Hop (Humulus lupulus L.) is grown mainly for the production of beer. The flowers of the female plant give it the bitter taste and pungent aroma. There are a large number of hop varieties differing in their α-acid content, essential oil levels and odor profiles. Aside from their use in brewing, more recently, hops have been used for the pharmacological properties of its derivatives that are of great importance to the pharmaceutical industry. Hop is known to have a fairly complex chemistry characterized by the presence of a variety of sesquiterpenoids, diterpenoids and triterpenoids, phytoestrogens and flavonoids. Additionally, considering the countless applications in the pharmacological sector in recent years, a chemical characterization of the different cultivars is essential to better identify the source of specific secondary metabolites. For this purpose, the dried inflorescences of two hop cultivars, Chinook and Cascade, were investigated using Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry (SPME-GC-MS and LC-MS-MS) to describe their metabolomic and proteomic profile. Furthermore, thanks to an in-depth statistical survey, it was possible to carry out a comparative study highlighting interesting implications deriving from this investigative study.

Background/Objectives: Natural products containing hydroxyanthracene derivatives (HADs) such as Cascara (Rhamnus purshiana), Frangula (Rhamnus frangula), Rhubarb (Rheum palmatum), and Senna (Cassia angustifolia) have long been used for their laxative properties, but also raise safety concerns due to reported genotoxic and carcinogenic potential. Most studies have focused on quantifying HADs, whereas the broader secondary metabolite landscape of these herbal drugs remains underexplored. We aimed to generate an untargeted metabolomic fingerprint of these four species and to explore their chemical diversity using AI-based structural classification. Methods: Four commercial botanical raw materials were extracted with 60% methanol and analysed by UPLC–HRMS/MS in positive and negative ion modes. Features were processed in Compound Discoverer and annotated by accurate mass and MS/MS matching against spectral databases, then assigned to structural classes using a graph neural network classifier. Multivariate analyses (PCA, HCA) were used to compare metabolic patterns across species. Results: In total, 93, 83, 83 and 51 metabolites were annotated in cascara, frangula, rhubarb, and senna, respectively, spanning flavonoids, anthraquinones, phenylpropanoids and other classes. Only four flavonoids were shared by all species, indicating marked biochemical divergence. Several putatively species-enriched features were observed, including pavine in cascara and frangula, vicenin-2 in senna, and piceatannol in rhubarb. Senna displayed the most distinct metabolic profile, whereas cascara and frangula clustered closely. Conclusions: This work provides a chemistry-centred metabolomic fingerprint of four HAD-containing herbal drugs using graph-based neural networks for natural product classification, supporting future studies on the pharmacological potential, bioavailability and safety of their metabolites.

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.