Deciphering the origin of total estrogenic activity of complex mixtures

Abstract

Introduction: Identifying compounds with endocrine properties in food is getting increasingly important. 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.

Methods: The present study addresses a promising approach using High Performance Thin-Layer Chromatography (HPTLC) coupled to bioassays were analyzed using the Liquid Chromatography Mass-Spectrometry (LC-MS). The estrogen receptor activation was assessed using the transcription activation Estrogen Receptor Alpha Chemical Activated LUciferase gene eXpression assay (ERα- CALUX) and the HPTLC coupled to the Estrogen Screen Yeast assay (p-YES).

Results: Seven isoflavones were identified in the soy isolates. Estrogen receptor activation was assessed for both, the identified isoflavones and the soy isolates with ERα-CALUX test. Correlation between the soy isolates extracts and the identified isoflavones was shown. Moreover, p-YES revealed the presence of an estrogenic bioactive zone. Analysis of the bioactive zone through LCHRMS 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.

Conclusion: Finally, 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. Altogether, these data suggest that the integration of HPTLC-bioassay together with chemical analysis is a powerful approach to characterize the endocrine activity of complex mixtures.

Keywords: High Performance Thin-Layer Chromatography (HPTLC); chemical identification; estrogenic activity; isoflavones; mixture assessment.

FIGURE 1

CALUX and p-YES estrogen receptor activation of soy isolates extracts. (A) Bioautograms of the two soy isolates under UV-light 366 nm after p-YES assay (region of interest shown). (B) Dose-response curves obtained with CALUX and HPTLC p-YES. CALUX results are expressed in μg or pg per well, HPTLC results are expression in μg or pg per band. The ERα threshold above which a response is considered positive is indicated with a black dashed line.

FIGURE 2

(A) Extracted ion chromatogram for naringenin (C₁₅H₁₂O₅; m/z 273.0757) and formononetin (C₁₆H₁₂O₄; m/z 269.0808) in a soy protein isolate sample. (B) Fragmentation spectrum of naringenin at NCE 50. (C) Fragmentation spectrum of formononetin at NCE 70.

FIGURE 3

Soy isolate isoflavone profiles. Soy isolate 1 (4 and 6 μL, line 1 and 2, respectively), soy isolate 2 (4 and 6 μL, line 3, and 4, respectively), genistein (1.3 μg, line 5), genistin (1 μg, line 6), daidzein (1.3 μg, line 7), daidzin (1 μg, line 8), glycitein (0.4 μg, line 9), glycitin (0.6 μg, 10), and biochanin A (1 μg, line 11). (A) Autogram under UV-light 254 nm. (B) Autogram after chemical revelation with NP/PEG and documentation under UV-light 366 nm.

FIGURE 4

p-YES and CALUX estrogen receptor activation of isoflavones identified in soy protein isolates. (A) Bioautograms of isoflavones under UV-light 366 nm after p-YES assay (region of interest shown). (B) Dose response curves obtained with CALUX (black triangle) and HPTLC p-YES (black circle). For liquid format, data are expressed in pg or ng/well. For p-YES, data are expressed in pg or ng/band. The ERα threshold above which a response is considered positive is indicated with a black dashed line.