - ++ ±
15 4’-O-methyl-Glabridin - - ++ ±
16 Hispaglabridin A - ++ + ±
17 3-hydroxy-Glabrol - ++ - + 18 Hispaglabridin B - - ++ ±
a Number refers to the compounds in Table 1.
- Fragment ion not determined, less than 0.1%.
± The relative abundance of this fragment ion is less than 5 %.
+ The relative abundance of this fragment ion is between 5% and 100%. ++ The relative abundance of this fragment ion is 100% (dominating fragment in the MSn spectrum).
n.d. not determined 1 Glabrene was tentatively assigned in NI mode. The [M-H]- parent had an m/z value of 321. In PI mode, however,
the [M+H]+ of the peak at the same retention time had an m/z value of 322, while 323 was expected. Additional experiments with high resolution MS showed that two compounds were co-eluting with an m/z value of 321 and
323. This resulted in an average m/z value of 322 in the UHPLC-MS chromatogram. It was not possible to
determine the fragmentation of glabrene.
Chapter 2
48
From the results shown in Table 2, it appeared that the degradation of a prenyl
chain in PI mode in MS2 preferably resulted in a neutral loss of 56, sometimes
accompanied by a minor loss of 42. In contrast, the degradation of the pyran ring
appeared to be characterised by a predominant neutral loss of 42 in combination with a
minor loss of 56. The loss of 56 is suggested to be attributed to the loss of C4H8, whereas
the loss of 42 corresponds with the loss of C3H6. This characteristic difference in
fragmentation of both prenyl groups is supported by observations of Stevens et al. [10] and
Da Costa et al. [29] in PI mode APCI. Based on our observations and the literature, a
proposed fragmentation pathway for both prenyl chain and pyran ring is shown in Figure
5.
Figure 5. Proposed degradation pathways of a prenyl chain and a pyran ring attached to the A-ring of a flavonoid
in PI mode. The same degradation pathways apply for a B-ring prenylation. Bold arrow indicates preferred
fragmentation pathway, dotted arrow indicates less likely pathway. The loss of 56 u is always observed in both
prenyl chain and pyran ring degradation. The loss of 42 u is always observed in the degradation of a pyran ring, but not necessarily in that of the prenyl chain.
LC-MS based screening method for prenylated flavonoids in licorice
49
The fragmentation of prenyl groups of prenylated polyphenolic compounds has
been reviewed by Takayama et al. [26]. This review focused on the fragmentation patterns
in electron ionisation (EI), PI mode FAB [9] and chemical ionisation (CI) and summarised the
neutral losses encountered upon fragmentation of the prenyl group (Table 3). This
overview shows that the neutral losses of 42 and 56 are commonly observed upon
fragmentation of prenylated flavonoids. In a more recent study using ESI-MS [4] a neutral
loss of 56 was also observed. These observations are consistent with our findings on the
fragmentation of prenylated flavonoids obtained with ESI-MS.
Table 3. Characteristic fragments for the fragmentation of a prenyl-group reported for different ionisation techniques in positive mode.
EI1 (+)FAB
1 (+)APCI
2, 3 (+)ESI
4
Neutral
Loss
Loss Chain Pyran Chain Pyran Chain Pyran Chain
42 C3H6 � �
43 C3H7• � �
54 C4H6 �
55 C4H7• �
56 C4H8 � � � � �
57 C4H9• �
68 C5H8 � �
69 C5H9• �
71 C5H11 �
EI – electron ionisation; APCI – atmospheric pressure chemical ionisation; ESI – electrospray ionisation 1 Takayama (1992); 2 Stevens (1997); 3 Da Costa (2000); 4 Zhang (2008)
Table 4. Number of individual peaks detected in 70% aq. EtOH, EtOH, and EA extracts and their classification
based on the screening method. The ‘total’ column summarises the total number of unique peaks in all three
extracts.
Group 70% EtOH EtOH EA Total
Total number of peaks 66 82 60 118 Screening for 56 and 42 45 58 48 88 - m/z < 305 (non-prenylated) 5 5 1 8
- Sugar loss detected in MS2 6 6 3 10
Total prenylated 34 47 44 70
Total non-prenylated 32 35 16 48
Mono-prenylated 26 34 33 52
- Prenyl chain 20 26 26 41
- Pyran ring 6 8 7 11
Di-prenylated 9 14 10 18
- Prenyl chain + Prenyl chain 5 10 6 12
- Prenyl Chain + Pyran Ring 4 4 4 6
Chapter 2
50
Screening method for prenylated flavonoids based on neutral losses 42
and 56
The screening method for prenylated flavonoids is based on the detection of neutral
losses. In addition to the detection of prenyl substituents, it is possible to discriminate
between prenyl chain and pyran ring, based on the relative abundances of the ions that
lose 56 and 42, respectively. From these relative abundances for the compounds shown in
Table 2 it appears that if the ratio (42:56) is <1.0, the prenyl group is a prenyl chain. If the
ratio is >1.0, the prenyl group is a pyran ring.
Neutral losses 42 and 56 have also been observed in the fragmentation of non-
prenylated flavonoid aglycones. The fragmentation of licochalcone B (7) was characterised
by a predominant neutral loss of 42, and in the MS2 of formononetin (9) a neutral loss of
56 was observed (Table 1). This indicated that the occurrence of these neutral losses in
the MSn spectra of flavonoids in PI mode do not exclusively point at the presence of prenyl
groups. Losses of 42 have been observed by Ma et al. [28] upon fragmentation of flavonoid
aglycones in the PI mode, and were attributed to the loss of C2H2O, originating from the C-
ring of the flavonoid aglycones. The neutral loss of 56 has been observed by Kuhn et al. in
the fragmentation patterns of different isoflavonoid aglycones [30]. In their study the
authors concluded that a double loss of two CO molecules accounted for the loss of 56.
To rule out the possibility that flavonoid aglycones were assigned as prenylated
flavonoids, a criterion was set to selectively exclude flavonoid aglycones on the basis of
their molecular weight. The prenylated flavonoids of the lowest molecular weight isolated
from Glycyrrhiza glabra are glabrene and 2',4'-dihydroxy-6'',6''-dimethylpyrano-
[2'',3'':7,8]isoflav-3-ene (both isoflav-3-enes) and have a molecular weight of 322
(C20H18O4) [20]. According to an extensive review on prenylated flavonoids by Barron and
Ibrahim, the smallest prenylated flavonoid isolated from a natural source is 7,8-(2,2-
dimethylchromeno) flavone, with a molecular weight of 304 (C20H16O3) [5]. The m/z of the
parent ion of this molecule in PI mode would be 305. To exclude non-prenylated flavonoid
aglycones from the screening method, an m/z threshold of 305 was set.
In plants, flavonoids often appear as O-glycoside conjugates. The O-glycoside
conjugates of non-prenylated flavonoid aglycones that lose 56 u upon fragmentation
exceed the m/z threshold of 305, and they might be inappropriately scored as prenylated
flavonoids: in MS2 the glycoside moiety is cleaved off, after which the aglycone loses 56 in
MS3. In order to prevent that non-prenylated flavonoid O-glycosides are assigned as
prenylated, the MS2 spectra are checked for neutral losses corresponding with the loss of
a sugar moiety. The predominant loss of 132 (pentose), 146 (deoxyhexose), 162 (hexose)
or 176 (uronic acid), or a loss of more than 264 (2 pentoses) in the MS2 spectrum is
diagnostic for a flavonoid O-glycoside, and these compounds are, therefore, assigned as
non-prenylated. The exclusion of flavonoid O-glycosides may consequently lead to the
possibility that prenylated flavonoid O-glycosides are not detected. Five prenylated
LC-MS based screening method for prenylated flavonoids in licorice
51
flavonoid O-glycosides have been described occurring within the plant families of the
Leguminosae, Rutaceae, Rhamnaceae and Pteridaceae [31]. However, most prenylated
flavonoid O-glycosides have been isolated from species within the genera Epimedium and
Vancouveria belonging to the family of Berberidaceae [5].
Our screening procedure, developed on the basis of fragmentation of the
prenylated flavonoids from Glycyrrhiza glabra in the PI mode, is summarised in Figure 6.
The first selection criterion is the presence of a neutral loss of 56, alone or in combination
with a neutral loss of 42 in the MS2 spectrum. If a neutral loss is not present in MS2, the
MS3 spectrum is analysed. The next step is to verify whether the m/z value of the parent
ion [M+H]+ is above 305 to rule out non-prenylated flavonoid aglycones. In case neutral
losses 56, or both 56 and 42, are detected in the MS3, the MS2 of this peak is analysed for
neutral losses corresponding to the loss of sugar moieties. If the MS2 spectra indicates loss
of glycosyl residues, the peak is considered non-prenylated. If these criteria are met, the
compound is considered to be prenylated.
Figure 6. Interpretation guideline for identification of prenylated flavonoids.
Chapter 2
52
The second prenyl group of doubly prenylated flavonoids can be determined as
well. The minimum molecular weight of a double prenylated flavonoids is 372, calculated
by the addition of a prenyl chain (68) to the single prenylated flavone, 7,8-(2,2-
dimethylchromeno) flavone (MW=304), described earlier. Therefore, if the m/z value of
the parent ion [M+H]+ is higher than 373, the MS3 spectrum is checked. This can only be
done in case the MS3 was performed on the [M+H-42]+ or the [M+H-56]+ ion.
Verification of the screening method proposed
To verify the screening method proposed, the 70% aq. EtOH, EtOH and EA extracts of
Glycyrrhiza glabra roots were screened for the presence of prenylated flavonoids in
addition to the tentatively assigned compounds 10-18. Based on the analysis of the MS
chromatograms of the three different extracts by Mass Frontier software, 118 individual
peaks were detected (see Table 4 and Appendix V). Out of these 118 peaks, 88 showed a
neutral loss of 56, alone or in combination with a neutral loss of 42, in either the MS2 or
MS3 spectrum. Of these 88 peaks, 8 were assigned as non-prenylated aglycones, because
their m/z value did not exceed 305. Another 10 peaks were assigned as flavonoid
glycosides, as their MS2 fragmentation indicated a loss of one or more sugar moieties. In
total, 70 unique peaks were assigned as prenylated flavonoids present in the 3 different
extracts of Glycyrrhiza glabra. Of these 70 peaks, 52 peaks were assigned as mono-
prenylated and 18 as di-prenylated. Based on the ratio (42:56), the majority of the 70
peaks have a prenyl chain (see Appendix V).
The EA extract appeared to selectively extract prenylated flavonoids, which may
be related to the non-polar properties of this solvent. For this reason, the EA extract was
chosen as an illustrative example in Table 5 and Figure 7. Table 5 shows an overview of
the 48 peaks in the MS chromatogram of the EA extract that exhibit a loss of 56, alone or
in combination with 42 in MSn. The 43 peaks finally assigned as being prenylated, are
shown in the chromatograms in Figure 7.
Roughly half of the peaks in Table 5 assigned as prenylated, had the [M+H-56]+
ion as their most abundant fragment in their MS2 spectrum and, in addition, most of the
peaks in Table 5 had a ratio (42:56) smaller than 1, indicating the presence of a prenyl
chain. Peak S21 was the only peak with a pyran ring determined by the MS2 spectrum. The
other peaks that were assigned having a pyran ring, were based on their ratio (42:56) in
their MS3 spectrum. This observation indicated that a pyran ring is more stable to MS
degradation than a prenyl chain, as the fragmentation of the prenyl chain mostly occurred
in MS2, whereas the pyran ring is mostly fragmented in MS3.
LC-MS based screening method for prenylated flavonoids in licorice
53
Figure 7. Screening results for neutral losses 42 u and 56 u in the PI mode MS2 and MS3 spectra. RP-UHPLC-MS base peak chromatogram in positive mode (A) of the EA extract of Glycyrrhiza glabra roots. The MS chromatograms were screened for neutral losses in MS2 of 42 u (B) and 56 u (C), and for neutral losses in MS3 of 42 u (D) and 56 u (E). The numbers in the figure relate to the number given in Table 5.
Table 5. Screening results for prenylated flavonoids in the MS chromatogram of the EA extract of Glycyrrhiza glabra in PI mode. (C - prenyl chain; P - pyran ring; np – non-prenylated).
MS2 MS
3
No* Rt
**
(min)
MS
Identification [M+H]+
Ne
utr
al
loss
42
(r.
a.)
Ne
utr
al
loss
56
(r.
a.)
Ra
tio
(4
2:5
6)
Pre
ny
l -t
yp
e
Ne
utr
al
loss
42
(r.
a.)
Ne
utr
al
loss
56
(r.
a.)
Ra
tio
(4
2:5
6)
Pre
ny
l -t
yp
e
S23 10.44 327 - - - - 3 1 3.0 P
S26 10.79 321 24 25 1.0 C
S27 10.89 Formononetin 269 - 33 0.0 np
S29 11.27 341 <1 100 <0.1 C
S31 11.38 339 <1 100 <0.1 C
S32 11.51 323 2 100 <0.1 C
S33 11.57 341 - 100 0.0 C
S34 11.67 323 1 100 <0.1 C
S35 11.79 355 41 100 0.4 C
S36 11.90 337 1 7 0.1 C
S37 12.10 355 <1 100 <0.1 C
S38 12.20 325 3 100 <0.1 C
S39 12.28 321 24 28 0.9 C
S41 12.53 322 - 7 0.0 C
S42 12.68 337 28 57 0.5 C
S43 12.72 339 5 17 0.3 C
S45 12.97 308 - - - - 3 4 0.8 C
S47 13.25 355 - 100 0.0 C
S48 13.31 341 - 100 0.0 C
S49 13.52 409 - 21 0.0 C
S50 13.64 Glabrone 337 100 6 16.7 P
S51 13.77 Glabridin 325 - <1 0.0 - 100 1 100 P
S53 13.91 321 18 64 0.3 C
S54 14.08 391 - 100 0.0 C 1 100 <0.1 C
S55 14.13 645 1 100 <0.1 C 21 10 2.1 P
S57 14.29 Glabrol 393 - 100 0.0 C 21 22 1.0 C
S58 14.49 409 - 100 0.0 C 19 100 0.2 C
S59 14.66 395 - 82 0.0 C
S60 14.72 647 - 60 0.0 C
S62 14.79 3’-hydroxy-4’-O-methyl-glabridin 355 - - - - 100 1 100 P
S64 14.89 409 - 100 0.0 C 30 20 1.5 P
S66 15.04 647 - 14 0.0 C
S67 15.08 661 - 100 0.0 C 1 35 <0.1 C
S69 15.27 391 - 18 0.0 C
S72 15.42 645 <1 100 <0.1 C 35 18 1.9 P
S74 15.52 4’-O-methyl-glabridin 339 - - - - 100 1 100 P
S76 15.73 631 <1 100 <0.1 C 34 6 5.7 P
S77 15.88 391 - 100 <0.1 C 6 7 0.9 C
S78 15.97 Hispaglabridin A 393 - 100 <0.1 C 21 3 7.0 P
S80 16.08 3-hydroxy-glabrol 409 - 100 0.0 C 1 9 0.1 C
S81 16.18 391 1 39 <0.1 C
S82 16.34 409 - 42 0.0 C
S83 16.44 643 2 63 <0.1 np
S84 16.53 393 - 100 0.0 C 10 8 1.3 P
S85 16.72 659 - 26 0.0 np
S86 16.81 469 - - - - 4 14 0.3 C
S87 16.89 388 1 2 0.5 C 11 6 1.8 P
S88 17.21 Hispaglabridin B 391 - 3 - - 100 1 100 P
r.a. relative abundance
< 1 - 0.5% tot 1.0% n.d. - not detected, <0.5% * the ‘S’ refers to a peak found with the screening method in the EA extract ** The retention time in MS has a delay of 0.20±0.02 min compared to the UV retention time
Chapter 2
56
Around 75 prenylated flavonoids have been isolated from the roots of Glycyrrhiza
glabra [13-22]. The application of the screening method described here indicated the
presence of 70 unique prenylated flavonoids in 3 different extracts of the roots of
Glycyrrhiza glabra without any purification or fractionation steps. This suggested that
many of the prenylated flavonoids present can be detected in a crude extract by the
application of the described screening method. A next step would be the application of
this screening method for the detection of prenylated flavonoids in other plant extracts
and, furthermore, to provide a greater number of definitive assignments of the prenylated
flavonoids detected in complex extracts using the current screening method.
CONCLUSIONS
A rapid screening method is described to screen for prenylated flavonoids. The method is
based on the fragmentation of prenyl substituents of flavonoids in PI mode MSn, involving
a neutral loss 56, alone or in combination with 42. In addition to the detection of prenyl
substituents, it is possible to discriminate between prenyl chain and pyran ring, based on
the relative abundances of the ions that lose 42 and 56 (42:56). A ratio (42:56) of <1.0
indicates the presence of a prenyl chain, a ratio >1.0 that of a pyran ring. The screening of
three different extracts of roots from Glycyrrhiza glabra resulted in the detection of 70
peaks that are likely to be prenylated.
ACKNOWLEDGEMENTS
This work was supported by the Food & Nutrition Delta of the Ministry of Economic
Affairs, the Netherlands.
REFERENCES
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[2] B. Botta, A. Vitali, P. Menendez, D. Misiti, G. D. Monache, Prenylated flavonoids: Pharmacology and biotechnology. Current Medicinal Chemistry 2005, 12, 713-739.
[3] P. Shen, S. P. Wong, J. Li, E. L. Yong, Simple and sensitive liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera. Journal of Chromatography B 2009, 877, 71-78.
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Reports 2008, 25, 555-611.
Chapter 3
Identification of prenylated pterocarpans
and other isoflavonoids in Rhizopus spp.
elicited soya bean seedlings by electrospray
ionisation mass spectrometry
Based on: Rudy Simons, Jean-Paul Vincken, Maxime C. Bohin, Tomas F.M. Kuijpers, Marian
A. Verbruggen, Harry Gruppen, Identification of prenylated pterocarpans and other
isoflavonoids in Rhizopus spp. elicited soya bean seedlings by electrospray ionisation mass
spectrometry, Rapid Communications in Mass Spectrometry 2011, 25, 55-65 Reprinted with permission
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ABSTRACT
Phytoalexins from soya are mainly characterised as prenylated pterocarpans, the glyceollins. Extracts of non-soaked and soaked soya beans, as well as that of soya seedlings, grown in the presence of Rhizopus microsporus var. oryzae, were screened for the presence of prenylated flavonoids with an LC-MS based screening method. The glyceollins I-III and glyceollidins I-II, belonging to the isoflavonoid subclass of the pterocarpans, were tentatively assigned. The formation of these prenylated pterocarpans was accompanied by that of other prenylated isoflavonoids of the subclasses of the isoflavones and the coumestans. It was estimated that approx. 40% of the total isoflavonoid content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans, whereas 7% comprised prenylated isoflavones and prenylated coumestans. The site of prenylation (A-ring or B-ring) of the prenylated isoflavones was tentatively annotated using positive-ion mode MS by comparing the 1,3A+ RDA fragments of prenylated and non-prenylated isoflavones. Furthermore, the fragmentation pathways of the 5 pterocarpans in NI mode were proposed, which involved the cleavage of the C ring and/or D ring. The absence of the ring-closed prenyl (pyran or furan) gave exclusively -H2Ox,yRDA fragments, whereas its presence predominantly the common RDA fragments.
Keywords: Prenylation, isoflavonoids, soya, phytoalexins, germination, pterocarpans
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INTRODUCTION The application of stress on plants is a well-known approach to induce changes in the chemical profile of secondary metabolites, including phytoalexins. This is a heterogeneous group of low molecular weight anti-biotic compounds that are de novo synthesized in response to stress, pathogens or chemical elicitors [1]. Phytoalexins increasingly gain scientific interest due to their bioactivity and potential to enhance the nutraceutical value of crops [2].
Soya (Glycine max) is an important source of isoflavonoids. Soya beans contain isoflavones, a subclass of isoflavonoids that are considered to be bio-active constituents responsible for some of the health-improving effects ascribed to soya [3]. The three main isoflavones in soya beans are daidzein, genistein and glycitein, and their respective glucosides, acetyl-glucosides and malonyl-glucosides [4]. Upon application of stress, such as fungal infection, soya seedlings produce coumestrol and glyceollins as the main phytoalexins [5]. Both coumestrol and glyceollins are derived from the precursor daidzein and belong to the isoflavonoid subclasses of the coumestans and pterocarpans, respectively. Another group of compounds known to be generated in black soya beans upon pathogen-stressed germination are keto-oxo-octadecadienoic acids (KODEs) [6]. KODEs belong to the class of oxylipins, a broad class of signalling molecules derived from the oxidation of polyunsaturated fatty acids, of which jasmonic acid is the most well-known representative. In the context of plant defence, oxylipins are reported to have antimicrobial effects, stimulate plant defence gene expression, and regulate plant cell death. Their exact contribution to each functionality is largely unknown [7-9]. The formation of glyceollins in soya seedlings involves the conversion of daidzein into glycinol, which is subsequently A-ring prenylated on the 4 or the 2 position leading to the glyceollidins I and II, respectively. Cyclisation of the prenyl chain results for glyceollidin I into glyceollin I, and for glyceollidin II into glyceollin II and III [5, 10-13]. Prenylation of isoflavonoids is a common modification in the formation of phytoalexins in Leguminosae [14]. Prenylation of flavonoids is thought to increase their lipophilicity, their affinity to biological membranes, and their interaction with target proteins [15]. In this study, the extract of soya beans germinated in the presence of the fungus Rhizopus microsporus var. oryzae was screened for the presence of prenylated flavonoids.
EXPERIMENTAL
Materials
Soya beans, Glycine max (L.) Merrill, were provided by Frutarom Belgium (Londerzeel, Belgium). Authentic standards of genistein, daidzein and coumestrol were purchased from Sigma Aldrich (St. Louis, MO, USA). UHPLC/MS grade acetonitrile (ACN) was purchased
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from Biosolve BV (Valkenswaard, The Netherlands). Water was prepared using a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other chemicals were purchased from Merck (Darmstadt, Germany). The fungus, Rhizopus microsporus var. oryzae (LU 581), was purchased from the Fungal Biodiversity Centre CBS (Utrecht, The Netherlands).
Soya bean germination and challenging with Rhizopus
The soya beans were surface-sterilised by soaking them in a 1% (w/v) hypochlorite solution (5 L kg-1 beans) under continuous stirring for 1 h at 20 °C. Subsequently, the soya beans were rinsed with sterile Milli-Q water and soaked for 4 h at 40 °C in sterile Milli-Q water. After soaking, the beans were germinated in 370 mL glass jars of which the bottom was covered with Whatman filter paper (Type 520 A), humidified with sterile Milli-Q water to prevent the beans from drying out. The lid of the jars was loosely closed, and parafilm was loosely wrapped around the lid to allow air passage. The beans were incubated for 4 days at 30 °C in the dark.
After germination, the beans were inoculated with Rhizopus microsporus var. oryzae (LU 581). For the fungal inoculation, a sporangiospore suspension was prepared by scraping off the sporangia from pure slant cultures of this fungus, grown on malt extract agar (CM59; Oxoid, Basingstoke, UK) for 7 days at 30 °C. Subsequently, the sporangia were suspended in sterile Milli-Q water with 0.85% (w/v) NaCl (108 CFU mL-1). The beans were inoculated with the sporangiospore suspension (0.2 mL g-1) and incubated for 4 days at 30 °C in the dark.
Soya bean extraction
Non-soaked soya beans, soaked soya beans and Rhizopus-challenged germinated soya beans were freeze-dried, after which they were milled with a Retsch Ultra Centrifugal Mill ZM 200 (Haan, Germany) using a 1 mm sieve. The meal was defatted by hexane extraction for 30 min in a sonication bath at 30 °C (0.04 g meal mL-1 hexane) followed by air-drying of the residue. The flavonoids were extracted with absolute ethanol (0.04 g meal mL-1 EtOH) by a two-step sequential extraction of the defatted powder, each for 30 min in a sonication bath at 30 °C. The extracts were centrifuged (2500 g, 15 min at 20°C). The dried extracts were obtained after evaporation and were subsequently, dried, re-solubilised in methanol (MeOH) to a concentration of 10 mg mL-1 and stored at -20 °C. All samples were thawed and centrifuged before analysis.
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63
RP-UHPLC analysis
Samples were analysed on a Thermo Accela UHPLC system (San Jose, CA, USA) equipped with pump, autosampler and PDA detector. Samples (1 µL) were injected on a Waters Acquity UPLC BEH shield RP18 column (2.1 x 150 mm, 1.7 µm particle size) with a Waters Acquity UPLC shield RP18 Vanguard pre-column (2.1 x 5 mm, 1.7 µm particle size; Waters, Milford, MA, USA). Water acidified with 0.1% (v/v) acetic acid, eluent A, and ACN acidified with 0.1% (v/v) acetic acid, eluent B, were used as eluents. The flow rate was 300 µL min-1, the column oven temperature was controlled at 25 °C, and the PDA detector was set to measure at a range of 200-400 nm. The following elution profile was used: 0-2 min, linear gradient from 10%-25% (v/v) B; 2-9 min, linear gradient from 25%-50% (v/v) B; 9-12 min, isocratic on 50% B; 12-22 min, linear gradient from 50%-100% (v/v) B; 22-25 min, isocratic on 100% B; 25-27 min, linear gradient from 100%-10% (v/v) B; 27-29 min, isocratic on 10% (v/v) B.
Electrospray ionization mass spectrometry (ESI-MS)
Mass spectrometric data were obtained by analysing samples on a Thermo Scientific LTQ-XL (San Jose, CA, USA) equipped with an ESI-MS probe coupled to the RP-UHPLC. Helium was used as sheath gas and nitrogen as auxiliary gas. Data were collected over an m/z-range of 150-1500. Data dependent MSn analysis was performed with a normalised collision energy of 35%. The MSn fragmentation was always performed on the most intense daughter ion in the MSn-1 spectrum. Most settings were optimised via automatic tuning using “Tune Plus” (Xcalibur 2.07, Thermo Scientific). To this end, the system was tuned with genistein in both positive ionisation (PI) and negative ionisation (NI) mode. For both modes, the ion transfer tube temperature was 350 °C and the source voltage 4.8 kV. Data acquisition and reprocessing were done with Xcalibur 2.07 (Thermo Scientific).
RESULTS AND DISCUSSION
Precursors of prenylated isoflavonoids
The isoflavone aglycones daidzein (12), genistein (14) and glycitein (11), their glucoside- (1, 3 and 2, respectively) and malonyl-forms (6, 9 and 5, respectively) were all assigned in the EtOH extracts of the non-soaked soya beans, soaked soya beans and Rhizopus-challenged soya seedlings using UHPLC-UV-MS (Figures 1 and 2; Table 1). Furthermore, two peaks were annotated as acetyl-daidzin (7) and acetyl-genistin (10). The non-soaked beans contained predominantly glycosylated and malonylated forms of daidzein and genistein. These forms were partly converted into their respective aglycones upon soaking. Of the isoflavone aglycones present in the soaked beans, daidzein (12) and
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genistein (14) were the most abundant, whereas glycitein (11) was only present in small amounts. This is consistent with previous data [4]. The changes in isoflavonoid profile upon germination and subsequent application of R. microsporus were characterised by a further decrease of the glycoside-conjugated isoflavonoids. Daidzein, genistein and glycitein became the predominant isoflavonoid compounds in the UV-profile. This has also been reported for soya seedlings that were inoculated with Aspergillus sojae
[16]. The decrease of the daidzin and malonyl-daidzin content caused by deglucosylation has been proposed to increase the pool of daidzein in the soya seedling, in addition to the de novo synthesis by flavonoid metabolism, which might facilitate glyceollin production [17].
