Prenylated isoflavonoids from soya and licorice : analysis, induction and in vitro estrogenicity

Prenylatedisoflavonoids are found in large amountsin soya bean (Glycine max) germinated under stress and in licorice (Glycyrrhiza glabra). Prenylation of isoflavonoids has been associated with modification of theirestrogenic 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 vitroagonistic/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 diagnosticfor 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 Rhizopusspp. 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 activityusing 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 bea 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, andin 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.