European Food Safety Authority (EFSA) concerns on botanicals: characterization and safety evaluation of anthraquinone and red yeast rice food supplements

The use of botanicals and natural products for health purposes has seen exponential growth in recent years, driving the need for accurate scientific evaluations to ensure quality, efficacy, and safety. This doctoral research work focused on two classes of botanical dietary supplements: those containing hydroxyanthracene derivatives (HADs) and the other ones concerning red yeast rice (RYR), both of them have been the subject of scientific opinions issued by EFSA in response to potential consumer health risks. The primary objective was to perform a detailed characterization of their chemical composition and to investigate their toxicological effects using integrated approaches in analytical chemistry and molecular biology. A total of twenty-nine HAD samples and twenty-seven RYR samples were analyzed using liquid chromatography coupled with mass spectrometry (LC-MS/MS), enabling the identification and quantification of key markers as well as the detection of potential contaminants. Phytochemical analyses revealed significant compositional variability among commercial samples, reflecting the lack of standardization in production. In vitro cytotoxicity studies on human cell lines demonstrated dose-dependent toxicity, with notable differences between isolated molecules and complete extracts. In the case of HADs, toxicity was primarily attributed to the genotoxic potential of compounds such as emodin and aloe-emodin, while for RYR, the attention was focused on the variable levels of monacolin K, secondary monacolins content and potential citrinin contamination. Proteomic analyses, performed on in vitro cell models, provided detailed insights into changes in cellular protein profiles, in particular toxicological pathways and biomarkers associated with adverse effects have been investigated. This data enhanced the understanding of the molecular mechanisms underlying the difference in the toxicity of botanical products in comparison to single molecules, highlighting the critical role of interactions between compounds in complete extracts. The results of this study were compiled into two scientific dossiers submitted to EFSA to support regulatory decision-making. This work represents a significant step forward in the understanding of botanical safety, promoting the adoption of more rigorous monitoring strategies and the development of safer dietary supplements for consumers.