Furthermore, the accumulation of three other isoflavonoids was induced by R.
microsporus. At a retention time of 6.23 min a peak (4) appeared that was annotated as glycinol, followed by peaks 8 and 13 that were tentatively annotated as 2’-hydroxydaidzein (7,2’,4’-trihydroxyisoflavone) and 2’-hydroxygenistein (5,7,2’,4’-tetrahydroxyisoflavone), respectively. Glycinol (3,6a,9-trihydroxypterocarpan, 4) is a pterocarpan that is formed from daidzein (12), with the 2’-hydroxylation of daidzein leading to compound 8 as first step [10].
Based on MS fragmentation analysis in positive mode, compound 13 was tentatively assigned as genistein with an extra hydroxyl group on the B-ring. The tentative assignment was supported by the comparison of the MS2 and MS3 spectra of compound 13 with those reported for 2’-hydroxygenistein [18]. Nevertheless, it should be mentioned that the MSn fragmentation of 3’-hydroxygenistein (orobol) has never been described. Therefore, it cannot be excluded that its fragmentation is very similar to that of 2’-hydroxygenistein. As a consequence, the extra hydroxyl group on 13 might be on either the 2’- or the 3’-position. Both hydroxylated forms of genistein have not been reported in soya. Regarding the occurrence of both genistein forms in the family of Leguminosae, the presence of orobol has been reported in only 2 species, whereas 2’-hydroxygenistein was found in more than 30 species [19]. Therefore, 13 most likely corresponds to 2’-hydroxygenistein.
The formation of 2’-hydroxygenistein might be related to the conversion of daidzein to 2’-hydroxydaidzein as the first step in the formation of glycinol, in which 2’-hydroxygenistein may be formed as a by-product. Whereas 2’-hydroxydaidzein is converted to different pterocarpans, 2’-hydroxygenistein appears to accumulate as it might not be a suitable precursor for pterocarpan biosynthesis. This is supported by a study in which the enzyme NADPH:2’-hydroxydaidzein oxidoreductase was characterised [20]. In pterocarpan biosynthesis, this enzyme performs the conversion of 2’-hydroxydaidzein into 2’-hydroxydihydrodaidzein and a low conversion of 2’-hydroxygenistein to the corresponding isoflavanone was observed.
Besides glycinol, another non-prenylated isoflavonoid, coumestrol (17) was formed. Coumestrol was assigned on the basis of the reference compound. In addition to the annotated isoflavonoids, the peak eluting at 10.3 min was tentatively assigned as naringenin, a non-prenylated flavanone.
Table 1. Compounds tentatively assigned in the extracts of Glycine max by UHPLC-ESI-MS. No Rt.
(min)
UVmax (nm) Identification Class Molecular
formula
[M-H]- MS
2 product ions
(relative abundance)
[M+H]+ MS
2 product ions
(relative abundance)
1 4.86 225,248 Daidzin Isoflavone C21H20O9 415 253(100) 417 255(100)
2 4.91 225,256 Glycitin Isoflavone C22H22O10 445 283(100) 447 283(100)
3 5.99 225,260 Genistin Isoflavone C21H20O10 431 431(100), 269(11) 433 271(100)
4 6.23 205,226,281 Glycinol Pterocarpan C15H12O5 271 256(34), 227(28), 161(100) 255* 199(100), 237(52)
5 6.27 209,225 Malonyl-glycitin Isoflavone C25H24O13 531 283(100) 533 285(100)
6 6.33 225,255 Malonyl-daidzin Isoflavone C24H22O12 501 253(100) 503 255(100)
7 7.04 208,225 Acetyl-daidzin Isoflavone C23H22O10 457 269(100) 459 271(100)
8 7.63 209,225,260 2'-Hydroxydaidzein Isoflavone C15H10O5 269 269(100) 271 215(55), 201(100), 137(36)
9 7.70 209,225,260 Malonyl-genistin Isoflavone C24H22O13 517 269(100) 519 271(100)
10 7.89 210,225,256 Acetyl-genistin Isoflavone C27H30O15 473 269(100) 475 271(100)
11 8.46 226,257 Glycitein Isoflavone C16H12O5 283 268(100) 285 270(100), 229(21), 225(16)
12 8.79 226,300 Daidzein Isoflavone C15H10O4 253 253(100) 255 227(49), 199(100), 137(70)
13 8.98 212,226,257 2'-Hydroxygenistein Isoflavone C15H10O6 285 217(100) 287 245(49), 217(100), 153(57)
14 11.33 224,260 Genistein Isoflavone C15H10O5 269 269(100) 271 153(100), 215(79)
15 11.52 209,227,285 Glyceollidin I/II Pterocarpan C20H20O5 339 324(45), 161(100) 323* 267(100)
16 12.01 211,227,289,354
Glyceollin III Pterocarpan C20H18O5 337 319(100), 293(8), 149(18) 321* 306(76), 297(79), 251(100)
17 12.14 226,303,343 Coumestrol Coumestan C15H8O5 267 267(100) 269 241(100), 225(22), 197(23)
18 12.35 211,228,283,307
Glyceollin II Pterocarpan C20H18O5 337 319(100), 293(24), 149(44) 321* 306(74), 297(100), 251(94)
19 12.48 210,230,282 Glyceollin I Pterocarpan C20H18O5 337 319(100), 293(34), 149(95) 321* 306(71), 303(100), 293(33)
20 16.98 212,227 9-Z,E/E,E-KODE Oxylipin C18H30O3 293 275(100), 235(17), 185(2), 171(9)
295 n.a.
21 17.22 211,228 13-Z,E/E,E-KODE Oxylipin C18H30O3 293 275(100), 249(12), 195(29), 179(11)
295 n.a.
22 18.65 211,227,274 13-Z,E/E,E-KODE Oxylipin C18H30O3 293 275(63), 249(100), 179(17), 113(54)
295 277(100)
23 18.80 211,228,271 9-Z,E/E,E-KODE Oxylipin C18H30O3 293 275(46), 249(17), 197(37), 185(100)
295 277(100)
24 18.88 211,227,343 Unknown KODE Oxylipin C18H30O3 293 n.a. 295 277(100)
25 19.06 211,228,268 9-Z,E/E,E-KODE Oxylipin C18H30O3 293 275(20), 249(11), 197(41), 185(100)
295 277(100)
n.a. – not available; * The [M+H-H2O]+ dominated in the MS1 compared to the [M+H]
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Figure 1. RP-UHPLC-UV profile of EtOH extracts of non-soaked soya beans (A), soaked soya beans (B), and Rhizopus-challenged soya bean seedlings (C). Peak numbers refer to compounds in Table 1.
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67
Oxylipins
Upon soaking and challenging of the soaked beans, several peaks appeared in the UV-profile eluting after 15 min (peaks 20-25; Figure 1; Table 1). These peaks were annotated as KODEs. These peaks were not observed in the non-soaked soya bean extract. Based on in-source ESI fragmentation in NI mode, it was possible to distinguish between 13-KODE and 9-KODE [6]. Peaks 20, 23 and 25 were assigned as 9-KODEs, whereas peaks 21 and 22 were assigned as 13-KODEs. Based on the MS2 spectrum of peak 24, it was not possible to determine the type of KODE.
Figure 2. Structures of isoflavonoids in soya beans and challenged soya seedlings.
Prenylated pterocarpans
The incubation of germinated soya beans with R. microsporus led to the accumulation of glyceollins and glyceollidins (Figure 1; Table 1). Peaks 16, 18 and 19 with an m/z value in NI mode of 337 were tentatively assigned as glyceollins. The area of peaks 16, 18 and 19 had a relative ratio of approximately 1:2:6. It has been reported that glyceollin I is the most abundant of the three in an extract of pathogen-challenged soya beans, followed by glyceollin II [21]. Based on this data and by comparison of their UV spectra with those reported in literature [22], peaks 16, 18 and 19 were tentatively assigned as glyceollins III, II and I, respectively.
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The three glyceollin peaks were preceded by peak 15 with an m/z value of 339 in NI mode, which was tentatively assigned as glyceollidin I and II, assuming that both glyceollidin I (precursor of glyceollin I) and II (precursor of glyceollin II and III) are likely to be present in an extract containing the three glyceollins, and because no other peak with this m/z value was observed close to peak 15. The mass spectra of peaks 4, 15, 16, 18 and 19 measured in PI mode were all characterised by a predominant [M+H-H2O]+ ion and the less abundant [M+H]+ parent ion. Loss of water of glyceollins I-III in such ESI-MS1 spectra has been reported earlier [6, 21]. We also observed that glyceollidin I/II (15) and glycinol (4) lost a water molecule upon protonation in the PI mode. The hydroxyl groups on positions 3 (4, 15) and 9 (4, 15, 16, 18 and 19) are part of the conjugated system of the aromatic ring via hyper-conjugation, unlike the 6a-hydroxyl. Therefore, the 6a-hydroxyl might be released from the parent ion as a neutral water molecule upon protonation in PI mode.
The confirmation of the assignments of the glyceollins, glyceollidins and glycinol was performed by the analysis of their characteristic MSn fragmentations, obtained in NI mode. The nomenclature for RDA fragmentations suggested for isoflavones and pterocarpans was partly adopted from Ma et al. and Fabre et al. (Figure 3) [23, 24]. Fragmentation of the pterocarpans involves C-ring and/or D-ring cleavage, each resulting in different retro-Diels Alder (RDA) fragments. RDA pathways are proposed for glyceollin I degradation in NI mode (Figure 4). Upon fragmentation of the parent ion [M-H]-, several RDA fragments were formed by cleavage of the C-ring (2,4A-, 2,4B-), the D-ring (5,6A-, 5,6B-) or both C-and D-ring (2,3,7A-, 2,3,7B-). Although less common for glyceollins, a water loss of the parent ion [M-H-H2O]- might initiate the formation of different RDA fragments in MS2, i.e. -H2O1,4A-, -H2O 6,7A-, -H2O 0,4B- and -H2O 2,4B- +2H. Furthermore, the loss of a methyl radical was observed in the MS2 spectra of glycinol and glyceollidin I/II. The observation of a methyl radical upon fragmentation of flavonoids is indicative for the presence of a methoxy-group [24]. The absence of a methoxy group in both glycinol and glyceollidin I/II suggests that the methyl radical originates from either the prenyl chain (glyceollidin I/II) or the carbon 6 of the pterocarpan skeleton.
Figure 3. Nomenclature of the different retrocyclization cleavages, illustrated for an isoflavone (daidzein), adopted from Ma et al. and Fabre et al. [23, 24], and for a 6a-hydroxyl-pterocarpan (glycinol) and its parent ion after a water loss.
Prenylated isoflavonoids in Rhizopus spp. elicited soya bean seedlings
69
The fragmentation behaviour of glyceollin I can be extrapolated to the other pterocarpans, as can be seen from the formation of RDA fragments and their relative abundances observed in NI mode MS2 spectra (Table 2). The fragmentation pathways of glyceollins I-III (16, 18 and 19) were characterised by a predominant water loss [M-H-H2O]- in combination with RDA fragment 2,4B-. On the other hand, the MS2 spectra of glycinol (4) and glyceollidin I/II (15) were dominated by RDA fragments -H2O0,4B- and -H2O6,7A- and no RDA fragments originating from the fragmentation of parent ion [M-H]- were observed. This indicated that the presence of the pyran or furan ring predominantly resulted in RDA fragments that originate from the parent ion [M-H]- and to a lesser extent in -H2Ox,yRDA fragments, whereas their absence only results in -H2Ox,yRDA fragments (see Appendix VI). Apparently, a ring-closed prenyl chain inhibited further fragmentation of the [M-H-H2O]- fragment ion, the mechanism of which remains unclear. Table 2. MS2 product ions obtained from the [M-H]- parent ion of pterocarpans 4, 15, 16, 18 and 19.
Pterocarpan Glycinol Glyceollidin I/II Glyceollin I Glyceollin II Glyceollin III
[M-H]- 271 (1)a 339 (1) 337 (4) 337 (5) 337 (2)
[M-H-CH3]•-
256 (37) 324 (46) 322 (1) 322 (1) -
[M-H-H2O]- 253 (4) 321 (5) 319 (100) 319 (100) 319 (100)
[M-H-CO]- 243 (5) 311 (7) 309 (3) 309 (2) 309 (2)
[M-H-CO2]- 227 (29) 295 (20) 293 (37) 293 (36) 293 (9)
2,4A
- - - 187 (5) 187 (14) - 2,4
B- - - 149 (98) 149 (43) 149 (18)
2,3,7A
- - - 215 (3) 215 (3) 215 (1) 2,3,7
B- - - 121 (3) 121 (1) 121 (1)
5,6A
- - - 229 (3) - - 5,6
B- 109 (2) - 109 (1) - -
-H2O0,4
B- 161 (100) 161 (100) 161 (20) 161 (7) 161 (3)
-H2O1,4
A- 109 (2) 177 (5) 175 (4) 175 (3) -
-H2O6,7
A- 161 (100) 229 (8) 227 (1) 227 (5) 227 (1)
-H2O2,4
B- + 2H - 133 (2) - - -
a m/z (relative abundance)
Assignment of other prenylated isoflavonoids
An LC-MS-based method was employed to screen for the presence of novel prenylated
isoflavonoids in soya beans [25]. This method is based on characteristic neutral losses (56 u
alone, or in combination with 42 u), which are related to the specific fragmentation of the
prenyl-moiety in MS2 and MS3 spectra measured in PI mode. The ratio of the MSn
fragments corresponding to neutral losses of 42 u and 56 u, [M+H-C3H6]+ and [M+H-
C4H8]+, respectively, indicates whether the prenyl-moiety is a prenyl chain (42:56<1) or a
pyran ring (42:56>1). Furthermore, compounds that have an m/z value of less than 305
are assigned as non-prenylated.
Chapter 3
70
Figure 4. Proposed fragmentation pathways of glyceollin I in NI mode. The neutral losses are given between parentheses.
Prenylated isoflavonoids in Rhizopus spp. elicited soya bean seedlings
71
Figure 5. Screening results for neutral losses 42 u and 56 u in the PI mode MS2 spectra. RP-UHPLC-MS base peak chromatogram in positive mode (A) of the EtOH extract of germinated soya bean (Glycine max) in combination with Rhizopus microsporus var. oryzae. The MS chromatograms were screened for neutral losses in MS2 of 42 u (B) and 56 u (C). The numbers in the figure correspond to those given in Table 3.
Based on the analysis of the chromatograms of non-soaked, soaked and
Rhizopus-challenged soya bean seedling, 25 peaks showed a clear neutral loss of 56 u, alone or in combination with 42 u upon fragmentation in MS2 (Table 3 and Figure 5). Of these 25 peaks, several were eliminated due to the m/z < 305 criterion or the low relative abundance of the [M+H-C4H8]+ fragment ion. Twelve peaks (S7-15, S21-22 and S25) were assigned as prenylated flavonoids. MS3 spectra did not yield additional candidate peaks.
Chapter 3
72
Peaks S11-S15, S21, S22 and S25 could be tentatively assigned based on the predominant fragment ions in the MS2 and MS3 spectra. Peaks S13 and S15 both had an m/z value of 323 in PI mode. This is 68 u more than daidzein, indicating that S13 and S15 might be prenylated daidzein molecules. A comparison of the MS3 spectra of peaks S13 and S15 with the MS2 spectrum of daidzein (12) showed that the spectra were quite similar: Neutral losses calculated from the ions in the MS2 spectrum of daidzein were also found in the MS3 spectra of the [M+H-C4H8]+ fragments of S13 and S15 (see Appendix VII). The MS3 fragmentation of [M+H-C4H8]+ resulted for S13 in a predominant RDA fragment with an m/z value of 149. This is 12 u more than the 1,3A+ fragment from daidzein (m/z 137). The difference of 12 u between the MS2 fragments of daidzein and the MS3 fragments of both A-ring and B-ring prenylated daidzein can be accounted for the remaining CH-group after the degradation of the prenyl group. The 1,3A+ fragment from S13 with an m/z value of 149 is in fact -C4H8
1,3A+, suggesting that S13 is prenylated on the A-ring. For peak S15, the fragmentation of [M+H-C4H8]+ in MS3 resulted in the formation of the predominant RDA fragment 1,3A+ with an m/z value of 137, leading to the tentative assignment of this peak as a B-ring prenylated daidzein. This approach was also applied to the other prenylated isoflavonoids leading to the tentative assignment of these peaks as A-ring prenylated 2’-hydroxydaidzein (S11), B-ring prenylated glycitein (S12), A-ring prenylated 2’-hydroxygenistein (S14), A-ring prenylated genistein (S21), and B-ring prenylated genistein (S22). The fragmentation pathways for these novel prenylated flavonoids are proposed in Figure 6. Our study does not allow us to conclude on the position of the prenyl chain on the A-ring (6- or 8-position) or on the B-ring (2’- or 3’-positions). NMR spectroscopic analysis would be required to establish this. Several of these prenylated isoflavones have been isolated from other plant species belonging to the Leguminosae; Neobavaisoflavone, a B-ring prenylated daidzein, was isolated from Psoralea ssp. and Pueraria ssp.; Isowighteone, a B-ring prenylated genistein, has been isolated from Cajanus ssp. and Piscidia ssp.; Lupiwighteone and wighteone, both A-ring prenylated genistein molecules, and 2,3-dehydrokievitone and luteone, both A-ring prenylated 2’-hydroxygenistein molecules, were isolated from several Lupinus species [26-
31]. This is the first report on the occurrence of these prenylated isoflavonoids in soya. Peak S25 was tentatively assigned as a prenylated coumestrol. The assignment
was based on the comparison of the UV and MS spectra of S25 with those of the authentic standard of coumestrol, in a similar way as described for prenylated daidzein. Since the fragmentation of coumestrol did not result in cleavage of the C-ring, assignment of the prenyl group to specifically the A-ring or B-ring based on MS alone was not possible. However, the only coumestrol molecule with a prenyl chain isolated from Glycine max is phaseol, 4-prenyl-coumestrol [32]. Thereofore, S25 was tentatively assigned as phaseol. A rough estimation, based on expressing the peak areas as daidzein equivalents, indicated that approx 40% of the total isoflavonoids content in Rhizopus-challenged soya bean seedlings were prenylated pterocarpans and 7% were prenylated isoflavones and prenylated coumestan, whereas the remainder were isoflavones.
Prenylated isoflavonoids in Rhizopus spp. elicited soya bean seedlings
73
Table 3. Screening results for prenylated flavonoids in the MS chromatograms of the EtOH extracts of non-soaked, soaked and Rhizopus-challenged soya bean seedlings (Glycine max) in PI mode. (C – prenyl-chain; P – pyran ring; np – non-prenylated).
MS2
No† Rt
‡
(min)
Identification Extract* [M+H]+ R.A.
neutral
loss 42
R.A.
neutral
loss 56
Ratio
(42:56)
Prenyl
type
S1 6.28 Glycinol RS 255 n.a.
S2 6.54 n.d. RS 443 - <1 - n.a.
S3 8.52 Glycitein NS/SB/RS 285 n.a.
S4 8.86 Daidzein NS/SB/RS 255 n.a.
S5 9.04 2’-hydroxygenistein RS 285 n.a.
S6 11.39 Genistein NS/SB/RS 271 n.a.
S7 11.57 Glyceollidin I/II RS 323 2 100 0.0 C
S8 12.06 Glyceollin III RS 321 80 29 2.8 **
S9 12.39 Glyceollin II RS 321 100 34 2.9 P
S10 12.56 Glyceollin I RS 321 28 24 1.2 P
S11 13.83 A-ring prenylated 2’-hydroxydaidzein
RS 339 - 100 0.0 C
S12 14.64 B-ring prenylated glycitein
RS 353 - 100 0.0 C
S13 15.17 A-ring prenylated daidzein
RS 323 - 100 0.0 C
S14 15.33 A-ring prenylated 2’-hydroxygenistein
RS 355 - 100 0.0 C
S15 15.49 B-ring prenylated daidzein
RS 323 - 100 0.0 C
S16 15.92 n.d. RS 353 1 <1 - n.a.
S17 16.67 n.d. NS/SB/RS 267 n.a.
S18 16.91 n.d. RS 277 n.a.
S19 17.14 n.d. RS 279 n.a.
S20 17.57 n.d. RS 279 n.a.
S21 17.91 A-ring prenylated genistein
RS 339 - 100 0.0 C
S22 18.08 B-ring prenylated genistein
RS 339 <1 100 0.0 C
S23 18.22 n.d. RS 279 n.a.
S24 18.52 n.d. RS 279 n.a.
S25 18.99 4-prenyl-coumestrol RS 337 - 100 0.0 C
*NS – non-soaked beans; SB – soaked beans; RS – Rhizopus-challenged soya seedlings; R.A. – Relative abundance of ion within MS2 spectrum. ** Whereas glyceollins I and II are substituted with a pyran ring, glyceollin III has a furan ring; apparently the ratio (42:56) is similar for pyran and furan rings † the ‘S’ refers to a peak found with the screening method in an extract. ‡ The retenlon lme in MS has a delay of 0.20 ± 0.02 min compared to the UV retenlon lme (Rt). < 1 – Relative abundance of ion within MS2 spectrum is 0.5% to 1.0%. - not detected, relative abundance of ion within MS2 spectrum < 0.5%. n.d. – not determined; n.a. – not applicable.
Chapter 3
74
CONCLUSIONS Using LC-MS, three subclasses of prenylated isoflavonoids were found to accumulate in soya bean seedlings upon challenging by Rhizopus microsporus var. oryzae: Five prenylated pterocarpans, seven prenylated isoflavones and one prenylated coumestan. The prenylated isoflavonoids were tentatively assigned as A-ring and B-ring prenylated daidzein, A-ring prenylated 2’-hydroxydaidzein, B-ring prenylated glycitein, A-ring prenylated 2’-hydroxygenistein, A-ring and B-ring prenylated genistein and 4-prenyl-coumestrol. Furthermore, the fragmentation pathways of the 5 pterocarpans in NI mode were proposed, which involves the cleavage of the C ring and/or D ring. The presence of a ring-closed prenyl group affects the fragmentation pathway of the molecules in that the -H2Ox,yRDA fragments are more stable than in the absence of this group.
Figure 6. Proposed fragmentation pathways of tentatively assigned prenylated isoflavones in PI mode. The numbers inside the parentheses refer to the compounds in Tables 1 and 3. The prenyl groups on the 6, 8, 2’ and 3’-position are indicated by 6P, 8P, 2’P and 3’P, respectively.
Prenylated isoflavonoids in Rhizopus spp. elicited soya bean seedlings
75
ACKNOWLEDGEMENTS This work was financially supported by the Food & Nutrition Delta of the Ministry of Economic Affairs, The Netherlands. We would like to thank Dr. Ir. M.J.R. Nout and Dr. Ir. P.J. Roubos-van den Hil (Wageningen University, the Netherlands) for supplying the fungal spores and their advice and Dr. H.W.M. Hilhorst (Wageningen University, the Netherlands) for his assistance in setting up the germination experiments.
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[26] M. Stobiecki, P. Wojtaszek, Applications of gas-chromatography mass-spectrometry to the identification of isoflavonoids in lupine root extracts. Journal of Chromatography 1990, 508, 391-398.
[27] G. A. Lane, O. R. W. Sutherland, R. A. Skipp, Isoflavonoids as insect feeding deterrents and antifungal components from root of Lupinus angustifolius Journal of Chemical Ecology 1987, 13, 771-783.
[28] G. A. Lane, R. H. Newman, Isoflavones from Lupinus angustifolius root. Phytochemistry 1987, 26, 295-300.
[29] J. L. Ingham, S. Tahara, S. Shibaki, J. Mizutani, Isoflavonoids from the root bark of Piscidia erythrina
and a note on the structure of piscidone. Zeitschrift Fur Naturforschung 1989, 44c, 905-913. [30] Y. Hashidoko, S. Tahara, J. Mizutani, Isoflavonoids of yellow lupin 1. New complex isoflavones in the
root of yellow lupin (Lupinus luteus L. cv barpine). Agricultural and Biological Chemistry 1986, 50, 1797-1807.
[31] S. J. Dahiya, Reversed-phase high-performance liquid chromatography of cajanus cajan phytoalexins. Journal of Chromatography A 1987, 409, 355-359.
[32] P. Caballero, C. M. Smith, F. R. Fronczek, N. H. Fischer, Isoflavones from an insect-resistant variety of soybean and the molecular structure of afrormosin. Journal of Natural Products 1986, 49, 1126-1129.
Chapter 4
Agonistic and antagonistic estrogens in
licorice root (Glycyrrhiza glabra)
Based on: Rudy Simons, Jean-Paul Vincken, Loes A.M. Mol, Susan The, Toine F.H. Bovee,
Teus J.C. Luijendijk, Marian A. Verbruggen, Harry Gruppen, Agonistic and antagonistic
estrogens in licorice root (Glycyrrhiza glabra), Analytical and Bioanalytical Chemistry,
2011, in press
Chapter 4
78
ABSTRACT
The roots of licorice (Glycyrrhiza glabra) are a rich source of flavonoids, in particular prenylated flavonoids, such as the isoflavan glabridin and the isoflavene glabrene. Fractionation of an ethyl acetate extract from licorice root by centrifugal partitioning chromatography yielded 51 fractions, which were characterized by LC-MS and screened for activity in yeast estrogen bioassays. One third of the fractions displayed estrogenic activity towards either one or both estrogen receptor (ERα and ERβ). Glabrene-rich fractions displayed an estrogenic response, predominantly to the ERα. Surprisingly, glabridin did not exert agonistic activity to both ER subtypes. Several fractions displayed higher responses than the maximum response obtained with the reference compound, the natural hormone 17β-estradiol (E2). The estrogenic activities of all fractions, including this so-called superinduction, were clearly ER-mediated, as the estrogenic response was inhibited by 20-60% by known ER antagonists, and no activity was found in yeast cells that did not express the ERα or ERβ subtype. Prolonged exposure of the yeast to the estrogenic fractions that showed superinduction did, contrary to E2, not result in a decrease of the fluorescent response. Therefore, the superinduction was most likely the result of stabilization of the ER, yEGFP, or a combination of both. Most fractions displaying superinduction were rich in flavonoids with single prenylation. Glabridin displayed ERα-selective antagonism, similar to the ERα-selective antagonist RU 58668. Whereas glabridin was able to reduce the estrogenic response of E2 by approximately 80% at 6 × 10-6 M, glabrene-rich fractions only exhibited agonistic responses, preferentially on ERα.
