Analysis, induction and in vitro estrogenicity Rudy Simons

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


Other members: Prof. Dr. Renger Witkamp


Dr. Nigel Veitch

Royal Botanic Gardens, Kew, United Kingdom

Prof. Dr. Robert Verpoorte

Leiden University

Dr. Henk Hilhorst


This research was conducted under the auspices of the Graduate School VLAG (Voeding,

Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid)

Prenylated isoflavonoids from

soya and licorice


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


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].


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].


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].


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.


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].


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


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


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


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]


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.


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)-


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)


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.


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.


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.


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


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.


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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[4] Y. Zhang, P. Zhang, Y. Cheng, Structural characterization of isoprenylated flavonoids from Kushen by electrospray ionization multistage tandem mass spectrometry. Journal of Mass Spectrometry 2008, 43, 1421-1431.

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liquid chromatography coupled to electrospray ionization tandem mass spectrometry and photodiode array detection. Rapid Communications in Mass Spectrometry 2008, 22, 1767-1778.

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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

Prenylated isoflavonoids in Rhizopus spp. elicited soya bean seedlings

61

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.


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.


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).


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)


formula

[M-H]- MS

2 product ions

(relative abundance)

[M+H]+ MS

2 product ions


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)


295 277(100)

24 18.88 211,227,343 Unknown KODE Oxylipin C18H30O3 293 n.a. 295 277(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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66

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.


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.


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.


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.


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.


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.

REFERENCES [1] U. Zähringer, J. Ebel, H. Grisebach, Induction of phytoaxelin synthesis in soybean - elicitor-induced

increase in enzume-activitiet of flavonoid biosynthesis and incorporation of mevalonate into glyceollin. Archives of Biochemistry and Biophysics 1978, 188, 450-455.

[2] S. M. Boué, T. E. Cleveland, C. Carter-Wientjes, B. Y. Shih, D. Bhatnagar, J. M. McLachlan, M. E. Burow, Phytoalexin-Enriched Functional Foods. Journal of Agricultural and Food Chemistry 2009, 57, 2614-2622.

[3] H. T. Adlercreutz, Phytoestrogens. State of the art. Environmental Toxicology and Pharmacology 1999, 7, 201-207.

[4] H. J. Wang, P. A. Murphy, Isoflavone content in commercial soybean foods. Journal of Agricultural and

Food Chemistry 1994, 42, 1666-1673. [5] J. L. Ingham, N. T. Keen, L. J. Mulheirn, R. L. Lyne, Inducibly-formed isoflavonoids from leaves of

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generation of oxooctadecadienoic acids in addition to glyceollins. Journal of Agricultural and Food

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mimics in soybean defense responses. Biological Chemistry 2003, 384, 437-446. [8] A. Mosblech, I. Feussner, I. Heilmann, Oxylipins: Structurally diverse metabolites from fatty acid

oxidation. Plant Physiology and Biochemistry 2009, 47, 511-517. [9] I. Prost, S. Dhondt, G. Rothe, J. Vicente, M. J. Rodriguez, N. Kift, F. Carbonne, G. Griffiths, M. T.

Esquerré-Tugayé, S. Rosahl, C. Castresana, M. Hamberg, J. Fournier, Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens. Plant Physiology

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2729-2733. [11] R. S. Burden, J. A. Bailey, Structure of the phytoalexin from soybean. Phytochemistry 1975, 14, 1389-

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Chemical Society, Chemical Communications 1976, 497-498. [13] U. Zähringer, E. Schaller, H. Grisebach, Induction of phytoalexin synthesis in soybean. Structure and

reactions of naturally-occurring and enzymatically prepared prenylated pterocarpans from elicitor-treated cotyledons and cell-cultures of soybean. Zeitschrift Fur Naturforschung C-a 1981, 36, 234-241.

[14] R. A. Dixon, The phytoalexin response: Elicitation, signaling and control of host gene-expression. Biological Reviews of the Cambridge Philosophical Society 1986, 61, 239-291.

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[17] T. L. Graham, J. E. Kim, M. Y. Graham, Role of constitutive isoflavone conjugates in the accumulation of glyceollin in soybean infected with Phytophthora megasperma Molecular Plant-Microbe

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extracts of lupine species with LC/ESI/MSn systems. Journal of Mass Spectrometry 2005, 40, 1088-1103.

[19] P. M. Dewick, in The flavonoids - Advances in research since 1986 (Ed.: J. B. Harborne), Chapman & Hall, London, 1986, pp. 117-238.

