Prenylated Isoflavonoids from Soya and Licorice

Prenylated isoflavonoids from soya and licorice Analysis, induction and in vitro estrogenicity Rudy Simons Thesis committee Thesis supervisor: Prof. Dr. Ir. Harry Gruppen Professor of Food Chemistry Wageningen University Thesis co-supervisor : Dr. Ir. Jean-Paul Vincken Assistent Professor, Laboratory of Food Chemistry Wageningen University Other members : Prof. Dr. Renger Witkamp Wageningen University Dr. Nigel Veitch Royal Botanic Gardens, Kew, United Kingdom Prof. Dr. Robert Verpoorte Leiden University Dr. Henk Hilhorst Wageningen University This research was conducted under the auspices of the Graduate School VLAG ( Voeding, Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid ) Prenylated isoflavonoids from soya and licorice Analysis, induction and in vitro estrogenicity Rudy Simons Thesis submitted in fulfilment of the requirements of the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr. M.J. Kropff in the presence of the Thesis Committee appointed by the Academic Board To be defended in public on Tuesday 28 June 2011 at 1.30 p.m. in the Aula. Rudy Simons Prenylated isoflavonoids from soya and licorice Analysis, induction and in vitro estrogenicity Ph.D. thesis, Wageningen University, Wageningen, the Netherlands (2011) With references, with summaries in Dutch and English ISBN: 978-90-8585-943-7 ABSTRACT Prenylated isoflavonoids are found in large amounts in soya bean ( Glycine max ) germinated under stress and in licorice ( Glycyrrhiza glabra ). Prenylation of isoflavonoids has been associated with modification of their estrogenic activity. The aims of this thesis were (1) to provide a structural characterisation of isoflavonoids, in particular the prenylated isoflavonoids occurring in soya and licorice, (2) to increase the estrogenic activity of soya beans by a malting treatment in the presence of a food-grade fungus, and (3) to correlate the in vitro agonistic/antagonistic estrogenicity with the presence of prenylated isoflavonoids. A reversed-phase UHPLC-DAD-MS n-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 37 UHPLC-ESI-MS in licorice root extracts Chapter 3 Identification of prenylated pterocarpans and other 59 isoflavonoids in Rhizopus spp. elicited soya bean seedlings by electrospray ionisation mass spectrometry Chapter 4 Agonistic and antagonistic estrogens in licorice root 77 (Glycyrrhiza glabra ) Chapter 5 Increasing soya isoflavonoid content and diversity by 95 simultaneous malting and challenging by a fungus to modulate estrogenicity Chapter 6 General discussion 117 Appendix 139 Summary 155 Samenvatting 159 Acknowledgements 165 Curriculum vitae 170 List of publications 171 Overvie w of completed training activities 172 Chapter 1 General introduction Chapter 1 STRUCTURAL CLASSIFICATION AND BIOSYNTHESIS OF FLAVONOIDS Flavonoids are a group of secondary metabolites that naturally occur throughout the plant [1] kingdom and are structurally characterised by a C 6-C3-C6 carbon framework . 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 C 6-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. 2 Figure 1 . Flavonoid subdivision into 4 main classes based on the linkage of the aromatic B-ring to the benzopyran moiety. Chapter 1 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

Load more