Keywords: Prenylation, isoflavonoids, estrogen, antagonism, licorice
Agonistic and antagonistic estrogens in licorice root
79
INTRODUCTION
Flavonoids are a broad class of phenolic compounds mainly found in plants with a wide
range of bioactivities [1]. Prenylated flavonoids in particular are of interest with respect to
bioactivity, as prenylation is considered to modulate the responses towards the estrogen
receptor [1, 2]. Prenyl-substitution of the flavonoid subclasses flavones, flavanones and
flavonols has been linked to an increased affinity to the estrogen receptor α ERα [3-5], e.g.
prenylation of the 8-position of the flavanone naringenin resulted in a 200-1000 higher
estrogenic activity [6]. Furthermore, the prenylation of isoflavonoids has been suggested to
induce antagonistic activity when binding to ERα [5, 7, 8].
Licorice roots (Glycyrrhiza glabra) are a rich source of prenylated flavonoids. They
might offer opportunities for the development of new food supplements related to e.g.
the alleviation of osteoporosis and menopausal complaints [9]. Approximately 75
prenylated flavonoids have been identified, mainly belonging to isoflavans, isoflavenes
and flavanones [10, 11].
Previous studies have shown that licorice root extracts have estrogenic activity
towards the ERα and ERβ [12, 13]. The key estrogenic compounds isolated from G. glabra
were identified as glabrene and glabridin, both prenylated isoflavonoids [14, 15]. The
estrogen-like activities of both compounds have been established by means of
competitive ligand binding assays, in vitro cell assays, and in vivo animal models [16, 17]. It
has been demonstrated that glabrene and glabridin bind to the ER with EC50 values of 5
×10-5 M and 5×10-6 M, respectively. These values were obtained using an MCF-7 cell line
that is known to express the ERα type mainly (no detectable ERβ amounts on the protein
level), indicating that both compounds were agonists [18, 19]. However, the specific
estrogenic potencies of glabrene and glabridin towards ERα and ERβ, and their potential
antagonistic activities, have not yet been investigated. Such information is vital for
understanding their specific estrogenic activity in the human body.
The aim of the present study was to determine the predominant estrogenic
compounds of licorice roots that are active on both ER-subtypes, and investigate their
agonistic and antagonistic potencies. To this end, fractions of a licorice root extract
obtained by centrifugal partitioning chromatography were characterized by LC-MS and
subsequently screened for (anti)estrogenic activity using yeast estrogen bioassays.
Chapter 4
80
EXPERIMENTAL
Materials
The roots of Glycyrrhiza glabra, collected in Afghanistan, were provided by Frutarom US
(North Bergen, NJ, USA). Estradiol was purchased from Sigma Aldrich (St. Louis, MO, USA),
glabridin from Wako Chemicals GmbH (Neuss, Germany). RU 58668 and R,R-diethyl-THC
(R,R-THC) were purchased from Tocris Bioscience (Bristol, United Kingdom). AR grade n-
hexane, acetone and absolute ethanol and ULC/MS grade acetonitrile (ACN) were
purchased from Biosolve BV (Valkenswaard, The Netherlands). Water was prepared using
a Milli-Q water purification system (Millipore, Billerica, MA, USA). Dimethylsulfoxide
(DMSO) and all other chemicals were purchased from Merck (Darmstadt, Germany).
Preparation of licorice extract
The roots were milled with a ZM 200 Retsch Ultra centrifugal mill (Haan, Germany) using a
1 mm sieve. The root powder was extracted with ethyl acetate (EA) in a ratio of 1 to 25
(w/w) for 2 h at 40 °C under continuous stirring. The extract was obtained by pressing the
mixture with a Fischer Maschinenbau hydraulic press type HP 5M (Gemmrigheim,
Germany) under 40 bar for 1 h. The dried extract was obtained after evaporation of the EA
under reduced pressure at 40 °C.
CPC fractionation of licorice extract
Centrifugal partitioning chromatography (CPC) was performed using a thermostatted
Kromaton FCPC machine (Angers, France) connected to an Armen AP 100 (Chromtech,
Boronia, Victoria, Australia) plunger pump. The two-phase solvent system used consisted
of n-hexane/acetone/water in a ratio of 5:9:1 (v/v/v). It was equilibrated under stirring at
22 °C for at least one hour. Small-scale fractionations as part of the method development
were done with a 200 mL rotor in ascending mode (i.e. lower phase is stationary phase) at
22 °C, a rotation speed of 1000 rpm, and a flow rate of 10 mL/min. The volume of
displaced stationary phase was approximately 83 mL. Eighty five mg dried extract was
dissolved in a mixture of upper and lower phase, 4 mL of each phase. The fractionation
process was monitored using a Jasco UV-2075 UV detector equipped with a 1 mm
preparative cell at a wavelength of 330 nm (absorbance is expressed as relative response
to the highest peak).
For the actual fractionation of the licorice root extract, a 1000 mL rotor was used
(22 °C; rotation speed 1100 rpm; flow rate 25 mL/min). The volume of displaced stationary
phase in the 1000 mL rotor was approximately 625 mL. Seven hundred fifty mg dried
Agonistic and antagonistic estrogens in licorice root
81
extract was dissolved in 28 mL of a mixture of upper and lower phase (1:1). Seven
subsequent runs were performed that resulted in 51 fractions per run; the fraction size
was 50 mL. Based on the CPC UV profile, corresponding fractions were combined and
evaporated in combination with lyophilization, in order to remove solvents. The combined
fractions were resolubilized in absolute ethanol (EtOH) and stored at -20 °C. All samples
were thawed and centrifuged before analysis. Fractions collected were analyzed by
UHPLC-MS at a concentration of 1 mg/mL.
RP-UHPLC
Samples were analyzed using an Accela UHPLC system (Thermo Scientific, San Jose, CA,
USA) equipped with pump, autosampler and PDA detector. Samples (1 µL) were injected
onto an Acquity UPLC BEH C18 column (2.1 x 150 mm, 1.7 µm particle size) with an
Acquity UPLC BEH C18 Vanguard pre-column (2.1 x 5 mm, 1.7 µm particle size; Waters,
Milford, MA, USA). Eluents were water acidified with 0.1% (v/v) acetic acid (eluent A), and
acetonitrile (ACN) acidified with 0.1% (v/v) acetic acid (eluent B). The flow rate was 300
µL/min and the PDA detector was set to measure at a range of 205-400 nm. The following
elution profile was used: 0-18 min, linear gradient from 10%-100% (v/v) B; 18-22 min,
isocratic on 100% B; 22-23 min, linear gradient from 100%-10% B 23-25 min, isocratic on
10% B.
Electrospray ionization mass spectrometry (ESI-MS)
Mass spectrometric data were obtained by analyzing samples on an ion trap LTQ-XL
(Thermo Scientific) equipped with an ESI-MS probe coupled to the RP-UHPLC. Helium was
used as sheath gas and nitrogen as auxiliary gas. Data were collected over an m/z-range of
150-1500. Data dependent MSn analysis was performed with a normalized collision energy
of 35%. The MSn fragmentation was always performed on the most intense daughter ion
in the MSn-1 spectrum. Most settings were optimized via automatic tuning using “Tune
Plus” (Xcalibur 2.0.7, Thermo Scientific). To this end, the system was tuned with glabridin
in both positive ionization (PI) and negative ionization (NI) mode. In both modes, the ion
transfer tube temperature was 350 °C and the source voltage 4.8 kV. Data acquisition and
reprocessing were done with Xcalibur 2.0.7. Mass spectral data interpretation and peak
determination were performed with Mass Frontier 5.0 (Highchem, Bratislava, Slovakia).
Determination of estrogenic activity
The protocols for the yeast estrogen bioassays were adopted from Bovee et al. [20] with
slight modifications. The genetically modified yeast strains have a strong constitutive
Chapter 4
82
expression vector stably integrated in the genome to express either the human estrogen
receptor α (ERα) or the human estrogen receptor β (ERβ). The yeast genome also contains
a reporter construct. This reporter construct contains an inducible yeast enhanced green
fluorescent protein (yEGFP) regulated by the activation of a minimal promoter with
estrogen responsive elements (EREs). Cultures of the yeast estrogen biosensor with either
ERα or ERβ were grown overnight at 30 °C with shaking at 200 rpm. At the late log phase,
the cultures of both estrogen receptors were diluted in the selective minimal medium
(MM) supplemented with either leucine (ERα) or histidine (ERβ) to an OD value (630 nm)
of 0.05±0.01 (ERα) and 0.15±0.05 (ERβ). For exposure, 200 μL aliquots of these diluted
yeast cultures were combined with 2 μL of test compound or extract (in various
concentrations) in a 96-well plate to test the agonistic properties of these compounds.
DMSO (blank) and control samples containing 17β-estradiol (E2) or genistein dissolved in
DMSO were included in each experiment. Dilution series of each sample were prepared in
DMSO and the final concentration of DMSO in the assay did not exceed 1% (v/v). Each
sample concentration was assayed in triplicate. Exposure was performed for 24 h or 6 h
for the ERα or ERβ asssay, respectively, at 30 °C and orbital shaking at 200 rpm.
Fluorescence and OD were measured at 0 and 24 h for the ERα and 0 and 6-8 h
for the ERβ in a Tecan Infinite F500 (Männedorf, Switzerland), using an excitation filter of
485 nm (bandwidth 20 nm) and an emission filter of 535 nm (bandwidth 35 nm). The
fluorescence signals of the samples were corrected with the signal obtained with the
diluted yeast suspension at t 0 h (background signal). In order to check the viability of the
yeast in each well, the absorbance was measured at 630 nm. Each fraction was tested in a
concentration series ranging from 0.1 - 100 µg/mL. EC50 calculations were performed in
Sigma Plot (8.02, SPSS Inc.). In a number of cases, a concentration of 10 µg/mL for the ERα
and 3 µg/mL for the ERβ resulted in decreased yeast growth during incubation of more
than 50% during incubation due to cytotoxicity. This cytotoxicity could be due to the anti-
microbial properties of licorice root constituents as reported before [21, 22].
A dilution-series of estradiol and genistein were used as reference controls in this
bioassay. The EC50 values in the ERα bioassay were 0.86 nM and 1.73 µM for estradiol and
genistein, respectively, and 0.12 nM and 9.1 nM in the ERβ bioassay, respectively. All EC50
values were in line with those reported previously [20].
For the determination of ER antagonism, the yeast cells were exposed to the EC70
(ERα) or the EC90 (ERβ) of estradiol in combination with different dilutions of a test
compound or fraction (measured in triplicates). As a positive control, the yeast cells were
exposed to the EC70 of estradiol in combination with the known ERα antagonist RU 58688 [23]. For ERβ antagonism, the yeast cells were exposed to the EC90 of estradiol in
combination with R,R-THC, a known antagonist on the ERβ [24].
In addition to the yeasts expressing the yEGFP reporter gene in combination with
either ERα or ERβ, a third yeast strain was used that only contained the reporter gene but
not the vector with the ER. This yeast strain was used as a negative control.
Agonistic and antagonistic estrogens in licorice root
83
RESULTS AND DISCUSSION
Estrogenic activity of CPC fractions for ERαααα and ERββββ.
CPC fractionation of the licorice root extract resulted in 51 fractions (Figure 1). After the
51st fraction no UV response was observed anymore. Fractions F1 to 5, F6 to 21 and F22
to 51 comprised ~25, ~40 and ~35% DW of the total extraction yield, respectively (see
Appendix VIII). Most fractions showed some estrogenicity on both ERs, indicating the
presence of phytoestrogens (Table 1).
A compound is considered a phytoestrogen, when it activates the ER at
concentrations ≤104 times than that of estradiol (E2) [25]. The EC50 value of E2 towards the
ERα in the yeast assay was determined to be 1.0-1.6×10-9 M, which corresponds to 2.7-
4.4×10-4 µg/mL. Therefore, only CPC fractions giving a response above the EC50 at a
dilution below 3 µg/mL were indicated in as active towards ERα in Figure 1. The
application of this threshold value for the ERα resulted in 9 active fractions out of 51 (see
Table 1).
The EC50 value of E2 towards the ERβ ranged from 1.1×10-10 to 2.1×10-10 M,
corresponding to an EC50 of 3.2-5.9×10-5 µg/mL. Therefore, only CPC fractions giving a
response above the EC50 at a dilution below 0.3 µg/mL were indicated as active towards
ERβ in Figure 1. The application of this threshold value for the ERβ resulted in 12 active
fractions out of 51 (Table 1).
Figure 1. UV-profile of the licorice root extract fractionation by CPC. Estrogenically active fractions are indicated.
Table 1. Estrogenic response of CPC-fractions from the EA extract of licorice roots. The estrogenicity towards the ERα and the ERβ was measured at two
concentrations. The activity was standardized to the maximum induced response of 2 µM estradiol (100%). Values are the mean ± StDev (n=3). Estrogenic
fractions are marked
3 µg/mL 10 µg/mL 0.3 µg/mL 1.0 µg/mL 3 µg/mL 10 µg/mL 0.3 µg/mL 1.0 µg/mL
Fr1 ERα ERα ERβ ERβ Fr ERα ERα ERβ ERβ
E2 100 100 F26* 38.7(±1.9) 93.7(±6.1) 95.5(±3.4) 151.3(±15.1)
F1 n.d.2 n.d. 0.0(±2.0) 0.0(±2.0) F27* 36.6(±0.3) 90.8(±2.1) 74.6(±5.9) 176.5(± 11.8)
F2 n.d. 1.1(±1.2) 0.0(±6.5) 0.0(±4.9) F28* 19.4(±1.9) 48.2(±2.7) 68.7(±4.0) 104.4(± 8.3)
F3 n.d. 4.9(±0.7) 1.6(±1.5) 0.0(±4.5) F29* 19.9(±1.3) 23.0(±3.9) 57.2(±12.5) 93.5(±0.6)
F4 n.d. 7.8(±0.8) 13.4(±5.0) 22.4(±7.7) F30* 23.8(±2.7) 22.4(±1.8) 57.3(±1.1) 48.2(±2.8)
F5 3.0(±2.3) 7.8(±0.3) 12.0(±5.1) 19.5(±2.2) F31 25.0(±1.5) 34.2(±1.9) 25.8(±8.5) 40.5(±17.8)
F6 n.d. n.d. n.d. n.d. F32* 26.8(±2.3) 34.5(±2.0) 57.4(±4.7) 47.4(±10.7)
F7 17.1(±3.0) 21.0(±2.7) 21.4(±5.6) 12.2(±2.2)† F33* 32.0(±1.4) 38.0(±0.4) 50.2(±9.5) 36.0(±3.2)
F8 10.5(±2.2) 21.8(±6.3) 17.2(±2.3) 58.3(±3.4) F34 29.6(±5.3) 44.9(±1.3) 27.7(±8.8) 46.1(±5.2)
F9 13.3(±1.5) 7.1(±6.4) 37.8(±10.0) 49.1(±21.5) F35 30.8(±3.1) 45.5(±4.2) 43.6(±7.6) 37.5(±21.0)
F10 9.7(±3.3) 9.3(±2.0) 23.0(±9.5) 57.3(±12.9) F36 42.8(±2.3) 42.9(±6.5) 19.9(±2.3) 45.3(±2.5)
F11 n.d. n.d. 46.3(±12.0) n.d. † F37 13.6(±1.6) 25.3(±2.5) 24.8(±2.6) 35.1(±9.9)
F12 15.6(±2.9) 15.7(±3.2)† 30.7(±0.1) 77.2(±7.3) F38 14.8(±1.7) 28.8(±0.5) 14.2(±6.8) 30.8(±15.4)
F13 n.d. n.d. 40.3(±5.0) 2.4(±4.9)† F39 19.8(±1.6) 32.4(±2.6) 21.1(±4.4) 45.6(±14.7)
F14 4.8(±4.7) n.d. 42.7(±6.6) 73.3(± 4.1) F40 25.6(±4.3) 33.9(±2.3) 26.5(±5.5) 35.4(±17.4)
F15 n.d. n.d. 28.4(±7.1) 77.7(± 1.8) F41* 159.9(±9.5) 186.9(±15.0) 28.3(±4.9) 34.9(±13.5)
F16* 74.8(±4.0) 20.4(±28.9)† 40.7(±1.0) 62.6(± 2.1) F42 21.6(±6.0) 26.2(±2.2) 19.8(±11.0) 53.8(±25.5)
F17* 109.1(±2.3) 49.3(±4.4)† 36.7(±9.5) 88.8(± 8.2) F43 26.5(±1.8) 24.7(±4.3) 25.4(±1.2) 38.9(±20.0)
F18* 105.3(±6.2) 104.6(±10.5)† 57.1(±9.4) 98.8(± 15.1) F44 18.9(±1.9) 24.6(±1.5) 22.7(±7.3) 33.8(±12.2)
F19* 89.1(±4.2) 131.2(±16.2) 48.3(±4.6) 123.8(±17.1) F45 20.9(±1.5) 21.0(±4.3) 19.6(±3.4) 66.3(±15.1)
F20* 60.2(±3.1) 140.5(±10.7) 60.5(±14.5) 116.7(±14.3) F46 16.9(±1.2) 20.5(±3.2) 18.0(±11.6) 73.8(±7.3)
F21* 71.9(±8.4) 125.3(±1.2) 45.2(±3.8) 145.9(±25.2) F47 21.2(±2.9) 23.3(±4.2) 31.0(±5.1) 79.4(±6.1)
F22* 101.4(±11.6) 134.1(±2.7) 87.0(±10.3) 144.4(±10.7) F48 23.2(±4.6) 21.7(±2.4) 26.8(±2.5) 49.1(±17.8)
F23 16.0(±0.9) 83.0(±9.9) 26.3(±8.7) 84.8(±7.0) F49 26.4(±0.6) 26.7(±0.3) 16.5(±3.0) 82.9(±4.9)
F24* 79.6(±1.6) 156.3(±14.4) 103.1(±2.3) 159.3(±23.1) F50 18.8(±2.7) 16.7(±3.2) 7.3(±9.3) 41.3(±3.1)
F25* 44.2(±5.7) 108.6(±3.9) 97.9(±10.6) 172.8(±20.7) F51 22.0(±3.2) 15.6(±2.5) 18.8(±2.7) 40.5(±18.3) 1 Fraction; 2 n.d. –not detected (estrogenicity values were zero or slightly negative) * Estrogenically active; † inhibited yeast growth due to cytotoxicity
Agonistic and antagonistic estrogens in licorice root
85
The screening for estrogenicity of the CPC-fractions on both receptor subtypes
showed that the estrogenic response of several fractions substantially exceeded the
maximum response of E2 (Table 1). This phenomenon has been referred to as
superinduction [26]. In our study, this superinduction was observed with both receptors
and appeared more pronounced for ERβ. The mechanism that leads to superinduction is
not well understood, but sometimes occurs with colored extracts. Such colored extracts
can disturb the fluorescent measurement, as due to a decrease of the pH during the
exposure period, the color can change as well.
To determine whether fractions gave an increased fluorescent response as a
result of acidification (change of pH 5.0 to pH 2.9) of the culture medium due to yeast
growth, six representative fractions (F4, F13, F22, F27, F30 and F44), with no, moderate or
high estrogenic activity, were measured at different pH values in the absence of yeast. No
altered fluorescent signals were observed compared to the blank, showing that the
observed superinduction was not related to altered fluorescent signals due to a drop in
pH.
In the next series of experiments, two subtype selective antagonists were used to
determine whether the observed estrogenic activities, including the superinduction, were
ER-mediated. First, RU 58668 (ERα-selective) [27] and R,R-THC (ERβ-selective) [24] were
tested in the yeast estrogen bioassays to confirm their antagonistic properties. Co-
incubation of E2 and RU 58668 showed that 6×10-6 M RU 58668 was able to decrease the
E2-induced response of ERα by ~60% (Figure 2A). RU 58668 itself showed a weak agonistic
activity of ~12% in concentrations above 1×10-5 M. RU 58668 is known as a pure ERα
antagonist, but its weak agonistic activity in the yeast estrogen bioassay with ERα is not
expected, as several other 11β-analogues of E2 were shown to be selective estrogen
receptor modulators (SERM) with both agonistic and antagonistic activity on the ERα in
the same yeast estrogen bioassay [23].
Table 2. Inhibition of estrogenic response of representative CPC-fractions by addition of a subtype-specific antagonist. Inhibition of the estrogenic response of 6 representative CPC fractions were determined by co-
incubation at 1 µg/mL with 6×10-6 M RU 58668 for the ERα, and 1 µg/mL with 2×10-8 M R,R-THC for the ERβ.
Values are the mean ± StDev (n=3).
Estrogenic
activity ERα
Inhibition by
RU 58668
Estrogenic
activity ERβ
Inhibition by
R,R-THC
E2 100 60% 100 55%
F4 n.d. - 13.4(±5.0) 70%
F13 n.d. - 40.3(±5.0) 37%
F22 101.4(±11.6) 52% 87.0(±10.3) 34%
F27 36.6(±0.3) 21% 74.6(±5.9) 61%
F30 23.8(±2.7) 21% 57.3(±1.1) 48%
F44 18.9(±1.9) 30% 22.7(±7.3) 70%
Chapter 4
86
Co-incubation of E2 and R,R-THC showed that 2×10-7 M R,R-THC was able to
decrease the E2-induced response of ERβ by ~55% (Figure 2B). Meyers and co-workers
reported an inhibition of ~100% at similar concentrations while using mammalian cell
based assays [24].
To determine whether the observed estrogenic responses of the fractions were
ER-mediated, selected fractions were co-incubated with the subtype-selective antagonists.
Besides, the fractions were tested in the yeast strain that expresses no estrogen receptor
and only contains the reporter construct. In all estrogen active fractions, the responses
were inhibited by either RU 58668 or R,R-THC and the fluorescence response was reduced
by up to 70%. This confirms that the estrogenic responses caused by the fractions on both
receptors were ER-mediated (Table 2). Also the controls with the yeast strain expressing
no ER confirmed that the observed responses were ER-mediated, as no fluorescent signal
was observed for any of the fractions or E2.
Superinduction by stabilization of ER-mediated response
The phenomenon of superinduction has been previously observed in several assay types
and the superinduction caused by genistein in human U2OS bone cells transfected with
the ERα and a luciferase reporter gene was intensively investigated [26]. It was concluded
that this superinduction was caused by a post-translational stabilization of the firefly
luciferase reporter enzyme by genistein and not by stabilization of the ERα. To verify the
hypothesis that superinduction in the yeast was caused by the stabilization of the ER
and/or the yEGFP, the yeast expressing ERβ was co-incubated with E2, genistein or the
representative fractions (F4, F13, F22, F27, F30 and F44) mentioned before. The
estrogenic responses were measured after 6 and 24 h (Figure 3). After 6 h, both E2 and
genistein showed the maximum estrogenic response, but as expected the estrogenic
response of E2 completely disappeared after 24 h. Also, the response of genistein
completely disappeared, whereas the estrogenic response of the fractions was similar or
even higher compared to their response measured after 6 h. This strongly indicates that
the responses, including the superinduction, of the fractions were stabilized. Our results
do not allow speculation on whether the ER, the yEGFP or both proteins were stabilized,
but the observed estrogenic responses were without doubt ER-mediated.
Agonistic and antagonistic estrogens in licorice root
87
Figure 2. Transcription activation by ERα (A) and ERβ (B) in response to E2 and sub-type selective antagonists
RU58668 (ERα) and R,R-THC (ERβ). The antagonistic activity of both receptor-specific antagonists were assayed in
the presence of the EC70 (0.8 nM) and EC90 (0.2 nM) E2 for ERα and ERβ, respectively. Both graphs were
normalized to E2. The response of R,R-THC on ERβ for every concentration was lower than the minimum response of E2. This was corrected by normalizing the lowest concentration of R,R-THC to 0%. Values are the
mean ± StDev (n=3).
Chapter 4
88
LC-MS characterization of licorice fractions
Because the estrogenic responses were ER-mediated, the licorice fractions were subjected
to characterization by LC-MS. Fractions F6-21 contained the main flavonoids previously
annotated in the EA extract of licorice roots (Table 3, Figure 4) [11]. These flavonoids have
been annotated based on UV and MSn spectra and, if possible, compared to spectra
published in the literature. The estrogenic activity of a number of active fractions (F20-
22,24-30,32-33,42) could not be traced back to individual components (see Appendix VIII).
Compositional analysis by LC-MS showed that the active fractions were complex mixtures,
indicating that further purification of CPC fractions is prerequisite for the identification of
the estrogenic compounds. In most cases, it was not possible to assign the identity of the
predominant peaks by UV and MSn. Furthermore, in most fractions the majority of the
compounds were prenylated, which might suggest a correlation between prenylation and
superinduction (Table 3, Appendix VIII).
Fractions F16-20, rich in glabrene, showed a predominant estrogenic activity on
the ERα. This is in agreement with the fact that glabrene is considered one of the principal
estrogenic components of licorice root. The estrogenic potency of glabrene for both ER-
subtypes, however, has not yet been established. Our results indicate that the glabrene-
rich fractions had a particularly high response towards ERα. Whereas phytoestrogens
generally have a more pronounced affinity to the ERβ compared to the ERα, the glabrene-
rich fractions showed the opposite behavior, similar to that of 8-prenyl naringenin [27].
Figure 3. Stabilizing effect of E2, genistein and several fractions obtained from the licorice root extract on the
relative activity measured after 6 and 24 h in the ERβ assay. E2, 2 × 10-10 M estradiol; Gen, 2 × 10-7 M genistein; F4
to F44, licorice root fractions obtained by CPC measured at 0.3 µg/mL. *, negative response.
Table 3. Fractions generated by CPC-fractionation and the presence of the main flavonoids in each fraction determined by UHPLC-MS.
1 4’-O-methyl-glabridin. 2 3’-hydroxy-4’-O-methyl-glabridin.
Fraction 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Yield (mg) 110 141 97 136 108 126 211 345 254 156 66 176 157 115 37
Number of peaks 20 6 12 25 22 18 7 4 6 6 15 14 17 30 42
Of which are:
- non-prenylated 3 1 2 4 2 1 0 0 0 0 0 1 2 4 9
- prenylated 17 5 10 21 20 17 7 4 6 6 15 13 15 26 33
Single prenylated 13 3 7 9 6 8 4 1 2 3 11 11 13 18 22
-Chain 7 2 4 6 4 5 2 0 1 2 7 5 9 13 21
-Pyran 6 1 3 3 2 3 2 1 1 1 4 6 4 5 1
Double prenylated 4 2 3 12 14 9 3 3 4 3 4 2 2 8 11
Characterization
Glabrene ++ ++ ++ ++ +
Glabrone + + ++ +
Glabridin + + ++ ++ + + +
Glabrol + ++ ++ + +
3’-OH-4’OMe-G1 + + ++ ++
4’-OMe-G2 + + ++ +
Hispaglabridin A + ++ ++
Hispaglabridin B ++ ++ +
Chapter 4
90
In addition to glabrene, the estrogenic activity of licorice roots extract has been
ascribed to the presence of glabridin and its derivatives. Despite the abundance of
glabridin in F11-15, no significant estrogenic response on both ER subtypes was observed.
