AUTHOR=Debon Emma , Gentili Bastien , Latado Hélia , Serrant Patrick , Badoud Flavia , Ernest Marion , Christinat Nicolas , Bessaire Thomas , Schilter Benoit , Marin-Kuan Maricel 

TITLE=Deciphering the origin of total estrogenic activity of complex mixtures

JOURNAL=Frontiers in Nutrition

VOLUME=Volume 10 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/nutrition/articles/10.3389/fnut.2023.1155800

DOI=10.3389/fnut.2023.1155800

ISSN=2296-861X

ABSTRACT=Identifying compounds with endocrine properties in food is getting increasingly important. This is complex since foods are mixtures containing thousands of chemicals, with many of them being unknown. Current chemical analysis methodology is mainly focused on the identification of known substances without bringing insight for biological activity. Recently, the application of bioassays has been promoted for their potential to detect unknown bioactive substances and to provide information on possible interactions between molecules. From the toxicological perspective, measuring endocrine activity cannot inform on endocrine disruption and/or health risks without sufficient knowledge on the nature of the responsible factors. To address this limitation, the present study addresses a promising approach using High Performance Thin-Layer Chromatography (HPTLC) coupled to bioassays to link analytical and biological activity data. Two soy protein isolates anticipated to contain estrogenic chemicals served as case studies. Seven isoflavones in the isolates were identified using Liquid Chromatography Mass-Spectrometry (LC-MS) analysis. Estrogen receptor activation was assessed for both, the identified isoflavones and the soy isolates using the transcription activation Estrogen Receptor Alpha Chemical Activated LUciferase gene eXpression assay (ER-CALUX). The use of chemical derivatization for natural products using HPTLC allowed the depiction of the correlation between the soy isolates extracts and the identified isoflavones. Moreover, coupling HPTLC with the Estrogen Screen Yeast assay (p-YES) revealed the presence of an estrogenic bioactive zone. Analysis of the bioactive zone through Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS) highlighted signals corresponding to several isoflavones already detected in the isolates as well as two additional ones. For all detected isoflavones, an estrogenic activity dose-response was established in both bioassays. The measured activity potencies ranged over several orders of magnitude. Genistein, daidzein, and naringenin were found as the most active substances. A concordance analysis integrating the analytical and bioassay data indicated that genistein and daidzein were the drivers of the estrogenic activity of these soy protein isolates.