Mycotoxins found in randomly selected commercial milk thistle dietary supplement were evaluated for their toxicity in silico and in vitro. Using in silico methods, the basic physicochemical, pharmacological, and toxicological properties of the mycotoxins were predicted using ACD/Percepta. The in vitro cytotoxicity of individual mycotoxins was determined in mouse macrophage (RAW 264.7), human hepatoblastoma (HepG2), and human embryonic kidney (HEK 293T) cells. In addition, we studied the bioavailability potential of mycotoxins and silibinin utilizing an in vitro transwell system with differentiated human colon adenocarcinoma cells (Caco-2) simulating mycotoxin transfer through the intestinal epithelial barrier. The IC₅₀ values for individual mycotoxins in studied cells were in the biologically relevant ranges as follows: 3.57–13.37 nM (T-2 toxin), 5.07–47.44 nM (HT-2 toxin), 3.66–17.74 nM (diacetoxyscirpenol). Furthermore, no acute toxicity was obtained for deoxynivalenol, beauvericin, zearalenone, enniatinENN-A, enniatin-A1, enniatin-B, enniatin-B1, alternariol, alternariol-9-methyl ether, tentoxin, and mycophenolic acid up to the 50 nM concentration. The acute toxicity of these mycotoxins in binary combinations exhibited antagonistic effects in the combinations of T-2 with DON, ENN-A1, or ENN-B, while the rest showed synergistic or additive effects. Silibinin had a significant protective effect against both the cytotoxicity of three mycotoxins (T-2 toxin, HT-2 toxin, DAS) and genotoxicity of AME, AOH, DON, and ENNs on HEK 293T. The bioavailability results confirmed that AME, DAS, ENN-B, TEN, T-2, and silibinin are transported through the epithelial cell layer and further metabolized. The bioavailability of silibinin is very similar to mycotoxins poor penetration.

Herbal-based dietary supplements have become increasingly popular. The extract from milk thistle (Silybum marianum), is often used for the treatment of liver diseases. However, serious concerns exist regarding the efficacy, composition, as well as the safety of these over-the-counter preparations. Therefore, the aim of the present study was to investigate the composition as well as chemical and biological safety of 26 milk thistle-based dietary supplements purchased from both the U.S. and Czech markets between 2016 and 2017. The study was focused on a determination of the composition of active ingredients, as well as analyses of possible contaminants including: mycotoxins, plant alkaloids, and pesticide residues, as well as the microbial purity. High-throughput analyses were performed using advanced U-HPLC-HRMS techniques. Large differences in the silymarin content were observed among individual milk thistle preparations, often in contrast with the information provided by the manufacturers. In addition, substantial inter-batch differences in silymarin content were also demonstrated. In all milk thistle preparations tested, large numbers and high concentrations of mycotoxins and several pesticides, as well as the substantial presence of microbiological contamination were detected, pointing to serious safety issues. In conclusion, our results strongly indicate the need for strict controls of the composition, chemical contaminants, as well as the microbiological purity of commercial milk thistle extracts used for the treatment of liver diseases. Poor definition of these preparations together with contamination by biologically active substances may not only account for the inconsistency of clinical observations, but also be responsible for possible herbal-based dietary supplements-induced liver injury.

Hulled, or ancient, wheats were the earliest domesticated wheats by mankind and the ancestors of current wheats. Their cultivation drastically decreased during the 1960s; however, the increasing demand for a healthy and equilibrated diet led to rediscovering these grains. Our aim was to use a non-targeted metabolomic approach to discriminate and characterize similarities and differences between ancient Triticum varieties. For this purpose, 77 hulled wheat samples from three different varieties were collected: Garfagnana T. turgidum var. dicoccum L. (emmer), ID331 T. monococcum L. (einkorn) and Rouquin T. spelta L. (spelt). The ultra high performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC-QTOF) metabolomics approach highlighted a pronounced sample clustering according to the wheat variety, with an excellent predictability (Q²), for all the models built. Fifteen metabolites were tentatively identified based on accurate masses, isotopic pattern, and product ion spectra. Among these, alkylresorcinols (ARs) were found to be significantly higher in spelt and emmer, showing different homologue composition. Furthermore, phosphatidylcholines (PC) and lysophosphatidylcholines (lysoPC) levels were higher in einkorn variety. The results obtained in this study confirmed the importance of ARs as markers to distinguish between Triticum species and revealed their values as cultivar markers, being not affected by the environmental influences.

Numerous in vitro assays are used to characterize the antioxidant properties of natural-based matrices. However, many of them generate contradictory and non-compliant results. In our study, we focused on the characterization of traditionally used biochemical (2,2′-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS), Oxygen Radical Absorption Capacity (ORAC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and cellular (CAA) antioxidant tests on a broad set of milk thistle dietary supplements containing silymarin. In addition to 26 commercially available preparations, also the natural silymarin extract available from Sigma Aldrich, St. Louis, MI, USA, and a model mixture of pure flavonoid/flavonolignans mimicking the silymarin composition were investigated as control samples. Significant differences in the antioxidant capacity of the supplements were observed. Unlike the DPPH, the results of the ABTS and ORAC methods correlated with the silymarin components determined by U-HPLC-HRMS/MS. The responses in CAA were considerably lower than in other assays. Silymarin exhibited a significantly higher antioxidant capacity than the artificially prepared flavonoid/flavonolignans mixture in all tests, indicating possible presence of other antioxidants of natural origin. The follow-up U-HPLC-HRMS/MS screening revealed the presence of tens of non-silymarin compounds with reported antioxidant activity (not only in the silymarin extract, but also in the milk thistle preparations). The sum of the total phenolics and the sum of the simple phenolics correlated with CAA results more than silymarin.