EC50 values of 5×10-6 M have been reported for glabridin, using different mammalian
proliferation assays [15-17]. Furthermore, several glabridin derivatives were shown to be
moderately estrogenic compared to glabridin [14]. In our study, the pure reference
standard of glabridin did not exert any estrogenic response in a concentration range of
1×10-7 to 1×10-4 M towards both ER subtypes (data not shown). In concentrations above
1×10-4 M glabridin was toxic to the yeast cells. Because glabridin had been shown to
interact with the ERs, and because it is known that different ER-based bioassays can
generate different output, glabridin as well as the glabrene-rich fraction F18 (due to the
lack of a glabrene reference standard) were tested for their antagonistic properties.
Figure 4. Main flavonoids identified in CPC fractions 6-21 obtained from the EA extract of licorice root.
Agonistic and antagonistic estrogens in licorice root
91
Antagonistic activity of glabridin and glabrene
Prenylation of isoflavonoids has been suggested to induce antagonism towards the ERα [5,
7, 8]. The glabrene-rich fraction F18 did not show antagonistic activity on both ER subtypes
(no further data shown), but increased the estrogenic response upon co-exposure with E2,
confirming its agonistic character.
The reference standard of glabridin did not have antagonistic properties towards
the ERβ (data not shown), but was shown to be an ERα-selective antagonist (Figure 5). At
a concentration of 6×10-6 M, glabridin was able to inhibit the E2 response by ~80% without
being toxic towards the yeast cells. The agonistic activity of glabridin in the MCF-7
proliferation assay, in in vivo animal models, and the ERα-selective antagonistic activity in
the yeast estrogen bioassay might imply that glabridin acts as a SERM. The estrogenic
activity of glabridin is similar to kievitone and phaseollin, a prenyl-chain substituted
isoflavanone and a pyran-ring substituted pterocarpan, respectively. Both kievitone and
phaseollin displayed agonistic activity in the MCF-7 proliferation assay and in the human
HEK 293 transactivation assay, but were antagonistic in the MCF-7 colony formation assay [28]. The assay-dependent mode of action of glabridin has also been observed with other
compounds. For example, both tamoxifen and 4-hydroxy-tamoxifen act as SERMs,
displaying both estrogenic and anti-estrogenic activities in mammalian breast and
endometrial cells, act as agonists in yeast estrogen bioassays [27]. As mentioned before, LC-
MS characterization showed that fractions F6-15 were rich in glabridin derivatives. These
compounds share prenyl-substitution on the A-ring with a pyran ring (Table 3, Figure 4). It
will be worthwhile to also test the purified glabridin derivatives for antagonistic activity in
the yeast estrogen bioassay.
Figure 5. Antagonistic activity of glabridin on the estrogenic response in the ERα yeast estrogen bioassay.
Chapter 4
92
ACKNOWLEDGEMENTS
This work was financially supported by the Food & Nutrition Delta of the Ministry of
Economic Affairs, The Netherlands. We would like to thank Daniel Piscitello, Florian
Wiegand and Miroslav Bolardt (Frutarom Switzerland Ltd., Wädenswil, Switzerland) for
their assistance in preparing the licorice extract and RIKILT-Institute of Food Safety for the
yeast estrogen bioassays.
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[14] S. Tamir, M. Eizenberg, D. Somjen, S. Izrael, J. Vaya, Estrogen-like activity of glabrene and other constituents isolated from licorice root. Journal of Steroid Biochemistry and Molecular Biology 2001, 78, 291-298.
[15] S. Tamir, M. Eizenberg, D. Somjen, N. Stern, R. Shelach, A. Kaye, J. Vaya, Estrogenic and antiproliferative properties of glabridin from licorice in human breast cancer cells. Cancer Research
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Chapter 4
94
Chapter 5
Increasing soya isoflavonoid content and
diversity by simultaneous malting and
challenging by a fungus to modulate
estrogenicity
Based on: Rudy Simons, Jean-Paul Vincken, Nikolaos Roidos, Toine F.H. Bovee, Martijn van
Iersel, Marian A. Verbruggen, Harry Gruppen, Increasing soy isoflavonoid content and
diversity by simultaneous malting and challenging by a fungus to modulate estrogenicity,
Journal of Agricultural and Food Chemistry, 2011, in press
Chapter 5
96
ABSTRACT
Soybeans were germinated on kilogram-scale, by application of malting technology used
in the brewing industry, and concomitantly challenged with Rhizopus microsporus var.
oryzae. In a time-course experiment, samples were taken every 24 h for 10 days and the
isoflavonoid profile was analyzed by RP-UHPLC-MS. Upon induction with R. microsporus,
the isoflavonoid composition changed drastically with the formation of phytoalexins
belonging to the subclasses of the pterocarpans and coumestans, and by prenylation of
the various isoflavonoids. The pterocarpan content stabilized at 2.24 mg daidzein
equivalents (DE) per g after ~9 days. The levels of the less common glyceofuran, glyceollin
IV and VI ranged from 0.18 to 0.35 mg DE/g, and were comparable to those of the more
commonly reported glyceollins I, II and III (0.22-0.32 mg DE/g) and glycinol (0.42 mg DE/g).
The content of prenylated isoflavones after the induction process was 0.30 mg DE/g. The
total isoflavonoid content increased by a factor 10-12 on DW basis after 9 days, which
suggested to be ascribable to de novo synthesis. These changes were accompanied by a
gradual increase in agonistic activity of the extracts towards both the estrogen receptor α
(ERα) and ERβ during the 10-day induction, with a more pronounced activity towards ERβ.
Thus, the induction process yielded a completely different spectrum of isoflavonoids, with
a much higher bioactivity towards the estrogen receptors. This, together with the over 10-
fold increase in potential bioactives, offers promising perspectives for producing more,
novel, and higher potency nutraceuticals by malting under stressed conditions.
Keywords: Prenylation, isoflavonoids, soya, estrogenicity, phytoalexins, antagonism
Increasing the isoflavonoid content and diversity of soya
97
INTRODUCTION
The Leguminosae (or Fabaceae) is an economically important family of flowering plants. It
comprises many edible legumes such as soya beans (Glycine max), beans (Phaseolus ssp.),
peas (Pisum sativum), chickpeas (Cicer arietinum), alfalfa (Medicago sativa), peanut
(Arachis hypogaea) and licorice (Glycyrrhiza glabra). Many edible legumes share the
feature of belonging to the subfamily of Papilionoideae. All species within the subfamily
Papilionoideae have in common that they accumulate isoflavonoids, a subclass of
flavonoids [1]. Soybean is considered to be one of the richest sources of isoflavones, a
subclass of isoflavonoids. The three main isoflavones in soya are daidzein, genistein and
glycitein. These isoflavones are present as aglycone, glucoside, acetyl-glucoside or
malonyl-glucoside [2]. Soya isoflavones are known for their weak estrogenic properties and
it is assumed that soya consumption is associated with health-beneficial effects towards
hormone-related conditions such as menopausal complaints and osteoporosis [3].
Isoflavones exert their in vitro estrogenicity by binding to the estrogen receptor (ER) and
are, therefore, often referred to as phytoestrogens.
Application of stress to soya during germination is known to induce changes in
the isoflavonoid profile, i.e. the formation of novel isoflavonoids, also known as
phytoalexins. Phytoalexins are defined as low molecular-weight, anti-microbial
compounds that are synthesized and accumulated in plants in response to e.g. pathogen
attack or application of chemical stimuli [4]. Soy phytoalexins comprise pterocarpans,
coumestans and isoflavones [5]. The formation of coumestans and pterocarpans is thought
to result from the conversion of daidzein into coumestrol and glycinol, respectively [6].
Prenylation is a common feature to these phytoalexins. Coumestrol can be converted into
phaseol by enzyme-catalysed substitution with a prenyl chain on the 4-position. Glycinol
can be enzymatically substituted with a prenyl chain to form glyceollidins I and II, which
can subsequently ring-closed into a pyran ring to form glyceollins I-III by the action of a
cyclase [4, 6, 7]. Furthermore, the occurrence of prenylated isoflavones in Rhizopus-
challenged soya seedlings has been reported recently [5].
Although the estrogenic activities of several soya phytoalexins have been
described, surprisingly neither the formation of phytoalexins nor the changes in estrogenic
activity in response to the altered isoflavonoid composition has been quantitatively
monitored in time. In this study, soya beans were germinated in the presence of the food-
grade fungus Rhizopus microsporus var. oryzae. Subsequently, both their isoflavonoid
profile and estrogenic activity towards ERα and ERβ were assessed during 10 days with 24
h intervals. The treatment was performed on kilogram-scale with malting technology
commonly employed in the brewing industry. The aim of this study is to quantify the
changes in isoflavonoid composition and the concomitant changes in the estrogenicity by
means of a yeast estrogen bioassay [8].
Chapter 5
98
EXPERIMENTAL
Materials
Soya beans, Glycine max (L.) Merrill, were provided by Frutarom Belgium (Londerzeel,
Belgium). The authentic standards of daidzein, genistein, coumestrol and estradiol (E2)
were purchased from Sigma Aldrich (St. Louis, MO, USA), both R,R-THC and RU 58668
were purchased from Tocris Bioscience (Bristol, United Kingdom). UHPLC/MS grade
acetonitrile (ACN) was purchased from Biosolve BV (Valkenswaard, The Netherlands).
Water was prepared using a Milli-Q water purification system (Millipore, Billerica, MA,
USA). All other chemicals were purchased from Merck (Darmstadt, Germany).
The fungus, Rhizopus microsporus var. oryzae (LU 581), was purchased from the
Fungal Biodiversity Centre CBS (Utrecht, The Netherlands).
Rhizopus-challenged germination of soya beans
The soya beans were subjected to a controlled malting protocol. This treatment consists of
3 stages: soaking, germination and challenging by Rhizopus, and is further referred to as
induction. All stages were performed in the absence of light. Samples were collected every
24 h and directly stored at -20 °C.
Soya beans were selected by visual screening. Damaged beans and debris were
discarded (2-3% w/w). Before starting the treatment, surface-sterilization was performed
by soaking the soya beans in a 1% (w/v) hypochlorite solution (5 L/kg beans) for 1 h at 20
°C. After surface-sterilization the soya beans were rinsed at least 3 times with (tap) water
(5 L/kg beans). The treatment was performed in a Joe White micro-malting system unit
90-102 (Perth, Australia) comprising 16 separate compartments. Compartments 1 to 3 and
14 to 16 were not used for the treatment. The micro-malting system was cleaned and
surface-sterilized before use, according to a cleaning in place protocol with the application
of the commercial cleaning product Chlorosept (Sopura S.A., Courcelles, Belgium). For
each time course experiment, a total of 4.0 kg of non-soaked soya beans was divided into
10 portions of 400 g each. The portions were distributed over compartments 4 to 13.
In the first stage, the beans were soaked for 24 h at 22 °C (air flow parameters:
30% air (100% = 3000 rpm of ventilator); 85% recirculation, 15% fresh air). After soaking,
the soaking water was drained, and the beans entered the second stage in which they
were germinated for 48 h at 30 °C under 100% RH (30% air; 85% recirculation). The
germination stage was followed by a third stage in which the soya seedlings were
inoculated with a spore suspension of R. microsporus. After inoculation, the germination
was continued for another 7 days at 30 °C, with adjusted air flow parameters (40% air;
25% recirculation).
Increasing the isoflavonoid content and diversity of soya
99
For the fungal inoculation, a sporangiospore suspension was prepared by
scraping off the sporangia from pure slant cultures of R. microsporus, grown on malt
extract agar (CM59; Oxoid, Basingstoke, UK) for 7 days at 30 °C. Subsequently, the
sporangia were suspended in sterile Milli-Q water with 0.85% (w/v) NaCl (108 CFU/mL).
The beans were inoculated with the sporangiospore suspension (0.2 mL/g) by gently
distributing the suspension manually through the germinated seeds.
Soya seedling extraction
The frozen samples were freeze-dried and subsequently milled in a Retch Ultra centrifugal
mill ZM 200 (Haan, Germany) using a 0.5 mm sieve. Ten grams of soya bean powder was
defatted with 250 mL hexane under continuous reflux conditions for 4 h. Defatted powder
(0.5 g) was subsequently extracted with 50 mL of EtOH by sonication (20 min) and shaking
(20 min; 250 rpm) at 20 oC in order to extract the isoflavonoids. The suspension was
centrifuged (2500 g, 10 min at 20 °C) and the pellet was rinsed with 10 mL of EtOH. This
procedure was repeated four times and the isoflavonoid contents of the supernatants was
monitored by UV absorption at 280 nm to ensure that all isoflavonoids were extracted.
The first four extracts and rinsing fractions were pooled, as the fifth supernatant did not
contain isoflavonoids, i.e. did not show any UV absorption at 280 nm. The ethanol was
removed under reduced pressure by a Savant ISS-110 SpeedVac concentrator (Thermo
Fisher Scientific, Waltham, USA). The dried extracts were dissolved in EtOH to a
concentration of 10 mg/mL and were stored at -20 oC. Prior to analysis, these stock
solutions were thawed at room temperature (RT), vortexed and centrifuged (5 min; 14000
rpm, RT).
RP-UHPLC-MS analysis
Samples were analyzed on a Thermo Accela UHPLC system (San Jose, CA, USA) equipped
with pump, autosampler and PDA detector. Samples (1 µL) were injected on an Acquity
UPLC BEH shield RP18 column (2.1 x 150 mm, 1.7 µm particle size) with an Acquity UPLC
shield RP18 Vanguard pre-column (2.1 x 5 mm, 1.7 µm particle size; Waters, Milford, MA,
USA). Water acidified with 0.1% (v/v) acetic acid, eluent A, and ACN acidified with 0.1%
(v/v) acetic acid, eluent B, were used as eluents. The flow rate was 300 µL/min, the
column temperature was controlled at 35 °C, and the PDA detector was set to measure at
a range of 200-400 nm. The following elution profile was used: 0-2 min, linear gradient
from 10%-25% (v/v) B; 2-9 min, linear gradient from 25%-50% (v/v) B; 9-12 min, isocratic
on 50% B; 12-22 min, linear gradient from 50%-100% (v/v) B; 22-24 min, isocratic on 100%
B; 24-25 min, linear gradient from 100%-10% (v/v) B; 25-27 min, isocratic on 10% (v/v) B.
Mass spectrometric data were obtained by analyzing samples on a LTQ-XL (Thermo
Chapter 5
100
Scientific) equipped with an ESI-MS probe coupled to the RP-UHPLC. Mass spectrometric
analysis in both positive (PI) and negative ion (NI) mode (data acquisition and
reprocessing) was performed as described previously [5]. Due to the lack of commercially
available references, daidzein was used as a generic standard to quantify the amounts of
isoflavonoids expressed as mg daidzein equivalents per g dry weight (mg DE/g). The
quantification was performed at 280 nm by means of Xcalibur (version 2.0.7, Thermo
Scientific).
Determination of estrogenic activity
The protocol for the yeast-based estrogen bioassay measurements was adopted from
Bovee et al. [8] with slight modifications. The yeast for this assay is genetically modified. It
has a strong constitutive expression vector, stably integrated in the genome, to express
either the human estrogen receptor α (ERα) or the human estrogen receptor β (ERβ). The
yeast genome also contains a reporter construct, with an inducible yeast-enhanced
fluorescent protein (yEGFP) regulated by the activation of a minimal promoter with
estrogen responsive elements (EREs). Cultures of the yeast estrogen biosensor with either
ERα or ERβ were grown overnight at 30 °C with shaking at 200 rpm. At the late log phase,
the cultures of both estrogen receptors were diluted in selective minimal medium (MM)
supplemented with either leucine (ERα) or histidine (ERβ) to an OD value (630 nm)
between 0.04 and 0.06 (ERα) and 0.1-0.2 (ERβ). For exposure, 200-μL aliquots of this
diluted yeast culture were combined with 2 μL of test compound or extract (in various
concentrations) in a 96-well plate to test the agonistic properties of these compounds.
DMSO (blank) and control samples containing 17β-estradiol (E2) or genistein dissolved in
DMSO were included in each experiment. Dilution series of each sample were prepared in
DMSO and the final concentration of DMSO in the assay did not exceed 1% (v/v). Each
sample concentration was assayed in triplicate. Exposure was performed for 24 h and 6 h
for the ERα and ERβ, respectively, in an orbital shaker (200 rpm; 30 °C).
Fluorescence and OD were measured at 0 and 24 h for the ERα and 0 and 6-8 h
for the ERβ in a Tecan Infinite F500 (Männedorf, Switzerland) using an excitation filter of
485 nm (bandwidth 20 nm) and an emission filter of 535 nm (bandwidth 35 nm). The
fluorescence signal of the samples was corrected with the signals obtained for the
background signal of the culture medium with yeast. In order to verify the viability of the
yeast in each well, the absorbance was measured at 630 nm. Each extract of the different
time points in the treatment of a representative Rhizopus-challenged germination
experiment was measured at 4 different dilutions, and every dilution was measured in
triplicates: 1, 3, 10 and 30 µg/mL in both the ERα and ERβ bioassay. EC50 calculations were
performed in Sigma Plot (8.02, SPSS Inc.). The yeast-based assay was validated with a
dilution-series of reference compounds, estradiol and genistein. The EC50 values in the ERα
bioassay were 0.86 nM and 1.73 µM for estradiol and genistein, respectively, and 0.12 nM
Increasing the isoflavonoid content and diversity of soya
101
and 9.1 nM in the ERβ bioassay. All EC50 values were in line with those reported in
literature [8].
For the determination of the potential antagonistic properties of the soya
extracts towards ERα and β, the yeast cells were exposed to the EC70 or EC90 of estradiol,
respectively, in combination with the 4 different extract concentrations (1, 3, 10 and 30
µg/mL; each measured in triplicates) of the same representative Rhizopus-challenged
germination experiment. For each ER antagonist test, a reference compound was used as
a positive control for antagonism, RU 58688 for ERα [9], and R,R-THC for ERβ [10]. All
experiments were performed in triplicates.
RESULTS
Growth parameters of soya seedlings
The percentage of germination of the soya beans and the average root length were
determined on 20 randomly picked seedlings from each of the 24 h interval-samples. Root
length was determined on the main root, excluding possible side-roots. Germinated beans
were defined as beans of which the root had a minimal length of 2 mm. The germination
was expressed as the percentage of the germinated beans of the total amount of beans
sampled. The averages (± standard deviation) of both percentage of germination and root
length in the time course experiment were calculated on the basis of 5 individual time
course experiments.
The different parameters of the soya bean treatment that were monitored are
shown in Table 1. The percentage of germination increased from 11% after 24 h to 90%
after 6-7 days. The root development was characterized by a steady growth in the first 5
days after which a plateau was reached of approximately 90 mm. Furthermore, side-root
formation was observed after 4 days of germination. These results are in line with similar
soya bean germination experiments in which an average germination percentage of 93%
after 4 days was reported [11]. The seedling development in time is illustrated in Figure 1.
A weight loss of ~10% DW compared to the non-soaked beans was observed after 10 days
(Table 1). To account for this loss, the isoflavonoid content was recalculated to g DW of
original soya bean material at t=0. Without this correction, the total amount of
isoflavonoid after germination would be overestimated.
Chapter 5
102
Table 1. Growth parameters of the soya beans/soya seedlings during the induction process, represented by the
average of 5 experiments. Data are mean ± SD values of measurements performed in five-fold.
Time
(d)
Stage in
treatment % of
germination
Root length
(mm)†
Absolute DW (g)¶ Relative DW
(g/100g)¶
0 Non-soaked* 0 0 366.3 (±0.2) 100
1 Soaking 11 (±14) 5.3 (±0.3) 341.2 (±20.1) 93.2 (±5.5)
2 Germination
53 (±9) 30.4 (±2.3) 343.9 (±29.2) 93.9 (±8.0) 3 63 (±11) 41.9 (±3.4) 322.0 (±4.6) 87.9 (±1.2)
4
Challenging
75 (±8) 54.1 (±13.8) 317.8 (±43.1) 86.8 (±11.8) 5 83 (±5) 67.2 (±6.9) 314.8 (±36.8) 86.0 (±10.1) 6 86 (±4) 82.0 (±8.5) 328.5 (±3.1) 89.7 (±0.9) 7 88 (±3) 77.5 (±9.0) 333.3 (±8.3) 91.0 (±2.3) 8 90 (±0) 78.2 (±1.4) 336.2 (±7.4) 91.8 (±2.0) 9 90 (±0) 90.5 (±1.8) 340.6 (±14.8) 93.0 (±4.0)
10 90 (±0) 87.7 (±7.7) 330.7 (±4.7) 90.3 (±1.3) * The weight of the experimental batch size of fresh beans was 400 g before drying. † The root length was defined as the length of the main root excluding possible side-roots. ¶ Absolute and relative DW refers to the whole seedling weight.
Identification of compounds
The UHPLC analysis of the extracts of non-soaked beans, seedlings and Rhizopus-
challenged seedlings revealed that the UV-profiles changed drastically during induction
(Figure 2). The identities of most peaks were established previously [5], whereas peaks 1, 8,
10, 13-15, 24 and 28 were now identified for the first time (Table 2). Figure 3 summarizes
most of the structures found in this study. Furthermore, the metabolite flow from
constitutive (isoflavones) to induced isoflavonoids (pterocarpans and coumestans) is
visualized. Prenylation is indicated as the most down-stream event in the biosynthetic
pathway, and relates to all three isoflavonoid subclasses mentioned.
Compound 1 was tentatively assigned as phenylalanine based on MS analysis. In addition, three other pterocarpans were tentatively assigned at retention times (Rt) of 8.08 (10), 13.85 (24) and 14.67 (28) min with MW of 354, 336 and 354, respectively. The UV spectra of compounds 10, 24 and 28 were characterized by two λmax values of 227(±2) nm and 288(±3) nm. Glycinol, glyceollidins I/II and glyceollins I, II and III showed the same two λmax values. In addition, the NI MS2 spectra of compounds 10, 24 and 28 displayed a characteristic fragment combination (-H2Ox,yRDA fragments) that is also observed in the MS2 spectra of glycinol, glyceollidins I/II and glyceollins I-III, but not in any other MS2 spectrum in the chromatogram [5]. Moreover, in PI mode, in-source fragmentation of compounds 10, 24 and 28 occurred, leading to the predominance of the [M+H-H2O]+ fragment ion in MS1. These observations strongly suggest that 10, 24 and 28 belong to the subclass of the 6a-hydroxypterocarpans. Prenylation of 24 and 28 was confirmed by the characteristic neutral loss of 56 in PI mode MS2 [12], whereas a loss of a methyl radical was
Increasing the isoflavonoid content and diversity of soya
103
Figure 1. Soya seedling development in time (t in days) illustrated by representative samples. The induction process starts with non-soaked beans (t=0) that were soaked (t=1), germinated (t=2,3) and challenged by Rhizopus (t=4-10).
observed in NI mode MS2 of compound 28. Based on all these findings,
compounds 24 and 28 were tentatively assigned and glyceollin V/VI and glyceollin IV,
respectively. The absence of the characteristic neutral loss of 56 in PI mode MS2 for
compound 10 could be explained by the presence of a hydroxyl-group on the furan ring
(hydroxyl-isopropenyl furan), as in glyceofuran. This hydroxyl-group altered its
fragmentation: instead of a loss of 42 [C3H6] or 56 [C4H8], a loss of 28 [CO] was observed.
Therefore, compound 10 was tentatively assigned as glyceofuran.
Five other compounds were observed. Compound 14 was tentatively assigned as
the flavanone naringenin based on comparison of the NI mode MS2 spectrum [13].
Compound 8 was assigned as biochanin A on the basis of comparative MS spectral analysis [14-16]. Two other unknown flavonoid peaks were observed at retention times 8.85 (13) and
10.33 (15). In the UV spectra of both 13 and 15, two λmax values were observed around 224
and 355 nm, resembling the UV spectrum of coumestrol that has λmax values at 221 and
343-360 nm (data not shown). Moreover, coumestrol, 13 and 15 have a predominant loss
of 28 (CO) in PI mode MS2 in common, and all three showed no retro-Diels-Alder (RDA)
fragments in both PI and NI mode MS2. Therefore, compound 13 was tentatively assigned
as C-methylated derivative of coumestrol, methyl-coumestrol. The predominant loss of a
methyl radical observed in NI mode MS2 of compound 15 led to its tentative assignment as
Chapter 5
104
a methyl ether of hydroxylated coumestrol. The absence of RDA fragments in MS2 does
not allow further speculation on the position of the C-methyl (13) or methoxyl (15) group.
The only methoxylated coumestrol isolated from soya is isotrifoliol, 3,9-dihydroxy-1-
methoxy-coumestan [17]. Compound 15 was, therefore, tentatively assigned as isotrifoliol.
Changes in the isoflavonoid composition during the induction process
Isoflavones
The quantification of all isoflavonoids annotated at each time point is given in Table 3. The
changes in the isoflavone profile upon soaking and germination were mainly characterized
by an increase of the aglycone, glucoside and malonyl-glucoside forms of daidzein,
genistein and glycitein. This increase was most pronounced after soaking (t=1). The total
isoflavone content increased from 0.37 to 1.54 mg DE/g during induction. The ratio of
total (free and conjugated forms of) genistein, daidzein and glycitein of approximately
0.45:0.45:0.10 remained constant throughout the whole process.
Pterocarpans
The pterocarpan pool consisted of glyceollins I-IV (19-21,28) and VI/V* (24), glyceollidin
I/II (17), glyceofuran (10) and glycinol (4). The pterocarpans represent the predominant
group of phytoalexins that was induced in the soya seedlings and increased throughout
the treatment, reaching a maximum level of 2.2 mg DE/g. The non-prenylated glycinol was
the only pterocarpan that steadily accumulated throughout the whole time course
experiment. The levels of all other pterocarpans appeared to stabilize at t=9.
Coumestans
The coumestans in the seedlings comprised coumestrol (18), C-methyl-coumestrol (13),
isotrifoliol (15) and 4-prenyl-coumestrol (31). The levels of coumestrol showed the same
trend as those of glyceollidin I/II, but they did not exceed the 0.16 mg DE/g. The total level
of coumestans reached a maximum of 0.41 mg DE/g at t=9. Similar to most pterocarpans,
the level of coumestans appeared to stabilize at t=9.
Estrogenic activity of extracts from induced soya beans
The estrogenic activity in response to the altered isoflavonoid profile in the extracts of
soya during induction is shown in Figure 4. With 1 and 3 µg/mL extract, ERα showed too
low responses, whereas a concentration of 30 µg/mL gave too high responses for most
extracts (comparable with the maximum response obtained with a high dose of 17β-
estradiol). With 10 µg/mL, a clear increase in estrogenic activity in the ERα bioassay in
time was observed. For ERβ, the three highest concentrations of extracts resulted in.
Increasing the isoflavonoid content and diversity of soya
105
Figure 2. RP-UHPLC-UV-profile of EtOH extracts of non-soaked soya beans (A), soya bean seedlings at t=1 (B), Rhizopus -challenged soya bean seedlings at t=4 (C) and at t=10 (D). Peak numbers refer to compounds in
Table 2.
Table 2. Compounds tentatively assigned by UHPLC-ESI-MS in the extracts of Glycine max after induction.
No Rt.
