title: Bioassay directed identification of natural aryl hydrocarbon-receptor agonists in marmalade.
abstract: Citrus fruit and citrus fruit products, like grapefruit, lemon and marmalade were shown to contain aryl hydrocarbon receptor (AhR) agonists, as detected with the DR CALUX bioassay. This is of interest regarding the role of the Ah-receptor pathway in the adverse effects of dioxins, PCBs and other aromatic hydrocarbons. So far it is unclear which compounds in citrus fruit are responsible for the AhR-mediated activity and whether regular exposure to these compounds can cause effects comparable to, e.g. dioxins. The present study aimed at developing a method for identifying unknown Ah-receptor agonists in citrus products based on bioassay directed analysis, using marmalade as a first target. Following extraction with hexane and purification on an aluminium oxide-column, the extract was fractionated by HPLC using a C-18 semi-preparative column. Fractions were extracted, solvent-exchanged into dimethylsulfoxide and subsequently tested with DR CALUX. Extracts were shown to contain primarily coumarins, furocoumarins (FCs) and polymethoxyflavones (PMFs). Identification of fractions most active in the bioassay via LC/MS revealed that bergapten (an FC) is the most important Ah-receptor agonist in marmalade. The approach and method developed resulted in the successful identification of the bioactive component. However, potential pitfalls of the procedure will be discussed.
authors.affiliation: RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands.
authors.forename: Karin
authors.initials: K
authors.lastname: van Ede
authors.forename: An
authors.initials: A
authors.lastname: Li
authors.forename: Elsa
authors.initials: E
authors.lastname: Antunes-Fernandes
authors.forename: Patrick
authors.initials: P
authors.lastname: Mulder
authors.forename: Ad
authors.initials: A
authors.lastname: Peijnenburg
authors.forename: Ron
authors.initials: R
authors.lastname: Hoogenboom
mesh_terms: D000078223:5-Methoxypsoralen; D001681:Biological Assay / Q000379:methods*; D001688:Biological Products / Q000032:analysis* / Q000494:pharmacology; D032084:Citrus sinensis / Q000737:chemistry*; D004121:Dimethyl Sulfoxide; D047309:Flavones / Q000737:chemistry; D005504:Food Analysis / Q000379:methods*; D008730:Methoxsalen / Q000031:analogs & derivatives; D015394:Molecular Structure; D018336:Receptors, Aryl Hydrocarbon / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012997:Solvents; D053719:Tandem Mass Spectrometry
chemical_list: D001688:Biological Products; D047309:Flavones; D018336:Receptors, Aryl Hydrocarbon; D012997:Solvents; D000078223:5-Methoxypsoralen; D008730:Methoxsalen; D004121:Dimethyl Sulfoxide