Chemical Profiling and In Vitro Cytotoxicity Assessment of Commercial Red Yeast Rice Ingredients

Red yeast rice (RYR) food supplements are widely used for cholesterol management due to their monacolin content, particularly monacolin K (MK), which shares the same chemical structure as lovastatin, a compound associated with several side effects. The European Food Safety Authority (EFSA) highlighted uncertainties regarding the compositional variability, characterization of bioactive constituents, and safety of RYR products, concluding that a safe level of monacolin intake could not be established. In this study, twenty-seven commercial semi-processed RYR ingredients for food supplements manufacturing were investigated through a combined chemical and biological approach to characterize their composition and evaluate potential safety concerns. Targeted LC-MS/MS analyses revealed substantial variability in MK, monacolin hydroxy acid (MKA), and dehydromonacolin K (DMK) levels among products. Exploratory qualitative untargeted metabolomics enabled the putative annotation of multiple secondary metabolites, including azaphilones and polyketides, highlighting differences in chemical profiles. Correlation analyses indicated that DMK content was associated with pigment distribution and cytotoxic responses in human hepatocellular carcinoma cells and primary human skeletal muscle cells, whereas MK and MKA showed no significant association with toxicity under the tested conditions. Exploratory proteomic profiling suggested distinct protein-abundance patterns between RYR- and simvastatin-treated cells, providing hypothesis-generating information on functional annotations associated with these patterns. These findings emphasize the importance of comprehensive chemical characterization and quality control strategies considering both monacolins and secondary metabolites to improve the safety assessment of RYR supplements.