(min)
UVmax (nm) Identification Class Molecular
formula
[M-H]- MS
2 NI product ions
(relative abundance)
[M+H]+ MS
2 PI product ions
(relative abundance)
1 1.63 224,290 Phenylalanine Amino acid C9H11NO2 164 147(100) 166 120(100)
2 4.46 225,248 Daidzin† Isoflavone C21H20O9 415 253(100) 417 255(100)
3 4.56 225,256 Glycitin† Isoflavone C22H22O10 445 283(100) 447 283(100)
4 5.56 205,226,281 Glycinol† Pterocarpan C15H12O5 271 256(34),227(28),161(100) 255* 199(100),237(52)
5 5.57 225,260 Genistin† Isoflavone C21H20O10 431 431(100),269(11) 433 271(100)
6 5.81 225,255 Malonyl-daidzin† Isoflavone C24H22O12 501 253(100) 503 255(100)
7 7.13 209,225,260 Malonyl-genistin† Isoflavone C24H22O13 517 269(100) 519 271(100)
8 7.74 224,257 Biochanin A Isoflavone C16H12O5 283 268(100) 285 270(100)
9 7.95 226,257 Glycitein† Isoflavone C16H12O5 283 268(100) 285 270(100),229(21),225(16)
10 8.03 225,257,291 Glyceofuran Pterocarpan C20H18O6 353 335(100),161(6),149(25) 337* 319(87),309(100),188(25)
11 8.14 226,297 Daidzein† Isoflavone C15H10O4 253 253(100) 255 227(49),199(100),137(70)
12 8.26 212,226,257 2'-OH-genistein† Isoflavone C15H10O6 285 217(100) 287 245(49),217(100),153(57)
13 8.80 225,286,360 C-methyl-coumestrol Coumestan C16H10O5 281 281(11),253(100) 283 255(100)
14 9.62 225,287 Naringenin Flavanone C15H12O5 271 177(23),151(100) 273 243(56),215(73),253(100)
15 10.30 225,351 Isotrifoliol Coumestan C16H10O6 297 297(25),282(100) 299 284(19),271(100),267(15)
16 10.52 224,260 Genistein† Isoflavone C15H10O5 269 269(100) 271 153(100),215(79)
17 10.93 209,227,285 Glyceollidin I/II† Pterocarpan C20H20O5 339 324(45),161(100) 323* 267(100)
18 11.15 226,303,343 Coumestrol† Coumestan C15H8O5 267 267(100) 269 241(100),225(22),197(23)
19 11.41 211,227,284 Glyceollin III† Pterocarpan C20H18O5 337 319(100),293(8),149(18) 321* 306(76),297(79),251(100)
20 11.72 211,228,283,307 Glyceollin II† Pterocarpan C20H18O5 337 319(100),293(24),149(44) 321* 306(74),297(100), 251(94)
21 11.85 210,230,282 Glyceollin I† Pterocarpan C20H18O5 337 319(100),293(34),149(95) 321* 306(71),303(100), 293(33)
22 12.92 225,292 Aprenyl-2’OH-daidzein† Isoflavone C20H18O5 337 337(37),293(100),282(84) 339 283(100)
23 13.80 225,257 Bprenyl-glycitein† Isoflavone C21H20O5 351 336(100) 353 297(100),285(11)
24 13.89 225,287,308 Glyceollin V/VI Pterocarpan C20H16O5 335 317(100),149(39) 319* 319(28),291(63),263(100)
25 14.35 225,251,305 Aprenyl-daidzein† Isoflavone C20H18O4 321 321(16),266(100) 323 267(100)
26 14.44 229,262 Aprenyl-2’OH-genistein† Isoflavone C20H18O6 353 285(95),284(100),267(37) 355 299(100)
27 14.62 225,257,301 Bprenyl-daidzein† Isoflavone C20H18O4 321 321(100),266(16),265(68) 323 267(100),255(8)
28 14.71 226,285 Glyceollin IV Pterocarpan C21H22O5 353 335(100),149(20),148(5) 337* 281(100),269(49)
29 17.12 229,265 Aprenyl-genistein† Isoflavone C20H18O5 337 337(34),283(12),282(100) 339 283(100)
30 17.30 229,260,334 Bprenyl-genistein† Isoflavone C20H18O5 337 337(100),282(16),281(51) 339 283(100),271(11)
31 18.03 229,307,343 4-prenyl-coumestrol† Coumestan C20H16O5 335 335(48),281(16),280(100) 337 281(100)
n.a. – not available; NI mode - negative ion mode; PI mode - positive ion mode. * The [M+H-H2O]+ dominated in the MS1 compared to the [M+H]+. † previously assigned by Simons et al. [5].
Figure 3. Biosynthetic pathway and structures of isoflavonoids in soya beans and Rhizopus-induced soya seedlings. * In the literature glyceollin V
is often referred to as glyceollin IIIb. This scheme was compiled from Veitch (2007, 2009), Banks and Dewick (1983), Simons et al. (2011) [1, 5, 6, 39].
Chapter 5
108
maximum responses in time, and no trend was observed. At 1 µg/mL of extract, saturation
in signal (i.e. maximal response) was not reached yet and an increase in estrogenicity for
the ERβ in time was observed. The most representative experiments with respect to the
increasing estrogenicity in time are shown in Figure 4. The estrogenicity towards the ERβ
was relatively higher compared to that towards the ERα, even more so as the ERβ bar
diagram was obtained with 10-fold lower extract concentrations than that of ERα. For 1
µg/mL the estrogenicity towards the ERβ appeared to reach a plateau value at t=5
The extracts were also tested for possible antagonistic activity towards both
estrogen receptors. These experiments showed that the extracts were unable to suppress
the agonistic response caused by the dose of 17β-estradiol that gave a 70 to 90% maximal
response. On the contrary, the responses only increased by these co-exposures, showing
additive agonistic effects, e.g. the addition of the extracts of t=4 to t=10 enhanced the
estrogenicity of estradiol by 20-25% (data not shown).
Figure 4. Estrogenic activity of the ethanol extracts of the soya bean seedlings at different time points measured in the yeast estrogen bioassays. The extracts were measured at 10 µg/mL in the ERα bioassay, and at 1 µg/mL in the ERβ bioassay. Data are mean ± SD values of measurements performed in triplicates. E2 is 1 µM 17β-estradiol.
Increasing the isoflavonoid content, diversity and estrogenicity of soya
109
DISCUSSION
By combining malting technology and fermentation with the food-grade R. microsporus,
the induction of phytoalexins in soya seedlings was successfully performed in terms of
increasing total isoflavonoid content, altering isoflavonoid composition and enhancing the
estrogenic potential of the soya material.
De novo synthesis of isoflavonoids during the induction-process
The isoflavonoid content of soya increased by a factor 10-12 suggesting de novo synthesis
of isoflavonoids. This is further corroborated by the appearance (t=1) of phenylalanine (1),
the basic precursor of flavonoid biosynthesis, and naringenin (14), a precursor of genistein
(16). The formation of the flavanone naringenin might thus explain the increase of the
genistein pool [18]. These findings are supported by several reports in which de novo
synthesis is suggested based on quantification of isoflavonoid content, up-regulation of
enzymes involved in isoflavonoid biosynthesis and incorporation of 14C-labeled
phenylalanine, daidzein, 2’OH-daidzein and glycinol into glyceollins I-III [6, 19-22]. Data on
accurate quantification of the total isoflavonoid content during the induction process are
very limited. It has been reported that levels ranging from 0.01 to 1 mg/g fresh weight
(FW) of glyceollins I-III can accumulate in fungus-challenged soya seedlings [23]. With an
approximate water content of 65-70% (w/w) [24], the glyceollin I-III levels in soya seedlings
range from 0.03 to 3 mg/g DW, whereas a content of 0.9 mg DE/g DW was found at 9 days
in our study (Table 3). In most studies, soya beans are wounded prior to inoculation. It has
been shown that this additional treatment can increase the glyceollin I-III levels of soya
beans inoculated with Aspergillus sojae by a factor 10 [25]. This extra wounding step might
explain the difference between our data and those in the literature. Moreover, a number
of other pterocarpans (1.3 mg DE/g DW) were found in our study (Table 3), suggesting
that the difference between our results and those in the literature might actually be
smaller than they appear.
The stabilization of the glyceollins I-III contents at t=9 might be due to the
inhibition of cyclase, the enzyme responsible for the last step in glyceollin biosynthesis, as
this enzyme is inhibited by its reaction products [26]. In line with this, the accumulation of
glycinol did not seem to level off.
Changes in isoflavonoid profile Although inoculation was performed at t=3, small amounts of pterocarpans and
coumestans already occurred at t=2. This might be due to incomplete sterilization.
Alternatively, the sterilization agent itself (hypochlorite) might have acted as a chemical
elicitor.
Chapter 5
110
Table 3. Quantitative analysis of the isoflavonoid profile during soya bean induction. The content of each flavonoid is expressed in mg daidzein equivalent (DE) per g DW, corrected for loss in dry weight during the induction process. Data are mean ± SD values of five independent Rhizopus-challenged germination experiments.
No Isoflavonoid Prenyl t=0 t=1 t=2 t=3
5 Genistin - 0.11(±0.00) 0.30(±0.07) 0.25(±0.04) 0.18(±0.03) 7 Malonyl-genistin - 0.06(±0.03) 0.33(±0.07) 0.29(±0.04) 0.29(±0.07) 12 Genistein - 0.01(±0.00) 0.05(±0.01) 0.05(±0.02) 0.05(±0.02) 16 2'-Hydroxygenistein - - 0.01(±0.00) 0.01(±0.00) 0.02(±0.00) 26 Aprenyl-2’OH-genistein Chain - - - 0.01(±0.01) 29 Aprenyl-genistein Chain - - 0.01(±0.00) 0.02(±0.01) 30 Bprenyl-genistein Chain - - - 0.01(±0.01) Genistein pool 0.18(±0.04) 0.70(±0.16) 0.60(±0.10) 0.58(±0.14)
3 Glycitin - 0.02(±0.01) 0.10(±0.02) 0.09(±0.01) 0.07(±0.02) 9 Glycitein - 0.01(±0.01) 0.06(±0.01) 0.05(±0.01) 0.06(±0.02) 23 Bprenyl-glycitein Chain - - - <0.01 Glycitein pool 0.03(±0.02) 0.16(±0.03) 0.14(±0.02) 0.13(±0.04)
2 Daidzin - 0.07(±0.03) 0.25(±0.04) 0.22(±0.04) 0.18(±0.03) 6 Malonyl-daidzin - 0.07(±0.04) 0.39(±0.07) 0.34(±0.04) 0.32(±0.08) 11 Daidzein - 0.02(±0.01) 0.05(±0.01) 0.05(±0.02) 0.07(±0.02) 22 Aprenyl-2’OH-daidzein Chain - - - 0.02(±0.01) 25 Aprenyl-daidzein Chain - - <0.01 0.02(±0.01) 27 Bprenyl-daidzein Chain - - <0.01 0.01(±0.00) Daidzein pool 0.16(±0.07) 0.69(±0.13) 0.63(±0.10) 0.62(±0.15)
8 Biochanin A - - - - - Isoflavone pool 0.37(±0.13) 1.54(±0.32) 1.38(±0.22) 1.32(±0.33)
4 Glycinol - - <0.01 <0.01 0.01(±0.00) 17 Glyceollidin I/II Chain - <0.01 <0.01 0.02(±0.01) 19 Glyceollin III Furan
1 - <0.01 0.01(±0.00) 0.02(±0.01)
20 Glyceollin II Pyran - - 0.00(±0.00) 0.01(±0.01) 21 Glyceollin I Pyran - <0.01 0.02(±0.00) 0.07(±0.04) 28 Glyceollin IV Chain - - <0.01 <0.01 24 Glyceollin V/VI Furan
2 - <0.01 <0.01 <0.01
10 Glyceofuran Furan3 - 0.07(±0.00) 0.06(±0.00) 0.06(±0.00)
Pterocarpan pool - 0.07(±0.00) 0.09(±0.01) 0.20(±0.09)
18 Coumestrol - - - 0.01(±0.00) 0.02(±0.01) 13 C-methyl-coumestrol - - - - - 15 Isotrifoliol - - - - 0.01(±0.00) 31 4-prenyl-coumestrol Chain - - - 0.01(±0.01) Coumestan pool - - - 0.03(±0.02)
Total isoflavonoids 0.37(±0.13) 1.61(±0.32) 1.48(±0.23) 1.54(±0.44)
Chain, 3,3 dimethylallyl (prenyl chain). Pyran, 2,2-dimethylchromeno. Furan1, 2’’-isopropenyl-dihydrofuran. Furan2, 2’’-isopropenyl-furan. Furan3, 2’’-(2-hydroxy-)isopropenyl-dihydrofuran.
Increasing the isoflavonoid content, diversity and estrogenicity of soya
111
t=4 t=5 t=6 t=7 t=8 t=9 t=10
0.23(±0.04) 0.23(±0.03) 0.20(±0.01) 0.23(±0.03) 0.19(±0.04) 0.26(±0.06) 0.19(±0.02) 0.30(±0.07) 0.25(±0.06) 0.25(±0.04) 0.26(±0.02) 0.22(±0.05) 0.31(±0.13) 0.20(±0.04) 0.05(±0.03) 0.05(±0.04) 0.06(±0.04) 0.06(±0.03) 0.09(±0.02) 0.09(±0.04) 0.06(±0.01) 0.02(±0.01) 0.02(±0.01) 0.02(±0.01) 0.02(±0.01) 0.02(±0.00) 0.08(±0.08) 0.02(±0.00) 0.02(±0.01) 0.03(±0.02) 0.04(±0.02) 0.04(±0.01) 0.04(±0.02) 0.04(±0.02) 0.03(±0.01) 0.03(±0.01) 0.03(±0.01) 0.04(±0.02) 0.05(±0.02) 0.05(±0.02) 0.06(±0.02) 0.03(±0.02) 0.02(±0.01) 0.03(±0.01) 0.04(±0.02) 0.04(±0.01) 0.04(±0.02) 0.05(±0.02) 0.03(±0.01) 0.66(±0.18) 0.64(±0.18) 0.65(±0.17) 0.70(±0.14) 0.65(±0.17) 0.89(±0.38) 0.56(±0.11)
0.07(±0.01) 0.07(±0.01) 0.06(±0.01) 0.07(±0.01) 0.05(±0.01) 0.06(±0.01) 0.05(±0.01) 0.08(±0.01) 0.06(±0.01) 0.05(±0.01) 0.06(±0.01) 0.06(±0.01) 0.07(±0.02) 0.05(±0.02) 0.01(±0.00) 0.01(±0.00) 0.01(±0.00) 0.01(±0.00) 0.01(±0.00) 0.01(±0.00) 0.01(±0.00) 0.15(±0.03) 0.13(±0.02) 0.12(±0.03) 0.13(±0.02) 0.12(±0.02) 0.15(±0.04) 0.10(±0.03)
0.19(±0.03) 0.19(±0.01) 0.16(±0.02) 0.18(±0.03) 0.14(±0.04) 0.18(±0.03) 0.12(±0.01) 0.32(±0.06) 0.31(±0.05) 0.27(±0.05) 0.27(±0.05) 0.22(±0.06) 0.28(±0.10) 0.21(±0.05) 0.09(±0.04) 0.09(±0.03) 0.11(±0.05) 0.13(±0.04) 0.13(±0.02) 0.16(±0.05) 0.10(±0.01) 0.04(±0.02) 0.05(±0.02) 0.07(±0.04) 0.07(±0.02) 0.08(±0.04) 0.09(±0.04) 0.06(±0.03) 0.03(±0.01) 0.03(±0.01) 0.04(±0.02) 0.03(±0.01) 0.03(±0.02) 0.04(±0.02) 0.03(±0.01) 0.01(±0.00) 0.01(±0.00) 0.01(±0.01) 0.01(±0.00) 0.01(±0.00) 0.01(±0.00) 0.01(±0.00) 0.68(±0.17) 0.68(±0.14) 0.66(±0.18) 0.69(±0.16) 0.62(±0.18) 0.76(±0.24) 0.53(±0.12)
0.01(±0.01) 0.01(±0.01) 0.02(±0.01) 0.02(±0.01) 0.02(±0.01) 0.02(±0.01) 0.01(±0.01) 1.50(±0.39) 1.46(±0.34) 1.46(±0.38) 1.54(±0.32) 1.40(±0.39) 1.83(±0.67) 1.21(±0.27)
0.04(±0.01) 0.10(±0.03) 0.17(±0.04) 0.25(±0.03) 0.27(±0.06) 0.35(±0.03) 0.42(±0.07) 0.06(±0.04) 0.12(±0.06) 0.16(±0.11) 0.18(±0.07) 0.20(±0.08) 0.22(±0.05) 0.23(±0.09) 0.07(±0.04) 0.12(±0.07) 0.18(±0.11) 0.21(±0.09) 0.29(±0.09) 0.32(±0.07) 0.29(±0.06) 0.04(±0.02) 0.08(±0.03) 0.12(±0.07) 0.17(±0.06) 0.21(±0.04) 0.24(±0.03) 0.22(±0.04) 0.17(±0.09) 0.22(±0.10) 0.24(±0.13) 0.29(±0.11) 0.31(±0.09) 0.33(±0.05) 0.32(±0.06) 0.02(±0.03) 0.08(±0.08) 0.16(±0.15) 0.25(±0.13) 0.27(±0.16) 0.33(±0.20) 0.35(±0.21) 0.03(±0.02) 0.07(±0.04) 0.13(±0.06) 0.18(±0.04) 0.22(±0.08) 0.24(±0.05) 0.22(±0.05) 0.04(±0.01) 0.06(±0.02) 0.08(±0.04) 0.10(±0.04) 0.15(±0.05) 0.17(±0.03) 0.18(±0.04) 0.47(±0.26) 0.86(±0.43) 1.24(±0.70) 1.63(±0.57) 1.91(±0.64) 2.21(±0.51) 2.24(±0.62)
0.06(±0.02) 0.08(±0.03) 0.10(±0.06) 0.12(±0.04) 0.15(±0.06) 0.16(±0.05) 0.13(±0.04) 0.01(±0.01) 0.03(±0.01) 0.06(±0.03) 0.09(±0.04) 0.15(±0.03) 0.16(±0.04) 0.15(±0.03) 0.01(±0.01) 0.03(±0.02) 0.05(±0.03) 0.06(±0.03) 0.09(±0.05) 0.10(±0.04) 0.08(±0.02) 0.01(±0.01) 0.03(±0.02) 0.05(±0.04) 0.05(±0.03) 0.07(±0.06) 0.09(±0.06) 0.06(±0.05) 0.09(±0.05) 0.18(±0.09) 0.26(±0.17) 0.33(±0.13) 0.45(±0.20) 0.51(±0.20) 0.42(±0.14)
2.06(±0.70) 2.49(±0.85) 2.95(±1.24) 3.50(±1.03) 3.77(±1.24) 4.54(±1.38) 3.87(±1.03)
Chapter 5
112
The ratio of the commonly found glyceollins I, II and III was 1.4:1:1.3 at t=9, respectively, and differed from the ratios of 6:1:2 previously reported [27]. More strikingly, the present study shows the additional formation of glyceofuran (10), glyceollin IV (28), glyceollin VI (24), coumestrol-derivatives (13,15,31), and several prenylated isoflavones (22,23,25-27,29,30). The simultaneous occurrence of these compounds has never been reported in Glycine max. However, glyceollin IV has been found in G. tomentella and glyceollin VI in G. clandestine and G. tabacina [28, 29]. The isomer of glyceollin VI has recently been isolated and given the trivial name glyceollin V [17]. However, the name glyceollin V was already used for the enantiomeric form of glyceollin III [28]. To avoid confusion, the two enantiomers of glyceollins III are indicated as glyceollin IIIa and IIIb in Figure 3, whereas the isomer of glyceollin VI is indicated by glyceollin V.
The occurrence of phaseol (31) and isotrifoliol (15) has been reported before in G.
max [30, 31]. The formation of prenylated isoflavones has recently been reported for G. max,
as well as in several other, often stress-induced, Leguminosae species [5]. After the 9 days
of induction, these compounds have accumulated to an impressive 32 % (w/w) of the total
isoflavonoids in the soya material.
Taken together, the more extensive changes in isoflavonoid composition as
observed in the present study compared to studies by others is most likely due to the
combination of prolonged induction times and more favorable growth conditions in the
micro-malting system. Consequently, the altered ratio between the most common
glyceollins and the formation of compounds might represent the outcome of events
occurring more downstream in the biosynthesis process. Possibly, omission of the
wounding pretreatment also plays a role.
Accumulation of phytoestrogens during the induction process
Figure 4 shows a gradual increase in estrogenicity of the extracts during the induction
process towards both the ERα and ERβ. It is likely that the increased proportion of
isoflavonoids and the change in isoflavonoid composition in the extract are responsible for
this increase of estrogenic activity. Deglycosylation of isoflavones by β-glucosidase might
be a driver behind this [32]. The proportion of glucosides (compounds 2, 3 and 5-7)
decreased from 90% (t=0) to approximately 20% (t=9). It is known that the glucosyl
residues hamper binding to the ER [8]. More importantly, other isoflavonoids were formed
of which several have been reported as potent agonists towards both ERs, particularly
glycinol and coumestrol [8, 33]. Differences in the outcome of various bioassays described in
the literature, and lack of data for binding of individual isoflavonoids for both ERs, do not
allow extrapolations concerning the contribution of each isoflavonoid to the estrogenicity
of the current extract.
Besides agonistic activity, it is likely that the extracts contain compounds with
antagonistic properties. For soya bean, this has been demonstrated for glyceollin I [34],
which comprised 8% (w/w) of the total isoflavonoids at t=9. To determine to what extent
Increasing the isoflavonoid content, diversity and estrogenicity of soya
113
phytoestrogens with a known EC50 value can account for the estrogenic activity of a tested
extract, the expected estrogenicity of the extract was calculated from the contribution of
each phytoestrogen (expressed in E2 equivalents, EEQ), using their relative estrogenic
potency (REP) and their quantity in the extract. This was performed for extracts of t=10
and t=4 with ERα and ERβ, respectively (see Appendix IX). The calculated values were
compared to those actually measured. The expected estrogenicity (expressed as the sum
of the EEQ of genistein, coumestrol and daidzein) was approximately 25% higher than the
EEQ corresponding to the response measured in the assay. The same calculation was
performed for the ERβ showing that the total expected estrogenicity was even a factor 7
higher than the EEQ of the response measured. Thus, the measured response in the assay
was lower than the one calculated, which hints at the presence of antagonists in the
extract. Even more so, as the calculated EEQ based on genistein, coumestrol and daidzein
(phytoestrogens with a known EC50) is most likely underestimated because glycinol, a
potent phytoestrogen on both ER subtypes with an estrogenic activity similar to that of
coumestrol, has not been taken into account. The data on glycinol available from the
literature were obtained with a bioassay different from ours [33]. As mentioned earlier,
glyceollin I has been reported to display antagonistic activity on the ERα. It has been
suggested that prenyl substituents are important structural attributes for inducing
antagonistic responses [35-38]. In Figure 5, the accumulation of prenylated isoflavonoids is
compared with that of non-prenylated isoflavonoids. It is clearly seen that longer
induction times promote a higher proportion of prenylated isoflavonoids, reaching similar
amounts as non-prenylated isoflavonoids at around t=7. This suggests that the
accumulation of prenylated isoflavonoids in time increases the antagonistic activity of the
extracts.
Figure 5. Quantitative analysis of the isoflavonoid profile in the time course experiments of the development of Rhizopus-elicited soya seedlings.
Chapter 5
114
In summary, we speculate that the increased agonistic ER activity of the extracts
is mainly due to the formation of the non-prenylated pterocarpan, glycinol, and the non-
prenylated coumestans, particularly coumestrol. Besides, the extracts might also be rich in
ER antagonists, as prenylation of isoflavonoids is known to correlate well with antagonistic
properties (e.g. glyceollin I). Although antagonism was not established directly in our
study, we found lower estrogenic response with the extracts than might be expected from
their amounts of agonists. Further investigation is necessary to substantiate this.
CONCLUSIONS
Malting of soya beans in the presence of a food-grade fungus yielded a completely
different spectrum of isoflavonoids, with a much higher bioactivity towards both estrogen
receptors subtypes than the starting material (non-soaked beans). Together with the over
10-fold increase in potential bioactives, this offers promising perspectives for producing
more, novel, and higher potency nutraceuticals by malting soya beans under stressed
conditions.
ACKNOWLEDGEMENTS
This work was financially supported by the Food & Nutrition Delta of the Ministry of
Economic Affairs, The Netherlands. We would like to thank Loes Mol (Wageningen
University, Wageningen, the Netherlands) for her contribution to the estrogenicity
experiments, RIKILT-Institute of Food Safety (WUR) for making the yeast estrogen
bioassays available, and Hennie van de Laar (Holland Malt, Lieshout, the Netherlands) for
his assistance in setting up the induction experiments.
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[35] Q. Jiang, F. Payton-Stewart, S. Elliott, J. Driver, L. V. Rhodes, Q. Zhang, S. Zheng, D. Bhatnagar, S. M. Boue, B. M. Collins-Burow, J. Sridhar, C. Stevens, J. A. McLachlan, T. E. Wiese, M. E. Burow, G. Wang, Effects of 7-O substitutions on estrogenic and anti-estrogenic activities of daidzein analogues in MCF-7 breast cancer cells. Journal of Medicinal Chemistry 2010, 53, 6153-6163.
[36] E. M. Ahn, N. Nakamura, T. Akao, T. Nishihara, M. Hattori, Estrogenic and antiestrogenic activities of the roots of Moghania philippinensis and their constituents. Biological and Pharmaceutical Bulletin
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Chapter 6
General Discussion
Chapter 6
118
In this thesis, the prenylated isoflavonoid profile of soya and licorice, and their relation to
estrogenicity, is described. First, a liquid chromatography / mass spectrometry-based
screening method was developed that enabled the identification of prenylated flavonoids
(including isoflavonoids), and that also discriminated between different kinds of prenyl
substitution (chain and pyran ring). This method was used to characterise the prenylated
isoflavonoid profile of Rhizopus-challenged soya seedlings and licorice roots. Secondly, the
estrogenic activity of soya was enhanced by induction of isoflavonoids by a combination of
germination and biotic stress (challenging with Rhizopus). Thirdly, we have tried to
correlate the kind of estrogenic activity (agonism, antagonism) in extracts from licorice
root and Rhizopus-challenged soya seedlings to prenylation of the constituent
isoflavonoids.
MASS SPECTROMETRY AS A TOOL TO DETECT PRENYLATION
Update of guideline for detecting prenylation in flavonoids
Based on the literature, we expected the presence of a broad range of different
isoflavonoids in the sources studied in this thesis. Furthermore, we also anticipated
encountering a range of different types of prenyl substituents. Hence, isolation of all
individual components would not be feasible. Mass spectrometry (MS), coupled to ultra-
high performance liquid chromatography (UHPLC), provided a powerful analytical
technique allowing rapid analysis of complex extracts. With the possibility to perform MSn
fragmentations, MS is particularly suitable for the tentative identification of flavonoids.