[20] D. Fischer, C. Ebenau-Jehle, H. Grisebach, Phytoalexin synthesis in soybean: Purification and characterization of NADPH:2'-hydroxydaidzein oxidoreductase from elicitor-challenged soybean cell cultures. Archives of Biochemistry and Biophysics 1990, 276, 390-395.

[21] S. M. Boué, C. H. Carter, K. C. Ehrlich, T. E. Cleveland, Induction of the soybean phytoalexins coumestrol and glyceollin by Aspergillus. Journal of Agricultural and Food Chemistry 2000, 48, 2167-2172.

[22] W. F. Osswald, High-performance liquid chromatography of isoflavonoid phytoalexins from soybean (Glycine max, L. Harosoy 63). Journal of Chromatography A 1985, 333, 225-230.

[23] N. Fabre, I. Rustan, E. de Hoffmann, J. Quetin-Leclercq, Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. Journal of the American Society for Mass Spectrometry 2001, 12, 707-715.

[24] Y. L. Ma, Q. M. Li, H. van den Heuvel, M. Claeys, Characterization of flavone and flavonol aglycones by collision-induced dissociation tandem mass spectrometry. Rapid Communications in Mass

Spectrometry 1997, 11, 1357-1364. [25] R. Simons, J.-P. Vincken, E. J. Bakx, M. A. Verbruggen, H. 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.

[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


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.


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.


83


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


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.


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.


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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subtype-selective ligands: Asymmetric synthesis and biological evaluation of cis- and trans-5,11-dialkyl-5,6,11,12- tetrahydrochrysenes. Journal of Medicinal Chemistry 1999, 42, 2456-2468.

[25] M. G. Glazier, M. A. Bowman, A review of the evidence for the use of phytoestrogens as a replacement for traditional estrogen replacement therapy. Archives of internal medicine 2001, 161, 1161-1172.

[26] A. M. Sotoca, T. F. H. Bovee, W. Brand, N. Velikova, S. Boeren, A. J. Murk, J. Vervoort, I. M. C. M. Rietjens, Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism. Journal of Steroid Biochemistry and

Molecular Biology 2010, 122, 204-211. [27] T. F. H. Bovee, W. G. E. J. Schoonen, A. R. M. Hamers, M. J. Bento, A. A. C. M. Peijnenburg, Screening

of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities. Analytical and Bioanalytical Chemistry 2008, 390, 1111-1119.

[28] S. M. Boué, M. E. Burow, T. E. Wiese, B. Y. Shih, S. Elliott, C. H. Carter-Wientjes, J. A. McLachlan, D. Bhatnagar, Estrogenic and antiestrogenic activities of phytoalexins from red kidney bean (Phaseolus

vulgaris L.). Journal of Agricultural and Food Chemistry 2011, 59, 112-120.

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].

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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).


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


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


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.


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)


formula

[M-H]- MS

2 NI product ions


[M+H]+ MS

2 PI product ions


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.


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


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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[13] N. Fabre, I. Rustan, E. de Hoffmann, J. Quetin-Leclercq, Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. Journal of the American Society for Mass Spectrometry 2001, 12, 707-715.

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[15] J. Kang, L. A. Hick, W. E. Price, A fragmentation study of isoflavones in negative electrospray ionization by MSn ion trap mass spectrometry and triple quadrupole mass spectrometry. Rapid Communications

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[17] H. J. Yuk, J. H. Lee, M. J. Curtis-Long, J. W. Lee, Y. S. Kim, H. W. Ryu, C. G. Park, T. S. Jeong, K. H. Park, The most abundant polyphenol of soy leaves, coumestrol, displays potent α-glucosidase inhibitory activity. Food Chemistry 2011, 126, 1057-1063.

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Chapter 5

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[34] M. C. Zimmermann, S. L. Tilghman, S. M. Boué, V. A. Salvo, S. Elliott, K. Y. Williams, E. V. Skripnikova, H. Ashe, F. Payton-Stewart, L. Vanhoy-Rhodes, J. P. Fonseca, C. Corbitt, B. M. Collins-Burow, M. H. Howell, M. Lacey, B. Y. Shih, C. Carter-Wientjes, T. E. Cleveland, J. A. McLachlan, T. E. Wiese, B. S. Beckman, M. E. Burow, Glyceollin I, a novel antiestrogenic phytoalexin isolated from activated soy. Journal of Pharmacology and Experimental Therapeutics 2010, 332, 35-45.

[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


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


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

160

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

Documents

Analysis, induction and in vitro estrogenicity Rudy Simons