As described in Chapter 2, fragmentation of prenylated flavonoids is
characterised by a predominant degradation of the prenyl group, evidenced by neutral
losses of 56 alone, or 42 and 56. It is known that the MS2 fragmentation of e.g. non-
prenylated isoflavones, such as genistein, often shows a neutral loss of 56, related to the
sequential loss of two CO moieties [1]. Therefore, we introduced a mass threshold to
exclude that non-prenylated flavonoid aglycones were assigned as being prenylated. The
mass threshold would filter out the isoflavone aglycones, but not glycosylated flavonoids,
which might result in false positives (Chapter 2, Figure 6).
It is known that several species outside the Leguminosae family such as the genus
Epimedium within the family of Berberidaceae contain glycosyl-conjugates of prenylated
flavones [2]. However, to our knowledge no data was available on the MSn fragmentation
of the glycosyl-conjugates of prenylated flavones in PI mode. A recent study on the
analysis of two glycosyl-conjugates of prenylated isoflavones from the wild Mexican lupine
(Lupinus reflexus) showed that both luteone 4’,7-O-diglucoside and 2,3-
didehydrokievitone 4’,7-O-diglucoside gave a predominant loss of one and two glucosyl
moieties in the MS2 spectra, respectively, in combination with a neutral loss of 56 with a
General discussion
119
relative abundance of ~50% [3]. In contrast, the MS2 spectra of non-prenylated isoflavonoid
glycosides were dominated by the neutral loss of the glycosyl substituent, whereas the
neutral loss of 56 was exclusively observed in MS3 spectra. Thus, prenylated isoflavone
glycosides seem to give a neutral loss of 56 in MS2 originating from the prenyl group
(C4H8), whereas non-prenylated isoflavone glycosides exclusively show a neutral loss of in
56 in MS3, which is related to the sequential loss of two CO moieties [1]. The necessity to
screen for prenyl group-related neutral losses in MS3 is still required, as e.g. prenylated
isoflavans from licorice root (Chapter 2) would otherwise not be detected.
In Figure 1, an updated version, incorporating the latest data [3], of the
interpretation guideline as proposed in Chapter 2 is shown. With this updated
interpretation guideline it is now possible to screen for prenylated flavonoids and
discriminate between prenyl chain and pyran ring, no matter whether they are
glycosylated or not.
Figure 1. Updated interpretation guideline based on the screening for prenylated flavonoids from Chapter 2. In
this guideline, the combination of a neutral loss of 56 or 42 and 56 in MS3 in combination with a neutral loss related to a loss of glycosyl residues in MS2 indicates the presence of a non-prenylated flavonoid glycoside.
Chapter 6
120
Detection of prenyl substituents other than chains and pyran rings
In Chapters 3 and 4, the tentative assignment of three pterocarpans, glyceollin III,
glyceollin VI and glyceofuran was described. These pterocarpans were substituted with
the less common prenyl groups, 2’’-isopropenyl-dihydrofurano, 2’’-isopropenyl-furano,
and 2’’-(2-hydroxy-) isopropenyl-dihydrofurano, respectively (Figure 2). The degradation in
PI mode of glyceollin III and VI was characterised by predominant losses of 42 (C3H6) and
56 (C4H8) in MS2. Whereas the MS2 spectrum of glyceollin III showed a predominant loss of
42 (80%) over 56 (30%), degradation of glyceollin VI was characterised by a major loss of
56 (100%) and a minor loss of 42 (10%). Apparently, the double bond of the furan group
played an important role in the degradation. It appeared that the 2’’-isopropenyl-
dihydrofuran-group (glyceollin III) shows pyran ring-like behaviour in fragmentation,
whereas 2’’-isopropenyl-furano-group (glyceollin VI) shows behaviour similar to the prenyl
chain.
It should be noted that the minor neutral losses of 42 (2%) and 56 (3%) in the MS2
spectrum of glyceofuran (having a hydroxylated prenyl group) were observed in
combination with the dominant loss of 28 (CO), which also may originate from the 2’’-(2-
hydroxy-)isopropenyl-dihydrofurano-group. The observation of the near absence of
fragment ions [M+H-42]+ or [M+H-56]+ in the degradation of the hydroxylated prenyl
group is corroborated by a similar observation for the hydroxylated prenyl group of
dehydrocycloxanthohumol hydrate (Tabel 1) [4]. It seems as if hydroxylation of the prenyl-
group alters the degradation pathway, as the characteristic losses of 42 and 56 were
observed only in trace amounts. Other examples of prenyl fragmentation were not
encountered in our research.
Figure 2. Three pterocarpans from Rhizopus-induced soya seedlings: glyceollin III, glyceollin VI and glyceofuran.
General discussion
121
Mass spectrometric analysis of prenylated flavonoids: PI or NI mode?
As mentioned in Chapter 2, analysis of flavonoids in NI mode is preferred over PI mode,
mainly due its higher sensitivity. However, it has been suggested that fragmentation of
prenyl groups is hindered in NI mode due to the nucleophilic nature of the prenyl group [4].
As a result, the neutral loss characteristic of prenyl degradation, C4H8, has not been
observed in NI mode [5]. Our data on the fragmentation of prenylated flavonoids in NI
mode suggests that the prenyl group is often degraded in both MS2 and MS3, albeit less
prominently than in PI mode (Table 1).
In our PI mode MS2 spectra, a loss of 68 was observed for three prenyl chain-
substituted isoflavones and one prenyl chain-substituted pterocarpan (Table 1). This loss
has also been found by others, and was assigned to the neutral fragment C5H8,
representing the whole prenyl chain [6]. In that same study, the loss of 68 was not
observed in double prenylated isoflavones and flavanones. Therefore, the neutral loss of
68 does not appear to be diagnostic for prenylation, suggesting that this loss is an
inappropriate screening criterion for prenylation.
Several characteristic neutral losses originating from the degradation of prenyl-
groups, including 15 (CH3•), 42 (C3H6), 55 (C4H7), 68(C5H8) and 69 (C5H9) have been
observed in NI mode. The observation of these neutral losses was corroborated by a
number of studies (Table 2). Although the loss of a methyl radical in MS2 was often
observed, this loss generally suggests the presence of a methoxyl group [7], and is,
therefore, not suitable as a screening criterion for prenylation. Furthermore, other neutral
losses that could be related to the degradation of the prenyl group, such as 42 and 55,
were observed in only few cases.
Despite the observation of several neutral losses in NI mode that could be related
to the degradation of the prenyl group, the occurrence of these neutral losses was limited
and inconsistent. The analysis of prenylated flavonoids is preferred in PI mode, as it results
in the consistent and abundant observation of diagnostic neutral losses 42 and 56. In
addition, analysis in PI mode allows the discrimination between prenyl chain and pyran
ring. As the same neutral losses were observed in the MSn spectra of less common prenyl-
substituents, it remains unclear whether discrimination of these prenyl-substituents is
possible.
Chapter 6
122
Table 1.Overview of neutral losses of prenylated flavonoids observed in PI mode MS2 and MS3.
MS2 (Relative abundance)
Compound Prenyl group Ring 15 42 55 56 68 Ref.
Literature
Xanthohumol Chain A 100 [4]
4,2’,4’,6’-Tetrahydroxy-3’-
prenylchalcone Chain A 100
[4]
5’-Prenylxanthohumol Chain A 61 [4]
Dehydrocycloxanthohumol Pyran A [4]
Dehydrocycloxanthohumol
hydrate Pyran2 A
[4]
Isoxanthohumol Chain A 171 [4]
6-Prenylnaringenin Chain A 41 [4]
8-Prenylnaringenin Chain A 21 [4]
Euchrestaflavanone B Chain + chain 100 [8]
Euchrestaflavanone C Chain + pyran A,B ~25 [8]
Osajaxanthone Pyran -3 ~25 [8]
Macluraxanthone Chain + pyran -3 ~15 [8]
This thesis
Glabrone Pyran B 100 6 Ch. 2
Glabridin Pyran A 1004 14 Ch. 2
Glabrol Chain+chain A,B 100 Ch. 2
3’-OH-4’-OMe-Glabridin Pyran A 1004 14 Ch. 2
4’-OMe-Glabridin Pyran A 1004 14 Ch. 2
Hispaglabridin A Pyran + chain A,B 100 Ch. 2
Hispaglabridin B Pyran + pyran A,B 1004 14 Ch. 2
Glyceollidin I/II Chain A 2 8 100 Ch. 3
Glyceollin I Pyran A 80 28 10 24 Ch. 3
Glyceollin II Pyran A 68 100 15 34 Ch. 3
Glyceollin III Furan5 A 80 80 9 29 Ch. 3
Glyceollin IV Chain A 6 100 52 Ch. 3
Glyceollin VI Furan6 A 12 11 14 100 Ch. 3
Glyceofuran Furan7 A 2 3 Ch. 3
Ap-2’-OH-Daidzein Chain A 2 100 Ch. 3
Bp-Glycitein Chain B 1 100 5 Ch. 3
Ap-Daidzein Chain A 100 Ch. 3
Ap-2’-OH-Genistein Chain A 100 Ch. 3
Bp-Daidzein Chain B 100 10 Ch. 3
Ap-Genistein Chain A 100 Ch. 3
Bp-Genistein Chain B 100 10 Ch. 3
Prenyl-coumestrol8 Chain A 100 Ch. 3 1 More abundantly observed in MS3.
2 2,2-dimethyl-3-hydroxy-pyran. 3 This polyphenolic compound is not a flavonoid, but belongs to the xanthones or benzophenones [9]. 4 Observed in MS3. 5 2’’-isopropenyl-dihydrofuran. 6 2’’-isopropenyl-furan. 7 2’’-(2-hydroxy) isopropenyl-dihydrofuran. 8 Tentatively assigned as 4-prenyl-coumestrol (Chapter 3).
General discussion
123
Table 2.Overview of neutral losses of prenylated flavonoids observed in NI mode MS2.
MS2 (Relative abundance)
Compound Prenyl group Ring 15 42 55 56 68 Misc. Ref.
NI mode – This thesis
Glabrene Pyran B 100 12 Ch. 2
Glabrone Pyran B 17 Ch. 2
Glabridin Pyran A 12 4 Ch. 2
Glabrol Chain + chain A,B Ch. 2
3’-OH-4’-OMe-Glabridin Pyran A 38 Ch. 2
4’-OMe-Glabridin Pyran A 52 Ch. 2
Hispaglabridin A Pyran + chain A,B 231 1001 69 Ch. 2
Hispaglabridin B Pyran + pyran A,B 1001 Ch. 2
Glyceollidin I/II Chain A 46 69 Ch. 3
Glyceollin I Pyran A 151 9 Ch. 3
Glyceollin II Pyran A 1 1001 851 9 Ch. 3
Glyceollin III Furan2 A 1 1001 4 Ch. 3
Glyceollin IV Chain A 5 Ch. 3
Glyceollin VI Furan3 A 5 4 Ch. 3
Glyceofuran Furan4 A Ch. 3
Ap-2’-OH-Daidzein Chain A 84 8 Ch. 3
Bp-Glycitein Chain B 100 801 Ch. 3
Ap-Daidzein Chain A 100 Ch. 3
Ap-2’-OH-Genistein Chain A 29 90 69 Ch. 3
Bp-Daidzein Chain B 100 Ch. 3
Ap-Genistein Chain A 100 Ch. 3
Bp-Genistein Chain B 100 Ch. 3
Prenyl-coumestrol5 Chain A 100 Ch. 3
NI mode - Literature
Luteone6 Chain B � [10]
Breviflavone A Chain7 B,B 708 [11]
Breviflavone B Chain9 B,B 8610 [11]
M311 Chain12 A 7013 [12]
Hedysarimcoumestan G Chain B � � 4313 [13]
Hedysarimcoumestan D Chain B � � 4313 [13]
Hedysarimcoumestan H Chain B � � 4313 [13]
Hedysarimpterocarpene B Chain B � � 4313 [13]
Gancaonin A Chain A � � 4313 [13]
Gancaonin M Chain A � � 4313 [13]
5,7-Dihydroxy-4’-O-methyl-
6,8-diprenylisoflavone
Chain A � � 4313 [13]
Licochalcone A Chain B � [14]
Desmethylicaritin Chain A � [15]
Misc – miscellaneous neutral loss observed in MS2; �Neutral loss was observed, but relative abundance was not reported. 1 More abundantly observed in MS3.
2 2’’-isopropenyl-dihydrofuran. 3 2’’-isopropenyl-furan. 4 2’’-(2-hydroxy-)isopropenyl-dihydrofuran. 5 Tentatively assigned as 4-prenyl-coumestrol (Chapter 3). 6 6-prenyl-2’OH-genistein. 7 2-Hydroxy-3-metyl-but-3-enyl. 8 C4H6O was proposed as the corresponding fragment. 9 2’’-(2-hydroxy-)isopropenyl-1’’-hydroxy-dihydrofurano. 10 C4H6O2 was proposed as the corresponding fragment. 11 Human liver microsomal metabolite of xanthohumol with hydroxylation of the prenyl chain. 12 2-Hydroxy-3-metyl-but-3-enyl. 13 C4H7 was proposed as the corresponding fragment.
Chapter 6
124
MS fragmentation of pterocarpans and coumestans
As mentioned in Chapter 1, very little is known about the fragmentation pathways of
pterocarpans and coumestans. Three other studies, investigating the NI fragmentation of
pterocarpans, did not report any C-ring or D-ring cleavages [13, 16, 17]. In Chapter 3, we
proposed a fragmentation pathway for pterocarpans in NI mode based on glycinol,
glyceollidins and glyceollins (Figure 3). The additional D-ring appeared to affect the
fragmentation pathway and an adapted nomenclature was proposed for the
fragmentation pathway of this subclass.
To our knowledge, only one study previously reported the C-ring cleavage of a
pterocarpan in PI mode [18]. In that study, fragment ions 1,3A+ and 2,3B+ were assigned after
fragmentation in PI mode of 3-hydroxy-9,10-dimethoxy-pterocarpan via an isoflavene-
intermediate. According to our proposed nomenclature for pterocarpan fragmentation
(Chapter 3), their fragment ions 1,3A+ and 2,3B+ would be annotated as 1,3,5A+ and 2,3,5B+,
respectively (Figure 3).
The fragmentation of glyceollins in PI mode was not investigated, because the
parent ion was not available. Ionisation of glyceollins in PI mode is known to induce an in-
source water loss, resulting in the predominance of [M+H-H2O]+ (Chapter 3). This would
result in a pterocarpene structure indicated in Figure 3, which cannot convert into an
isoflavene. The presence of the 6a-hydroxyl group may, therefore, result in a different
fragmentation pattern. Apart from the 2 fragment ions previously proposed [18], no generic
fragmentation model has been suggested for pterocarpans in PI mode. Whereas the
occurrence of 6a-hydroxy-pterocarpans is mostly confined to Glycine spp., pterocarpans
are more widespread throughout the Leguminosae. This is illustrated by their occurrence
in the economically important legumes such as the phaseollins that are found in Phaseolus
ssp., Cicer ssp., Medicago ssp. and Pisum ssp. This warrants that further investigations of
the fragmentation of pterocarpans, in both PI and NI mode.
From our MSn spectra of coumestrol in NI and PI mode, no A-ring or B-ring
fragment ions resulting from C-ring or D-ring cleavage were observed. The fragmentation
of coumestrol is mainly characterised by neutral losses of 28 (CO), 44 (CO2) and 72 (CO-
CO2). Our observations were similar to those of a study in which the same neutral losses
were observed for several different coumestans [13]. Coumestans are highly conjugated
systems which makes them very stable. In MS fragmentation, this stability appears to lead
to a limited number of neutral losses and no apparent A-ring or B-ring fragment ions due
to C-ring or D-ring cleavage.
General discussion
125
Figure 3. Fragmentation pathway of the parent ion and parent ion after water loss of 6a-hydroxy-pterocarpans in
NI mode, based on Figure 3 from Chapter 3.
LICORICE SPENT AS ALTERNATIVE SOURCE FOR PRENYLATED
ISOFLAVONOIDS
As mentioned in Chapter 1, licorice is commonly used in the tobacco, confectionary and
pharmaceutical industries mainly as a sweetening agent [19, 20]. In order to extract the
major sweet component glycyrrhizin from licorice root, the industrial processing of licorice
generally involves the extraction of ground roots with water. After filtration, the water
extract is concentrated into syrup. The syrup primarily contains the triterpenoid
glycosides, including glycyrrhizin. The by-product after water extraction is often referred
to as spent.
The spent stripped from its polar compounds still contained relatively large
amounts of non-polar constituents, like prenylated flavonoids (Figure 4). We expect
relatively high amounts of glabridin in spent compared to root and, therefore, we
considered spent as an interesting source for developing a phytoestrogen-rich health
ingredient. These compounds might be recovered with solvents such as ethanol, methanol
or ethyl-acetate (EA).
Roots and spent were extracted on lab-scale with EA and the extracts were
analysed on RP-UHPLC to profile and quantify their flavonoid content (Figure 4). The
glabridin contents for root and spent were 3.81 and 1.75 mg/g, respectively. This might
suggest that glabridin and glycyrrhizin are co- extracted with water during industrial
processing, in accordance with Tian et al. [21]. Of the solvents tested in that study, the
extraction of glycyrrhizin was optimal when performed with water, whereas ethanol
appeared to be the best solvent for glabridin extraction. However, water extraction also
resulted in an extraction of ~20% of the total amount of glabridin from roots. In our study,
we found that spent contained only ~55% of the amount of glabridin compared to that of
Chapter 6
126
roots, suggesting that glabridin losses with water extraction might be larger than
suggested by Tian et al. [21].
Furthermore, it was observed that glabrene did not occur in the UV-profile of the
spent extract but in that of the syrup. This is consistent with the postulation that glabrene
is more polar than of glabridin. Thus, water extraction of glycyrrhizin during industrial
processing seems to co-extract glabrene and, to a lesser extent, glabridin. This observation
is of importance, as glabridin and glabrene are the major estrogenic licorice root
constituents. Successful exploitation of spent as a source of phytoestrogen-rich health
ingredients might depend on minimising losses in glabridin and glabrene upon extraction
of glycyrrhizin. Depending on the market price of glycyrrhizin and a phytoestrogen-rich
extract, process innovations can be considered to avoid these losses. Several options
might be explored: (1) a reversal of the extraction order in which the roots are first
extracted with a non-polar solvent such as ethyl acetate to retrieve all phytoestrogens, or
(2) modifying the pH to enable a more specific recovery of glycyrrhizin. With respect to (1),
roots were extracted on lab-scale with 100% ethanol and 100% EA. Quantitative analysis
showed that both 100% ethanol and 100% EA extracted almost all glabridin from the root.
Furthermore, based on MS-analysis hardly any glycyrrhizin was present in the EA extract,
whereas glycyrrhizin was found to be predominantly present in both 70% and 100% EtOH
extracts. Since EA is a less polar solvent than 100% ethanol, it might provide a good
alternative to more selectively extract prenylated flavonoids and not glycyrrhizin.
Figure 4. UV-profiles of the RP-UHPLC analyses of EA extracts of licorice root (A) and spent (B).
General discussion
127
DEVELOPMENT OF NOVEL PHYTOESTROGEN-ENRICHED SOYA EXTRACTS
Pre-treatment required for optimal induction of phytoalexins?
In glyceollin research, the response of soya to the pathogenic fungus Phytophthora ssp.
mostly studied, and generally high levels of glyceollins are reported. More recently other
fungi, in particular food-grade fungi, were proven to be effective elicitors [22-25]. Different
food-grade microorganisms such as Aspergillus spp., white rice yeast and Rhizopus
oligosporus were compared regarding their ability to induce glyceollin formation [25]. All
microorganisms were able to induce glyceollin accumulation, with Rhizopus oligosporus
being the most effective.
The maximum levels of 0.9 mg (daidzein eq.)/g DW of glyceollins I-III reached in
our experiments were determined to be within the range of 0.03 to 3 mg/g DW reported
in literature but not as high as we expected (Chapter 5) [26]. Interestingly, in addition to
glyceollins I-III, a broad range of other phytoalexins accumulated in the Rhizopus-
challenged soya seedlings in our experiments, such as glycinol, glyceollin IV, glyceollin
VI/V* and glyceofuran, in similar levels as glyceollins I-III. This led to a total pterocarpan
level of up to 2.2 mg (daidzein eq.)/g DW, suggesting that the difference between our
results and those from the literature might be smaller than they appear. Furthermore,
other novel phytoalexins were identified, mainly prenylated isoflavones and prenylated
coumestans. These phytoalexins were reported for the first time (Chapter 3) in fungi-
challenged soya seedlings, leading to a total phytoalexin content of 2.9 mg (daidzein eq.)/g
DW.
In many studies, the exposure of soya to an elicitor did not last longer than 2 to 3
days and often the soya beans, its cotyledons or hypocotyls, were not germinated prior to
elicitation. In our study, soya seedlings were pre-treated for 3 days by soaking and
germination, after which the seedlings were exposed to Rhizopus spp. for a 7-day period.
Our data showed that the glyceollin content reached its maximum after 6 days of
exposure at day 9 of the induction process. In another study, the glyceollin content of soya
cotyledons that were not pre-germinated was monitored over a 6 day period [23]. In that
study, it was concluded that the maximum glyceollin content was reached after 3 days,
after which the levels stabilised or slightly decreased.
A major difference between our induction experiments and those reported in
literature is the application of wounding to the soya tissue. It has been suggested that
wounding plays an important role in the induction of glyceollins [22]. The absence of
wounding prior to inoculation might, therefore, have resulted in underproduction of
glyceollin I-III, when our results are compared to the highest values reported in the
literature. Besides the content of glyceollins, wounding might also speed up the elicitation
process, consequently reducing the time necessary to reach maximum levels of
phytoalexins.
Chapter 6
128
Growth conditions and elicitors
An important difference between our induction experiments at lab-scale (Chapter 3) and
those performed in kilogram-scale in the micro-malting system (Chapter 5) is the
difference in growth conditions. Whereas the soya beans on lab-scale are grown in
temperature-controlled climate chambers, the kilogram-scale germination of the soya
bean was performed with malting technology used in the brewing industry. Apart from a
temperature-controlled environment, malting of soya in a micro-malting system provides
air circulation, controlled in-flow of fresh air and controlled humidity. Compared to our
own lab-scale experiments, the soya beans grown in micro-malting system showed a
faster and more even growth and less spoilage. The optimised growth conditions provided
by the micro-malting system might, therefore, also affect the induction of glyceollins.
The ratio of glyceollins described in Chapter 5 was roughly 6:2:1 in the first days
of induction. Towards the end of the induction experiment (t=9) a ratio of approx. 1:1:1
was observed. This ratio has not been reported before for seedlings. In lab-scale
experiments, as described in Chapter 3, soya beans were pre-germinated for 4 days
followed by a 4-day Rhizopus-challenged germination period. The isoflavonoid profile at
the end of the 8-day induction process was characterised by a ratio of glyceollin I, II and III
of 6:2:1. Furthermore, only small amounts of glyceollin IV and VI/V*, prenylated
isoflavones and prenylated coumestans were observed in the lab-scale experiment.
Reproduction of the lab-scale induction-process in the micro-malting system with the
same soya bean batch resulted in an isoflavonoid profile similar to the lab-scale induction
process regarding diversity, but with different ratios of glyceollin I, II and III (Figure 5). In
addition, the amounts of glyceofuran, glyceollin IV and VI/V* increased relative to
glyceollin I-III, in the micro-malting system. The same observation was made for the
groups of prenylated isoflavones and coumestans. Possibly, the improved germination
conditions provided by the micro-malting system have made the elicitation process more
efficient, making pre-germination prior to elicitation obsolete. Furthermore, it would be
worthwhile to investigate the combination of wounding with micro-malting to see
whether this combination results in higher levels of phytoalexins, in particular glyceollins.
This combination might accelerate the induction process.
General discussion
129
Figure 5. RP-UHPLC-UV profile of isoflavonoids extracted from Rhizopus-challenged soya seedlings grown on lab-scale (A) and performed in the micro-malting system (B). Ap, A-ring prenylated; Bp, B-ring prenylated; Mal,
malonyl glucoside.
PRENYLATION OF ISOFLAVONOIDS IN RELATION TO ER-ANTAGONISM
The induction of phytoalexins in soya seedlings (Chapter 5) increased the total
isoflavonoid content. Concomitant to this increase in isoflavonoids, an increase in
estrogenic activity on both ER-subtypes was observed. However, this increase in
estrogenic activity was lower than what might be expected based on the calculation of the
activity in estradiol equivalents. The difference between expected and measured
estrogenicity was observed for both ER-subtypes and was more pronounced for the ERβ.
This might suggest the presence of antagonists in the soya extracts that bind both ERα and
ERβ. It has been suggested that the beneficial effects of phytoestrogens might also be
related to their antagonistic effect on the ERβ [27]. It has been hypothesised that the ERβ
plays an important role in the prevention of hyper-proliferation and carcinogenesis in the
breast, prostate and gastrointestinal tract [28]. The difference in tissue distribution led to
Chapter 6
130
the hypothesis that there are potential differences in biological function and tissue-
selective actions of the two receptors. In literature, studies mainly focus on ERα-
antagonism. The discovery of new ERα-antagonists forms the basis of the development of
alternative drugs in hormone replacement therapy and cancer prevention [29]. Therefore,
the relationship between prenylation of isoflavonoids and ERα-antagonism was further
addressed.
As mentioned before, the discrepancy in estrogenicity measured and calculated
might be explained by the presence of antagonists. For soya, this has been demonstrated
for glyceollin I [30], which comprised 8% (w/w) of the total isoflavonoids at t=9. Moreover,
it was shown that glyceollin I docks in the ERα cavity occupying the same position as the
side chain of 4-hydroxy-tamoxifen, a well-known ERα antagonist in breast cells (Figure 6).
This side-chain is responsible for its antagonism [30]. No interaction between His524 of
ERα, binding most agonists, and glyceollin I was observed. In contrast, glycinol (similar to
glyceollin I, except for the prenyl group) binds His524, and does not follow the binding
path of the side chain of 4-hydroxy-tamoxifen. Thus, prenylation might be responsible for
the different docking poses in ERα and the kind of activity (agonist vs. antagonist) on ERα.
Investigation of the estrogenic properties of licorice isoflavonoids (Chapter 4) also
suggested that prenylation of isoflavonoids might induce antagonism.
Figure 6. Structures of E2 and 4-hydroxy-tamoxifen.
In Chapter 5, the accumulation of prenylated isoflavonoids is compared with that
of non-prenylated isoflavonoids. It is clearly seen that longer induction times promote a
higher proportion of prenylated isoflavonoids, reaching similar amounts as non-prenylated
isoflavonoids at around t=7. Although prenylation seems related to ER-antagonism, it does
not mean antagonism per se, as the prenylated glyceollin II and III, as well as
alpinumisoflavone (a prenylated isoflavone), did not seem to have such activity [30, 31]. In
Chapter 4, glabrene also did not show any antagonistic activity. Table 3, and in more
condensed form Figure 7, summarises the literature data on the correlation of prenylation
of isoflavonoids and kind of estrogenic activity (agonism / antagonism). To our knowledge,
the literature does not provide evidence that non-prenylated isoflavonoids show
antagonism, no matter which isoflavonoid subclass they belong to.
Figure 7. Summary of the relationship of the position of prenylation on the isoflavonoid skeleton and ERα activity (agonist/antagonist), based on the overview in Table 3. According to IUPAC, the carbon numbering of isoflavones differs from that of pterocarpans and coumestans. Therefore, the 4 different prenylation
positions are indicated by positions α, β, γ and δ. Weak and strong agonists are distinguished (+…+, respectively). For antagonists, the potency is more difficult to derive from the literature (+), but it is clear that the potency can vary between compounds. A number of molecules are diprenylated. It is unclear whether
prenylation at both sites is essential for antagonistic activity. In case of diprenylation with two different prenyl groups, the + symbols are connected with a dotted line. X, not applicable; ?, no information available. (1) Antagonism not tested; (2) Results on antagonism are ambiguous; 3 out of 4 compounds were
antagonists, 1 out of 4 was neither agonistic nor antagonistic; (3) results on the agonistic activity of glabrene require confirmation.
Chapter 6
132
Table 3. The influence of prenyl substituents, and their position, on the agonist/antagonist activity towards the
ERα of various isoflavonoids.
Isoflavonoid Prenyl Position Second
prenyl
Position Type1 Ref.
Pterocarpans
Glycinol - - Agonist [32]
Glyceollin I Pyran α Antagonist [30, 33]
Glyceollin II Pyran β No antagonist [30]
Glyceollin III Furan2 β No antagonist [30]
Bitucarpin A Chain α Weak antagonist [34]
Phaseollin Pyran δ Antagonist [35]
Isoflavones
Genistein - - Agonist [27, 31]
8-Prenyl-genistein Chain3 α Antagonist [36 ]
Ebenosin Chain α Weak agonist4
[37]
Wighteone Chain β n.d.5 [36 ]
6,8-Diprenyl-orobol Chain α Chain β Antagonist [31, 36]
Isoerysenegalensein E Chain α Chain6 β Antagonist [31]
Erysenegalensein E Chain6 α Chain β No antagonist [31]
Millewanin G Chain α Chain6 β Antagonist [38]
Millewanin H Chain6 α Chain β Antagonist [38]
7,8-(2,2-dimethyl-pyran)-Daidzein
Pyran α Antagonist [39]
Alpinumisoflavone Pyran β No antagonist [31]
Warangalone Chain α Pyran β Weak antagonist [31]
Auriculasin Chain α Pyran β Weak antagonist [31, 36]
Furowanin A Chain α Furan7 β Antagonist [31]
Furowanin B Furan7 α Chain β Antagonist [38]
2’,5’-Diprenylorobol Chain γ Chain δ Antagonist [36]
2’-Prenyl-4’5’-(2,2-
dimethyl-pyran)orobol
Chain γ Pyran δ Antagonist [36]
Isoflavanones
Kievitone Chain α Antagonist [35]
Isoflavans
Glabridin Pyran α Antagonist Chapter 4
Isoflavenes Glabrene Pyran δ Agonist Chapter 4
Coumestans
Coumestrol - - Agonist [27, 40]
Glycyrol Chain β Agonist8 [41] 1
Antagonists are referred as weak antagonist, if specified as such in reference. 2 2’’-isopropenyl-furan. 3 1,1-dimethyl allyl. 4 Antagonistic activity not determined. 5 Not determined in yeast assay due to cytotoxicity. 6 2-hydroxy-3-methylbut-3-enyl. 7 2’’-(2-hydroxy-)isopropenyldihydrofuran.
8 >500 times weaker than coumestrol, antagonism not determined.
General discussion
133
Prenylation at the αααα -position seems to promote antagonism more than that at
the β-position. The kind of prenylation (chain or pyran) seems to be of secondary
importance. Antagonism is not confined to α-ring prenylation. Prenylation at positions γγγγ
and δδδδ also seemed to promote antagonism, but this relationship is less clear because of
less representatives. Furthermore, it is unclear whether double prenylation amplifies the
antagonistic effect. It appears that single prenylation, at least at the δδδδ-position, would also
trigger the effect. On the one hand this is corroborated by the ERα-antagonist phaseollin
(Table 3). On the other hand, from our study on the prenylated isoflavonoids from licorice
(Chapter 4), glabrene appeared to be an agonist. Clearly, this needs to be confirmed by
purification.
In summary, prenylation of isoflavonoids seems to promote antagonistic activity
on the ERα. The antagonism might be related to the presence of a bulky side chain,
hindering the correct conformation of the ER [39]. In two studies, the effect of prenylation
of isoflavonoids on their estrogenic activity was investigated. Although antagonism was
not studied, generally a loss of estrogenicity was observed upon prenylation of genistein [42]. In another study, prenylation of genistein was shown to induce antagonistic activity [36]. This might suggest a trade-off between agonistic and antagonistic activities, in which
the loss of agonism indicates the induction of antagonism.
We speculate that the increased agonistic ER activity of the soya extracts is
mainly due to the formation of the non-prenylated pterocarpan, glycinol, and the non-
prenylated coumestans, particularly coumestrol. It is expected that the extracts are also
rich in ER-antagonists, as prenylation of isoflavonoids seems to correlate well with
antagonistic properties (e.g. glyceollin I). However, due to the presence of strong agonists,
it is impossible to determine antagonistic activity of these extracts in the bioassays, and
further fractionation is a prerequisite. Furthermore, the antagonistic properties of purified
glyceollidin I, glyceollin VI, A-ring prenylated isoflavones, and 4-prenyl coumestrol, in
particular, should be determined. These compounds have potentially the right molecular
signature for antagonistic activity with prenylation at position α. In line with this, licorice
root and spent might provide a rich source of the ERα-selective antagonists. In addition to
glabridin, glabridin derivatives might also potentially possess antagonistic activity as they
comply with the structural prerequisites for antagonism. This warrants further
investigation regarding their antagonistic activity.
Chapter 6
134
ANTI-MICROBIAL ACTIVITY OF SOYA ISOFLAVONOIDS
As defined in Chapter 1, phytoalexins generally possess anti-microbial activity. This was
out of the scope of this thesis. Predominant phytoalexins in the soya extracts such as
glycinol and coumestrol have been reported to have strong anti-bacterial activity against
bacteria such as Erwinia carotovora, Pseudomonas glycinea, Escherichia coli,
Xanthomonas phaseoli, and Bacillus subtilis [43, 44]. Growth inhibition of food-borne
pathogenic bacteria would provide an additional application of soya phytoalexins in food
products.
As a preliminary experiment, the ethanol extract of Rhizopus-induced soya
seedlings, prepared at kilogram-scale, was screened for anti-microbial activity against
pathogenic bacterial strains. In this screening assay, 5 strains (Staphylococcus aureus ATCC
25923, Listeria monocytogenes EGD-e, Salmonella typhimurium S2505, Bacillus cereus
ATCC 14579, and Escherichia coli K12) were grown in Mueller Hinton broth at 30 °C in the
absence or presence of the soya extract. The growth of the bacteria was monitored by
measuring the optical density (OD) at 600 nm over a period of 24 h. The soya extract was
applied to the bacteria at a final concentration of 50 mg extract per L culture medium.
As shown in Figure 8, the soya extract showed a complete inhibition of growth of
Bacillus cereus ATCC 14579 during the experimental time frame. Similarly, also for
Staphylococcus aureus ATCC 25923 the addition of soya extract resulted in a bacteriostatic
effect (data not shown). The growth of Listeria monocytogenes EGD-e was also inhibited
as indicated by a delayed growth (Figure 8). The extracts did not have any inhibitory effect
on the growth of Salmonella typhimurium S2505 and Escherichia coli K12. These results
suggest that the soya extract is able to inhibit the growth of Gram-positive bacteria but
not Gram-negative bacteria. The anti-bacterial properties of soya extract might provide
possibilities for its application in food products to inhibit growth of Gram-positive
pathogens. Despite the promising results, it should be taken into account that anti-
bacterial activity of the active components are most likely attenuated by the food matrix
due to e.g. binding to protein. Further investigation is, therefore, imperative to determine
the effectiveness of the soya extract in real food conditions.
General discussion
135
Figure 8. The inhibitory effects of soya extract on the growth of (A) Bacillus cereus ATCC 14579 and (B) Lysteria
monocytogenes EGD-e.
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138
Appendix
140
Appendix I. Proposed fragmentation pathway of glabrene (10) in NI mode. The number
(bold) inside the parenthesis refers to the compound in Table 1, Chapter 2.
141
Appendix II. Proposed fragmentation pathway of glabrone (11) in NI mode. The number (bold) inside the parenthesis refers to the compound in Table 1, Chapter 2.
142
Appendix III. Proposed fragmentation pathway of glabrol (13) and 3-hydroxy-glabrol (17) in NI mode. The numbers (bold) inside the parenthesis refer to the compounds in Table 1, Chapter 1.
143
Appendix IV. Proposed fragmentation pathway of glabridin (12), 3’-hydroxy-4’-O-methyl-glabridin (14), 4’-O-methyl-glabridin (15), hispaglabridin A (16) and B (18) in PI mode. The numbers (bold) inside the parenthesis refer to the compounds in Table 1, Chapter 2.
Appendix V. Screening results for prenylated flavonoids in the MS chromatograms of the 70% aq. EtOH, EtOH and EA extracts of Glycyrrhiza glabra in PI mode. All peaks in this table show a neutral in their MSn spectra of 56 u, alone or in combination with a loss of 42 u. (C - prenyl chain; P - pyran ring; np - non prenylated).
MS2 MS
3
No* Rt
** (min)
MS
Identification [M+H]+
R.A
. n
eu
tra
l
loss
42
u
R.A
. n
eu
tra
l
loss
56
u
Ra
tio
(4
2:5
6)
Pre
ny
l -t
yp
e
R.A
. n
eu
tra
l
loss
42
u
R.A
. n
eu
tra
l
loss
56
u
Ra
tio
(4
2:5
6)
Pre
ny
l -t
yp
e
S1 6.21 257 4 - - - 6 3 2.0 np
S2 6.41 257 3 - - - 5 3 1.7 np
S3 6.90 257 4 - - - 6 2 3.0 np
S4 7.19 271 12 3 4.0 - - 7 0.0 np
S5 7.76 563 - - - - <1 32 0.0 np
S6 7.88 556 - - - - <1 36 0.0 np
S7 8.08 431 - - - - <1 33 0.0 np
S8 8.44 255 3 100 <0.1 np
S9 8.67 517 - - - - <1 34 0.0 C
S10 8.80 285 - 6 0.0 np
S11 9.02 315 - 4 0.0 - <1 38 0.0 C
S12 9.11 709 - - - - <1 32 0.0 np
S13 9.18 739 - - - - - 32 0.0 np
S14 9.37 469 <1 2 <0.1 - 2 3 0.7 C
S15 9.45 339 4 6 0.7 C
S16 9.51 355 - - - - 20 17 1.2 P
S17 9.57 473 - - - - - 36 0.0 C
S18 9.97 410 - 3 0.0 C - - - -
S19 10.09 285 5 100 <0.1 np
S20 10.16 410 - 100 0.0 C - - -
S21 10.27 424 - 2 0.0 C - - -
S22 10.36 339 <1 100 <0.1 C
S23 10.44 327 - - - - 3 1 3.0 P
S24 10.52 453 2 1 2.0 - 6 5 1.2 P
S25 10.56 327 - - - - 3 1 3.0 P
S26 10.79 321 24 25 1.0 C
S27 10.89 Formononetin 269 - 33 0.0 np
S28 11.18 453 1 - - - 23 24 1.0 C
S29 11.27 341 <1 100 <0.1 C
S30 11.33 371 36 49 0.7 C
S31 11.38 339 <1 100 <0.1 C
S32 11.51 323 2 100 <0.1 C
S33 11.57 341 - 100 0.0 C
S34 11.67 323 1 100 <0.1 C
S35 11.79 355 41 100 0.4 C
S36 11.90 337 1 7 0.1 C
S37 12.10 355 <1 100 <0.1 C
S38 12.20 325 3 100 <0.1 C
S39 12.28 321 24 28 0.9 C
S40 12.40 323 - 100 0.0 C
S41 12.53 322 - 7 0.0 C
S42 12.68 337 28 57 0.5 C
S43 12.72 339 5 17 0.3 C
S44 12.88 353 - 100 0.0 C
R.A. – Relative abundance of ion within MSn spectrum < 1 – between 0.5% and 1.0% n.d. - not detected, <0.5% * the ‘S’ refers to a peak found with the screening method in any of the 3 extracts ** The retention time in MS has a delay of 0.20±0.02 min compared to the UV retention time
(Appendix V. continued)
MS2 MS
3
No* Rt
** (min)
MS
Identification [M+H]+
R.A
. n
eu
tra
l
loss
42
u
R.A
. n
eu
tra
l
loss
56
u
Ra
tio
(4
2:5
6)
Pre
ny
l -t
yp
e
R.A
. n
eu
tra
l
loss
42
u
R.A
. n
eu
tra
l
loss
56
u
Ra
tio
(4
2:5
6)
Pre
ny
l -t
yp
e
S45 12.97 308 - - - - 3 4 0.8 C
S46 13.16 321 63 48 1.3 P
S47 13.25 355 - 100 0.0 C
S48 13.31 341 - 100 0.0 C
S49 13.52 409 - 21 0.0 C
S50 13.64 Glabrone 337 100 6 16.7 P
S51 13.77 Glabridin 325 - <1 0.0 - 100 1 100 P
S52 13.88 389 2 100 <0.1 C 49 51 1.0 C
S53 13.91 321 18 64 0.3 C
S54 14.08 391 - 100 0.0 C 1 100 <0.1 C
S55 14.13 645 1 100 <0.1 C 21 10 2.1 P
S56 14.18 353 - 16 0.0 C
S57 14.29 Glabrol 393 - 100 0.0 C 21 22 1.0 C
S58 14.49 409 - 100 0.0 C 19 100 0.2 C
S59 14.66 395 - 82 0.0 C
S60 14.72 647 - 60 0.0 C
S61 14.73 407 - 100 0.0 C <1 100 <0.1 C
S62 14.79 3’-hydroxy-4’-O-methyl-glabridin
355 - - - - 100 1 100 P
S63 14.80 627 2 7 0.3 np
S64 14.89 409 - 100 0.0 C 30 20 1.5 P
S65 14.91 643 2 100 <0.1 C 16 67 0.2 C
S66 15.04 647 - 14 0.0 C
S67 15.08 661 - 100 0.0 C 1 35 <0.1 C
S68 15.14 391 - 100 0.0 C <1 100 <0.1 C
S69 15.27 391 - 18 0.0 C
S70 15.30 389 2 100 <0.1 C 100 68 1.5 P
S71 15.33 407 - 100 0.0 C 5 30 1.9 P
S72 15.42 645 <1 100 <0.1 C 35 18 1.9 P
S73 15.46 627 - 3 0.0 C
S74 15.52 4’-O-methyl-glabridin 339 - - - - 100 1 100 P
S75 15.62 645 <1 57 <0.1 np
S76 15.73 631 <1 100 <0.1 C 34 6 5.7 P
S77 15.88 391 - 100 <0.1 C 6 7 0.9 C
S78 15.97 Hispaglabridin A 393 - 100 <0.1 C 21 3 7.0 P
S79 16.00 407 - 100 0.0 C 6 16 0.4 C
S80 16.08 3-hydroxy-glabrol 409 - 100 0.0 C 1 9 0.1 C
S81 16.18 391 1 39 <0.1 C
S82 16.34 409 - 42 0.0 C
S83 16.44 643 2 63 <0.1 np
S84 16.53 393 - 100 0.0 C 10 8 1.3 P
S85 16.72 659 - 26 0.0 np
S86 16.81 469 - - - - 4 14 0.3 C
S87 16.89 388 1 2 0.5 - 11 6 1.8 P
S88 17.21 Hispaglabridin B 391 - 3 - - 100 1 100 P
R.A. – Relative abundance of ion within MSn spectrum < 1 – between 0.5% and 1.0% n.d. - not detected, <0.5% * the ‘S’ refers to a peak found with the screening method in any of the 3 extracts ** The retention time in MS has a delay of 0.20±0.02 min compared to the UV retention time
148
Appendix VI. Schematic representation of the different fragmentation pathways of glycinol, glyceollidin I/II and glyceollins I-III in NI mode MS2. The weight of the arrow indicates the likelihood of the pathway.
149
Appendix VII. MS2 spectra of daidzein and 2'-hydroxy-daidzein and the MS3 spectra of A-ring and B-ring prenylated daidzein. The difference of 16 u between the MS2 fragments of 2'-hydroxydaidzein and daidzein are explained by the presence of an additional hydroxyl-group in 2'-hydroxydaidzein. Similarly, the difference of 12 u between the MS2 fragments of daidzein and the MS3 fragments of both A-ring and B-ring prenylated daidzein can be accounted for by the remaining CH group after the degradation of the prenyl group.
Appendix VIII. Fractions generated by CPC-fractionation and the presence of the main flavonoids in each fraction determined by UHPLC-MS
Fraction 1-5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Yield (mg) 1422 110 141 97 136 108 126 211 345 254 156 66 176 157 115 37 16 94 16 67 86 44 71 80
Peaks n.d.1 20 6 12 25 22 18 7 4 6 6 15 14 17 30 42 36 30 25 24 18 33 31 22
Non-prenylated - 3 1 2 4 2 1 0 0 0 0 0 1 2 4 9 3 4 2 2 2 4 4 2
Prenylated - 17 5 10 21 20 17 7 4 6 6 15 13 15 26 33 33 26 23 22 16 29 27 20
Single - 13 3 7 9 6 8 4 1 2 3 11 11 13 18 22 21 20 23 19 16 22 20 16
-Chain - 7 2 4 6 4 5 2 0 1 2 7 5 9 13 21 17 18 20 16 14 19 17 15
-Pyran - 6 1 3 3 2 3 2 1 1 1 4 6 4 5 1 4 2 3 3 2 3 3 1
Double - 4 2 3 12 14 9 3 3 4 3 4 2 2 8 11 12 6 0 3 0 7 7 4
Characterization
Glabrene ++ ++ ++ ++ +
Glabrone + + ++ +
Glabridin + + ++ ++ + + +
Glabrol + ++ ++ + +
3’-OH-4’OMe-G2 + + ++ ++
4’-OMe-G3 + + ++ +
Hispaglabridin A + ++ ++
Hispaglabridin B ++ ++ +
1 n.d. – not detected. 2 4’-OMe-glabridin 3 3’-OH-4’OMe-glabridin
(Appendix VIII. continued)
Fraction 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
Yield (mg) 80 84 9 6 87 84 54 62 16 51 32 42 5 24 35 27 28 103 90 94 142 88 233 71
Peaks 22 22 12 16 12 9 19 20 15 14 18 21 28 50 45 13 9 5 36 14 28 30 23 24
Non-
prenylated 2 3 0 0 0 1 4 5 4 4 4 4 4 11 8 4 3 4 21 10 21 28 22 23
Prenylated 20 19 12 16 12 8 15 15 11 10 14 17 24 39 37 9 6 1 15 4 7 2 1 1
Single 16 17 9 13 9 6 12 12 10 8 12 16 22 33 30 7 5 1 15 4 7 1 1 1
-Chain 15 17 9 13 9 5 9 7 7 5 8 11 13 22 21 5 5 1 14 2 5 0 0 0
-Pyran 1 0 0 0 0 1 3 5 3 3 4 5 9 11 9 2 0 0 1 2 2 1 1 1
Double 4 2 3 3 3 2 3 3 1 2 2 1 2 6 7 2 1 0 0 0 0 1 0 0
152
Appendix IX. Calculation of the estradiol equivalents (EEQ)
To determine to what extent phytoestrogens with known EC50 value can account for the
estrogenic activity of a tested extract, the measured estrogenic response of the extract
was compared to the estrogenic response calculated from the contribution of all known
phytoestrogens in the extract. This was performed for extracts of t=10 and t=4 with ERα
and ERβ, respectively. Both extracts were chosen because they exhibited an estrogenic
response between 50 and 90% of the maximum response (the steep part of the E2
standard curve).
Measured at 10 µg/mL, the extract at t=10 displayed a relative response on ERα
of 60% compared to the maximum response of E2. This response corresponds to a
concentration of 7.8×10-10 M E2. Based on the known contents of genistein, coumestrol
and daidzein in the t=10 extract, 5.0, 4.2, and 5.6 µg/mg, respectively, the concentration
of genistein, coumestrol and daidzein could be calculated in M, based on the
concentration of the extract in the assay (10 mg/L) and the molecular weight (MW) of the
three isoflavonoids. Their concentrations in the assay were 1.8×10-7, 1.6×10-7, and 2.2×10-7
M, respectively. In order to determine their E2 equivalents in M, the concentration (M) of
each phytoestrogen was multiplied by its relative estrogenic potency (REP). The sum of
the E2 equivalents of genistein, coumestrol and daidzein was 9.8×10-10 M, approximately
25% higher than the E2 equivalents measured in the assay. The same calculation was
performed for the ERβ showing that the total E2 equivalents contributed by genistein,
coumestrol and daidzein was a factor 7 higher than the E2 equivalents measured in the
assay.
(Appendix IX. continued) The estrogenic potential of the extracts of t=10 and t=4 on ERα and ERβ, respectively, measured in the assay and calculated on the basis of their genistein, coumestrol and daidzein contents (expressed in E2 equivalents), phytoestrogens with known EC50.
ERαααα
(t=10)
Content in
mg/g DW
beans*
Extraction
yield1
(mg/g)
Content in
extract
(µg/mg)
Final conc. of
extract in assay
(mg/L)
Conc. in assay
(g/L)
MW Conc.
(M)
REP2 E2 equivalents
(M)
Genistein 0.22 5.0 5.0×10-5 270 1.8×10-7 5.0×10-4 9.2×10-11
Coumestrol 0.18 43.9 4.2 10 4.2×10-5 268 1.6×10-7 5.7×10-3 8.9×10-10
Daidzein 0.24 5.6 5.6×10-5 254 2.2×10-7 -3 n.a.
Total 9.8×10-10
Actual measurement 7.8×10-10
ERββββ
(t=4)
Content in
mg/g DW
beans*
Extraction
yield1
(mg/g)
Content in
extract
(µg/mg)
Final conc. of
extract in assay
(mg/L)
Conc. in assay
(g/L)
MW Conc.
(M)
REP2 E2 equivalents
(M)
Genistein 0.32 6.7 6.7×10-6 270 2.4×10-8 1.1×10-2 2.7×10-10
Coumestrol 0.09 47.4 1.9 1 1.9×10-6 268 7.1×10-9 2.7×10-2 1.9×10-10
Daidzein 0.32 6.7 6.7×10-6 254 2.6×10-8 1.1×10-4 2.9×10-12
Total 4.7×10-10
Actual measurement 6.6×10-11
* Values represent results of a single induction experiment. 1 The amount of extract (mg) that is recovered from beans DW (g) 2 Relative estrogenic potency; REP is normalized to E2 (REPEstradiol = 1.0) (1) 3 The EC50 of daidzein was not determined due to low binding affinity MW, molecular weight n.a., not applicable 1. Bovee, T. F. H.; Helsdingen, R. J. R.; Rietjens, I. M. C. M.; Keijer, J.; Hoogenboom, R. L. A. P., Rapid yeast estrogen bioassays stably expressing human
estrogen receptors a and b, and green fluorescent protein: A comparison of different compounds with both receptor types. J. Steroid Biochem. Mol. Biol.
2004, 91, (3), 99-109.
154
Summary
156
Epidemiological and clinical studies have shown that the dietary intake of phytoestrogens
is associated with beneficial health effects in the prevention or the treatment of hormone-
dependent deficiencies including osteoporosis, cancer, cardiovascular diseases and
menopausal symptoms. A large part of the dietary intake of phytoestrogens is contributed
by legumes such as soya, either as vegetable, as health ingredient in functional foods, or
as dietary supplement (nutraceuticals). Many phytoestrogens belong to the isoflavonoid
subclasses of isoflavones, pterocarpans and coumestans, although also other phenolic
compounds, such as lignans and stilbenes, are known to display estrogenic activity.
Prenylation refers to the substitution with an C5-isoprenoid or prenyl group. In a
broad sense, prenylation can also refer to the substitution with other groups, including
C10-isoprenoid (geranyl) or C15-isoprenoid (farnesyl). This thesis will be limited to C5-
prenylation and derivatives thereof. Prenylation of isoflavonoids has been associated with
modification of their structure-activity relationship with respect to induction of estrogenic
responses. Prenylated isoflavonoids are predominantly found within the Leguminosae,
and are mostly inducible metabolites as part of the plant’s defensive strategy. Licorice
(Glycyrrhiza glabra) is a well-known legume, the roots of which are also rich in prenylated
isoflavonoids, mainly represented by the isoflavan and isoflavene subclasses. Although
soya (Glycine max) is known as a rich source of non-prenylated isoflavonoids, germination
of soya beans under biotic stress (i.e. in the presence of fungus) can induce the formation
of prenylated isoflavonoids.
To determine whether licorice and soya extracts that are rich in prenylated and
non-prenylated isoflavonoids can lead to novel and unique phytoestrogen-rich health
ingredients, research on their characterisation, induction and estrogenic activity is
imperative. Therefore, the aims of this thesis were (1) to provide a characterisation of
isoflavonoids, in particular of the prenylated isoflavonoids occurring in soya and licorice,
(2) to increase the estrogenic activity of soya beans by the application of malting in the
presence of a food-grade fungus, and (3) to correlate the agonistic/antagonistic
estrogenicity of extracts, and fractions of the extracts, with the presence of prenylated
isoflavonoids.
An overview of the family of flavonoids is presented in Chapter 1. This overview
mainly focuses on the isoflavonoid class, its structural classification and biosynthesis. The
identification of the various flavonoids by mass spectrometry is reviewed and the various
kinds of prenyl-substitution of flavonoids are addressed. Flavonoids are known to exhibit
estrogenic activity and a large part of the dietary intake is contributed by economically
important legume species such as soya and licorice. Both soya and licorice are also known
as rich sources of prenylated isoflavonoids. Special attention is paid to methods that
enhance the estrogenic potential of soya including the challenging of germinated soya
beans.
Although many prenylated flavonoids have been described in the literature, there
were no methods available that specifically screen for their presence in complex mixtures,
157
prior to purification. In Chapter 2, a method is presented that allows such screening. This
method was based on the combined chromatographic separation and mass spectrometric
analysis (RP-UHPLC/MS), and was applied to an ethyl acetate extract of licorice root. The
prenylated flavonoids showed a preferential degradation of the prenyl-group in positive-
ion (PI) mode MS2 or MS3, resulting in the characteristic neutral losses 42 (C3H6) and 56
(C4H8). It appeared that the ratio between the relative abundances of [M+H-42]+ and
[M+H-56]+ could be used for determining the kind of prenylation, i.e. chain and ring-closed
(pyran ring).
The application of biotic stress on soya seedlings is known to induce the
formation of glyceollins, which are estrogenically active prenylated pterocarpans. The
process in which these bioactive pterocarpans are formed in soya seedlings is called
induction, and consisted of 3 steps: soaking, germination and fungus-challenged
germination. Chapter 3 describes the characterisation of novel prenylated isoflavonoids
that were induced in addition to prenylated pterocarpans (glyceollins I, II and III) in
Rhizopus-challenged soya seedlings grown on laboratory scale. By means of the screening
method described in Chapter 2, it was shown that in addition to glyceollins, comprising
~40% of the total isoflavonoid content, also prenylated isoflavones and prenylated
coumestans were formed, comprising ~7% of the total isoflavonoid content. The
prenylated isoflavones and coumestans were substituted with a prenyl-chain on either the
A-ring or the B-ring. Furthermore, the fragmentation pathway for 6a-hydroxy-
pterocarpans in negative ion (NI) mode was proposed, showing A-ring and B-ring fragment
ions resulting from C-ring and D-ring cleavages. The presence of a ring-closed prenyl chain
(pyran or furan ring) yielded predominantly x,yRDA fragments; its absence gave exclusively
-H2Ox,yRDA fragments.
In Chapter 4, fractions obtained from the fractionation of a licorice root extract
by centrifugal partitioning chromatography were screened for estrogenic activity using an
in vitro yeast estrogen receptor (ER) bioassay. About one third of all tested fractions were
considered estrogenically active on one or both ER-subtypes (α and β). Some of the
fractions displayed superinduced responses, being higher than that of the 17β-estradiol
(E2) standard. This superinduction observed did not appear to be an artefact as co-
incubation with an antagonist attenuated the estrogenic response indicating that the
signal was ER-mediated. The observed superinduction was most likely caused by
stabilisation of the ER, the yeast-enhanced green fluorescent protein or a combination of
both. The estrogenic activity of some fractions was associated with the presence of
glabrene, a prenylated isoflavene. Although the predominant phytoestrogen of licorice
root, glabridin, did not show any agonistic activity on both ER-subtypes, glabridin was
found to be a potent ERα antagonist, reducing the response of E2 by approximately 80% at
a concentration of 6 × 10-6 M.
To accurately monitor the changes in both isoflavonoid profile as well as in
estrogenic activity, the induction experiment was performed at kilogram-scale by
158
application of malting technology used in the brewing industry (Chapter 5). In a time-
course experiment, performed in five-fold, the total isoflavonoid content was shown to
increase by a factor 10-12 on dry weight (DW) basis in 9 days. Also, a substantial increase
in the diversity of the isoflavonoid profile was observed. The pterocarpan content
stabilized at 2.24 mg daidzein equivalents (DE) per g after 9 days. In addition to the
glyceollins I, II and III, other less common pterocarpans, glycinol, glyceollidin I/II,
glyceofuran, glyceollin IV and VI, were formed. All pterocarpans were formed in similar
amounts ranging from 0.18 to 0.42 mg DE per g after 10 days. The content of prenylated
isoflavones and prenylated coumestans reached up to 0.30 and 0.06 mg daidzein
equivalents (DE) per g, respectively, after 10 days. These changes in the chemical
composition were accompanied with changes in bioactivity, as the extracts showed a
gradual increase in agonistic activity towards both the ERα and ERβ during the 10-day
induction process. The measured estrogenicity of the extracts on both ERs was lower than
the theoretically calculated estrogenicity, indicating the presence of antagonists. The
antagonistic activity of the extract was associated with the presence of prenylated
isoflavonoids.
In the last chapter (Chapter 6), the fragmentation of prenyl-groups in both PI and
NI mode MSn are discussed and an update of the interpretation guideline, presented in
Chapter 2, was provided. In addition, the fragmentation of prenyl groups other than the
prenyl chain and pyran ring was addressed, as well as the current knowledge on the
fragmentation of pterocarpans and coumestans. Regarding the induction of prenylated
isoflavonoids in soya, specific attention is focussed on the growth conditions of challenged
soya seedlings. Speculations on the choice of elicitor and pre-treatment in relation to
optimal induction of phytoalexins are given. Apart from its estrogenic activity, the anti-
bacterial activity of the extract from Rhizopus-challenged soya seedlings was briefly
addressed. The by-product of industrial licorice processing, spent, is evaluated as an
alternative source for prenylated isoflavonoids with estrogenic activity. Furthermore,
prenylation of isoflavonoids and its effect on estrogenic activity are discussed. An
overview is presented, based on literature and data from this thesis, on the association
between prenylation of isoflavonoids and agonism / antagonism. We hypothesize that
prenylation of isoflavonoids reverses the agonistic activity into antagonism. The
implications of employing prenylated isoflavonoid-enriched extracts from licorice and
induced soya-seedlings in the development novel phytoestrogen-enriched health
ingredients are discussed.
Samenvatting
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Epidemiologische en klinische studies hebben aangetoond dat de dagelijkse inname van
phytoestrogenen in verband kan worden gebracht met gezondheidseffecten met
betrekking tot de preventieve behandeling van hormoongerelateerde condities zoals
bijvoorbeeld botontkalking, kanker, hart- en vaatziekten en menopausale klachten. Het
leeuwendeel van de phytoestrogenen in ons dieet zijn afkomstig van peulvruchten zoals
sojabonen, geconsumeerd als groente, maar ook als gezondheidsingredient in zgn.
functional foods of in voedingssupplementen. Veel phytoestrogenen behoren tot de
isoflavonoide subklassen isoflavonen, pterocarpanen en coumestanen, maar ook andere
phenolische verbindingen, zoals lignanen en stilbenen, staan bekend om hun estrogene
activiteit.
Prenylering verwijst naar de substitutie met een C5-isoprenoid of prenyl groep. In
de brede zin van het woord, omvat prenylering ook de substitutie met andere groepen
zoals de C10-isoprenyl (geranyl) of C15-isoprenoid (farnesyl). Dit proefschrift zal zich
beperken tot C5-prenylering en afgeleiden daarvan. Prenylering van isoflavonoïden
worden in verband gebracht met veranderingen met betrekking tot hun structuur-functie
relatie betreffende de inductie van estrogene activiteit. Geprenyleerde isoflavonoïden
worden voornamelijk aangetroffen in de Leguminoseae (Vlinderbloemigen) en zijn over
het algemeen induceerbare stoffen die fungeren als onderdeel van het afweer-
mechanisme van de plant. Zoethout (Glycyrrhiza glabra) is een bekende plantensoort
binnen de Vlinderbloemigen, waarvan de wortels rijk zijn aan geprenyleerde
isoflavonoïden, met name vertegenwoordigd door de isoflavan en isoflaveen subklassen.
Hoewel soja (Glycine max) bekend staat als een rijke bron van niet-geprenyleerde
isoflavonoïden, leidt de ontkieming van soja in de aanwezigheid van een biologische vorm
van stress tot de vorming van geprenyleerde isoflavonoïden.
Om te bepalen of extracten van zoethoutwortel en soja, rijk aan zowel
geprenyleerde als niet-geprenyleerde isoflavonoïden, geschikt zijn als basis voor de
ontwikkeling van nieuwe, innonatieve phytoestrogeen-rijke gezondheidsingredienten, is
onderzoek naar karakterisering, inductie en de bepaling van estrogene activiteit een
vereiste. Dit resulteerde in de volgende doelen van het proefschrift (1) de karakterisering
van isoflavonoïden, met name geprenyleerde isoflavonoïden uit soja en zoethout, (2) het
verhogen van de estrogene activiteit van sojabonen door middel van vermouting in de
aanwezigheid van een voedselveilige schimmel, en (3) verband leggen tussen de
estrogene en antiestrogene activiteit van zowel de extracten als de fracties ervan met de
aanwezigheid van geprenyleerde isoflavonoïden.
Hoofdstuk 1 wordt ingeleid met een overzicht van de familie der flavonoïden. Dit
overzicht richt zich met name op de klassen van de isoflavonoïden, hun classificatie op
basis van structuur en biosynthese. Vervolgens wordt ingegaan op de identificatie van de
verschillende flavonoïden door middel van massa spectrometrie (MS), alsmede de
identificatie van de verschillende prenyl-substituties van flavonoïden. Flavonoïden staan
erom bekend dat zij estrogeen actief kunnen zijn en dat ze in grote mate aanwezig zijn in
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onze dagelijkse voeding in de vorm van economische belangrijke gewassen zoals zoethout
en soja. Zowel zoethout als soja staan bekend als rijke bronnen van geprenyleerde
isoflavonoïden. Nadruk wordt met name gelegd op methoden die het estrogene
potentieel van soja kunnen verhogen inclusief het onder stress ontkiemen van sojabonen.
Ondanks hun uitgebreide beschrijving in de wetenschappelijke literatuur, bestaan
er geen methoden die het mogelijk maken om te screenen voor de aanwezigheid van
geprenyleerde flavonoïden in complexe mengsels alvorens ze op te zuiveren. Hoofdstuk 2
beschrijft een methode die deze screening mogelijk maakt. Deze methode is gebaseerd op
een combinatie van chromatografische scheiding met massa spectrometrische analyse
(RP-UHPLC/MS), toegepast op een ethylacetaat extract van zoethoutwortels. De prenyl-
groepen van de geprenyleerde flavonoïden breken bij voorkeur af in positive ion (PI) mode
MS2 of MS3, hetgeen leidde tot de vorming van karakteristieke neutral losses 42 (C3H6) en
56 (C4H8). Op basis van de verhouding tussen de relatieve hoeveelheid karakteristieke
fragmenten [M+H-42]+ en [M+H-56]+ kon worden bepaald of de betreffende prenyl-
substitutie een zgn. chain of een pyraan-ring (een ring-gesloten chain) was.
Het is bekend dat de toepassing van biotsche stress op soja zaailingen leidt tot de
vorming van glyceollinen. Glyceollinen zijn estrogene pterocapranen die gesubstitueerd
zijn met een prenyl-groep. Het process dat leidt tot de vorming van deze bioactieve
pterocarpanen wordt ook wel inductie genoemd en bestaat uit 3 stappen: weken,
ontkieming en ontkieming in de aanwezigheid van een schimmel (fungus-challenged
germination). Hoofdstuk 3 beschrijft de inductie van sojabonen op laboratoriumschaal
met behulp van Rhizopus microsporus var. oryzae waarbij de vorming van nieuwe
geprenyleerde isoflavonoïden wordt beschreven, naast de vorming van de bekende
geprenyleerde pterocarpanen glyceollin I, III en III. Met behulp van de screeningsmethode
die wordt beschreven in Hoofdstuk 2, kon worden aangetoond dat behalve de
glyceollinen, die ~40% van het totale isoflavonoïden gehalte uitmaken, ook geprenyleerde
isoflavonen alsmede geprenyleerde coumestanen worden gevormd, die samen ~7% van
het totaal uitmaken. De geprenyleerde isoflavonen en coumestanen bleken
gesubstitueerd met een prenyl-chain op óf de A-ring óf de B-ring. Daarnaast werd een
overzicht van MS fragmentatie routes voorgesteld voor de fragmentatie van 6a-hydroxy-
pterocarpanen in negative ion (NI) mode, die de vorming laat zien van verschillende A-ring
en B-ring fragmenten via de splijting van zowel C als D-ring. De substitutie met een pyraan
of furaan ring resulteerde in voornamelijk x,yRDA fragmenten terwijl de afwezigheid van
zo’n substituent uitsluitend resulteerde in -H2Ox,yRDA (retroDiels-Alder) fragmenten.
Het screenen van estrogeniciteit van fracties van het zoethout extract die werden
verkregen na fractionering van het extract middels centrifugal partitioning
chromatography (CPC) wordt beschreven in Hoofdstuk 4. De estrogeniciteit van de
fracties werd bepaald door middel van een in vitro estrogeniciteitsassay bestaande uit
transgene gistcellen die de estrogeen receptor (ER) bevatten. Een derde van de fracties
bleek estrogeen te zijn op ERα, de ERβ of beide receptoren. Sommige fracties bleken een
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hogere response te genereren dan de standaard 17β-estradiol (E2). Deze zgn.
superinductie bleek geen artefact te zijn aangezien de estrogene activiteit met behulp van
een antagonist (gedeeltelijk) kon worden uitgedoofd. Dit toonde aan dat de activeteit tot
stand kwam door middel van binding aan de ER. Een logische verklaring is dat de stoffen
uit de geteste fracties in staat waren de stabiliteit van de receptor en/of het reporter-gen
te vergroten. De estrogene activiteit van bepaalde fracties werd in verband gebracht met
de aanwezigheid van glabreen, een geprenyleerde isoflaveen. Alhoewel de meest
voorkomende phytoestrogeen in de zoethoutwortel, glabridin, geen estrogene activiteit
vertoonde op beide estrogeen receptoren, bleek glabridin een sterke antagonist op de
ERα waarbij de response van E2 met ~80% werd verminderd met een concentratie van 6 ×
10-6 M.
Om de veranderingen in het isoflavonoïden profiel alsmede de estrogene
activiteit nauwkeurig te volgen, werd het soja inductie experiment uitgevoerd op
kilogramschaal door de toepassing van vermoutingstechnologie die wordt gebruikt in
bierbrouwerijen (Hoofdstuk 5). In een tijdsverloopexperiment, dat werd uitgevoerd in
vijfvoud, bleek het totale isoflavonoïdengehalte toe te nemen met een factor 10-12 in 9
dagen op basis van drooggewicht. Verder was er een significante toename in de diversiteit
van het isoflavonoïden profiel. Het gehalte aan pterocarpanen stabiliseerde rond de 2.24
mg daidzein equivalenten (DE) per g drooggewicht bonen na 9 dagen. Behalve glyceollinen
I, II en III werden ook andere, minder bekende pterocarpanen zoals glycinol, glyceollidin
I/II, glyceofuran, glyceollinen IV en VI gevormd. De gehaltes van deze minder bekende
pterocarpanen varieerden tussen de 0.18 en 0.42 mg DE per g drooggewicht bonen na 10
dagen. Het gehalte aan geprenyleerde isoflavonen en coumestanen behaalden waardes
van, respectievelijk, 0.30 en 0.06 mg DE per g drooggewicht bonen na 10 dagen. Deze
veranderingen in samenstelling van isoflavonoïden leidde tot een toename van de
bioactiviteit, hetgeen zich vertaalde in een toename in estrogeniciteit van de extracten op
beide estrogeen receptoren tijdens het 10-daagse inductie experiment. Desondanks werd
er een lagere estrogeniciteit gemeten dan verwacht op basis van de theorische berekende
estrogeniciteit, hetgeen de aanwezigheid van antagonisten suggereerde. De
antagonitische activiteit van het extract werd in verband gebracht met de aanwezigheid
van geprenyleerde isoflavonoïden.
In het laatste hoofdstuk (Hoofdstuk 6) wordt de MS fragmentatie van prenyl
groepen in zowel PI en NI mode bediscussieerd en wordt er een bijgewerkte versie van de
zgn. interpretation guideline uit Hoofdstuk 2 besproken. Daarnaast werd tevens de
fragmentatie van minder voorkomende prenylgroepen besproken alsmede de huidige
stand van zaken omtrent de kennis van fragmentatie van pterocarpanen en coumestanen.
Wat betreft de inductie van geprenyleerde isoflavonoïden in soja wordt er met name
ingegaan op de ontkiemingscondities van gestresste soja zaailingen. Er wordt
gespeculeerd over de beste keuze voor (a)biotische stimulatie en ontkiemingscondities die
leiden tot de optimale inductie van phytoalexinen. Behalve de estrogene activiteit wordt
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ook de antiestrogene activiteit besproken van de extracten van sojabonen die zijn
ontkiemd in de aanwezigheid van Rhizopus microsporus var. oryzae. Ook wordt het spent,
het bijproduct van industriële extractie van zoethoutwortel, geëvalueerd als alternatieve
bron van geprenyleerde isoflavonoïden met estrogene activiteit. Verder wordt het effect
van prenylering van isoflavonoïden op hun estrogene activiteit besproken. Op basis van
een literatuuroverzicht wordt een mogelijk verband tussen prenylering en
agonisme/antagonisme samengevat en besproken. Voortvloeiend uit dit overzicht stellen
wij de hypothese dat prenylering van isoflavonoïden leidt tot een omkering van agonisme
in antagonisme. De gevolgen hiervan voor het gebruik van extracten die rijk zijn aan
geprenyleerde isoflavonoïden uit zoethout en geïnduceerde soja zaailingen in de
ontwikkeling van nieuwe, innovatieve phytoestrogeenrijke gezondheidsingredienten
worden verder uiteengezet.
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Acknowledgements
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At first I considered a PhD program as a one-person’s project supervised by very clever people. I’ve come to realise that the further you tumble down the rabbit hole the more you appreciate and need the input and help from the people around you, both personally and professionally. First and foremost I offer my sincerest gratitude to Marian and Jean-Paul. Thank you Marian for having the faith and confidence in me by offering me this wonderful opportunity to become a PhD. Your comments during every meeting we’ve had always helped me to stay focused. Jean-Paul, I consider myself fortunate to have you as my co-promoter. Our meetings were often a combination of science, humour and inspiration. Your ideas, often born from a different point of view, helped me to think out of the box and set my priorities. I could always count on you and walk in your office, especially the past few months when I was under constant pressure of many deadlines. Both you and Marian have been a source of inspiration for me. Harry, as my promoter you were always guarding the quality of my work as well as my deadlines and planning. Although sometimes confronting, you made me realise some of my weak points. However, both you and Jean-Paul were always ready to explain and advise me but also to challenge me to re-define myself. My work on the soya germination experiments wasn’t without help. Petra, Judith, Ingrid, Heidy and Rob from the 4th floor (Food Microbiology), thanks for the hands-on and expertise. Hennie, your explanation, enthusiasm, patience and care were indispensable during the malting experiments at Holland Malt. I always enjoyed having pleasant conversations in Lieshout or on the phone. Nikos, without you I could never have pulled off all those malting experiment at Holland Malt. You were a quick learner and a hard worker. The estrogenicity screening would not have been possible without the help of Toine, Gerrit en Richard. Thanks for patiently teaching me how to master the yeast assay at the RIKILT. Teus, Johan, Sam, Tom, Karel, Martijn, as members of the FND project team, thanks for all the valuable discussions we’ve had the past 4 years. Daniel, Florian and Miroslav, my dear colleagues from Frutarom Wädenswil, thanks for making my life a little easier making those nice licorice and soya extracts. I’m looking forward to our next challenge! At Food chemistry I’ve made many wonderful colleagues with whom I could share my happiness over successful experiments and my frustrations and struggles with things like the UHPLC-MS system. Thanks Karin and Maaike for the heads-up, tips and tricks in the beginning of my glorious Ph.D. career. Maaike, my meetings with Jean-Paul were never really complete without you behind your computer, commenting on our discussion topics guarding the quality of the discussions. Roomies from 509! Koen, Takao, Lieke, Marijn, Stéphanie, Jianwei, Jun, Anne and Uttara, thanks for all the wonderful times and lovely talks we’ve had the past 4 years. I’m going to miss you a lot! Koen, thanks for the
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(pep)talks and support. Marijn, I will never forgot all our talks on Utrecht CS, in the train to Ede-Wageningen (or visa versa) and on the bike to Wageningen and back. Weer of geen
weer, het was nooit een saai ritje op de fiets met jou! Tomas, Maxime, Olga, Erika, Susan and Loes, I enjoyed working together with you on my projects. As my precious (former) students, you’ve taught me a great deal and I appreciate all the hard the work and dedication. Koos, Stefan, Yvonne V, Yvonne W, organising and leading our Ph.D. study-trip to China was one of the most extraordinary experiences in the past 4 years. From the walking through the Forbidden City, climbing the Great Wall to eating noodle soup from a plastic bag in the streets of Beijing: I’m glad to have shared it together with you. Jolanda, als spil van de afdeling was je mijn rots in de branding and oase van rust in al het tumult. Raluca, Carlos, Wibke, Tomas, Maxime and Yannick, I cherish our magnificent phytonutrient meetings on Monday morning. Those time are like the twist of lemon in my martini. Milou good luck with the follow up of my project as a Ph.D. student and thanks for critically reading my thesis. Tomas and Maxime, you have a special place in my heart. Starting as my first, respectively, second student kickstarting my soya research, becoming my dear colleagues and laboratory mates and being my paranymphs. Thanks for all the good times and being a true friend. Winter and Young. As my elder brothers, you were there when I was standing on the crossroads of my life. Your words of wisdom have been and always will be important for me. Thank you for everything. I’m grateful that I can call you my family. Patrick and Bayu, my fellow martial artist masters! Thanks for being there during the good and bad days. You brought balance and feedback allowing me to let go of life’s hardships and enjoy the moment. Yu, our talks about life over lunch were my breathing room, my moment of rest in the day. You helped me to relativise things in life. I’ll be in Beijing next year to attend your wedding! Family. To have them is one of the most important things in the world. Mama and Donny, you have been very important for me before, during and after my PhD work. We have been though a lot together and it strengthened our bonds. Thank you for understanding all the times I said I was ‘too busy’. In the end I know I can always count of you. Thank you mama for giving me a life full of chances and choices. Thank you for your endless support and love. Mootje, ich bin ech heel blie mit dich. Joop. Papa. Despite our differences and fights we are more alike than we would like admit. During my life as a PhD, I was also confronted with my strengths, strengths of which I know some were yours as well. Even after your passing, you were able to push me and stimulate me to excel. Thank you for everything and bringing out the good in me.
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Carlos. As my step-father you helped me to open my eyes. You’ve helped me through hard and difficult times with your advice and care. You were a true father to me teaching me as a son. In the difficult moments of my PhD time I often recollect our conversations and the lessons you’ve taught me. Despite the fact that you and Joop had to leave this life too early, I’m grateful for everything that I’ve learned from you both. Ye-Ji, 자기야, thanks baby for all your love, support and patience. We’ve been through a lot together, you and me. You’ve been there from the beginning and followed me throughout my journey. With your background in linguistics, a passion for languages and no affinity for science, you always tried your best to understand me when I was talking about science (again). Sorry for the times I tried to explain molecular ionisation by electrospray of isoflavonoids in mass spectrometry to you ^^;; You always believed in me and never doubted for a second. When I needed you, you were always there. I admire your courage and I am grateful for your trust in me, in us. 잘 들어 그리고 꼭 기억해. 난
죽을 때까지 너만 선택해... Looking back, forward and where I am right now: without a doubt, I consider myself a fortunate and rich man. Thanks everyone, Rudy
About the author
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CURRICULUM VITAE
Ruud (Rudy) Simons was born on the 11th on June 1980 in Gouda. Starting his secondary education at the Goudse Scholen Gemeenschap in Gouda in 1992, he graduated from the Kalsbeek College in Woerden in 1998. After his graduation he directly started his study biology at Leiden University, specialising in molecular biology and analytical chemistry. After obtaining the degree of Master of Sciences (Doctorandus) in September 2003, he started working at TNO in the department of Applied Plant Sciences.
In 2005, Rudy started working for Frutarom Netherlands as a Product Technologist. In March 2007 he was appointed as PhD candidate at the Laboratory of Food Chemistry of Wageningen University. The results of this project, under the auspices of the Food and Nutrition Delta, are summarised in this thesis.
From the 5th of April 2011, Rudy continued his work at Frutarom Netherlands in Veenendaal as a member of the Medical Science and Regulatory Affairs team.
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LIST OF PUBLICATIONS Arno Hazekamp, Ruud Simons, Anja Peltenburg-Looman, Melvin Sengers, Rianne van Zweden and Robert Verpoorte, Preparative isolation of cannabinoids from Cannabis sativa by centrifugal partition chromatography, Journal of Liquid Chromatogromatography and
Related Technologies, 2004, 27, 2421-2439 Rudy Simons, Jean-Paul Vincken, Edwin J. Bakx, Marian A. Verbruggen and Harry Gruppen, A rapid screening method for prenylated flavonoids with ultra-high-performance liquid chromatography/electrospray ionisation mass spectrometry in licorice root extracts, Rapid
Communications in Mass Spectrometry, 2009, 23, 3083–3093 Rudy Simons, Jean-Paul Vincken, Maxime C. Bohin, Tomas F.M. Kuijpers, Marian A. Verbruggen and Harry Gruppen, Identification of prenylated pterocarpans and other isoflavonoids in Rhizopus spp. elicited soya bean seedlings by electrospray ionisation mass spectrometry, Rapid Communications in Mass Spectrometry, 2011, 25, 55–65 Rudy Simons, Jean-Paul Vincken, Loes A.M. Mol, Susan A.M. The, Toine F.H. Bovee, Teus J.C. Luijendijk, Marian A. Verbruggen, Harry Gruppen, Agonistic and antagonistic estrogens in licorice root (Glycyrrhiza glabra), Analytical and Bioanalytical Chemistry, 2011, in press Rudy Simons, Jean-Paul Vincken, Nikolaos Roidos, Toine F.H. Bovee, Martijn van Iersel, Marian A. Verbruggen, Harry Gruppen, Increasing the isoflavonoid content, diversity and estrogenicity of soy upon simultaneous malting and challenging by fungus, Journal of
Agricultural and Food Chemistry, 2011, in press
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OVERVIEW OF COMPLETED TRAINING ACTIVITIES
Discipline specific activities
Courses
Metabolomics, Wageningen, the Netherlands (2007)
Mass Frontiers, Dreieich, Germany (2008)
Ecophysiology of the gastro-intestinal tract, Helsinki, Finland (2009)
Conferences and meetings
XXIV International Conference on Polyphenols, Salamance, Spain (2008)
IUFOST 15th World Congress on Food Science and Technology, Shianghai, China (2008)
Food Allergy Symposium, Hangzhou, China (2008)
Jiangnan University Doctoral Symposium, Wuxi, China (2008)
Ghent University Food Chemistry Symposium, Ghent, Belgium (2009)
International Conference on Polyphenols and Health, Harrogate, United Kingdom (2009)
Food Chemistry colloquia, Wageningen, the Netherlands (2007-2010)
Phillip Morris International Symposium, Lausanne, Switzerland (2010)
Frutarom Workshop, Wädenswil, Switzerland (2010)
XXV International Conference on Polyphenols, Montpellier, France (2010)
General courses
Vitafoods Europe, Geneva, Switzerland (2006)
CTP, CEP and Active Substance Master File, Heidelberg, Germany (2006)
International Soy and Health Conference, Düsseldorf, Germany (2006)
Project and Time Management, Wageningen, the Netherlands (2007)
Ph.D. Introduction Week, De Bilt, the Netherlands (2007)
Science, the Press and the General Public, Wageningen, the Netherlands (2009)
Mobilising your - Scientific - Network, Wageningen, the Netherlands (2009)
Additional activities
Preparation Ph.D. research proposal, Wageningen, the Netherlands (2006)
Phytonutrient meetings, Wageningen, the Netherlands (2007-2011)
Preparation of Ph.D. study trip China, Wageningen, the Netherlands (2008)
Ph.D. study trip China (2008)
Ph.D. study trip Belgium (2009)
Ph.D. study trip Switzerland and Italy (2010)
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Research presented in the Ph.D. dissertation was financially supported by the Food &
Nutrition Delta of the Ministry of Economic Affairs, the Netherlands.
Printing: Ponsen & Looijen b.v., Ede
Cover design: Diana van Houten, www.dianavanhouten.nl
Rudy Simons, 2011