Sulfation is an important reaction in nature, and sulfated phenolic compounds are of interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be prepared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work was to obtain a large library of sulfated phenols, phenolic acids, flavonoids, and flavonolignans and optimize their HPLC (high performance liquid chromatography) analysis. Four new sulfates of 2,3,4-trihydroxybenzoic acid, catechol, 4-methylcatechol, and phloroglucinol were prepared and fully characterized using MS (mass spectrometry), 1H, and 13C NMR. The separation was investigated using HPLC with PDA (photodiode-array) detection and a total of 38 standards of phenolics and their sulfates. Different stationary (monolithic C18, C18 Polar, pentafluorophenyl, ZICpHILIC) and mobile phases with or without ammonium acetate buffer were compared. The separation results were strongly dependent on the pH and buffer capacity of the mobile phase. The developed robust HPLC method is suitable for the separation of enzymatic sulfation reaction mixtures of flavonoids, flavonolignans, 2,3-dehydroflavonolignans, phenolic acids, and phenols with PDA detection. Moreover, the method is directly applicable in conjunction with mass detection due to the low flow rate and the absence of phosphate buffer and/or ion-pairing reagents in the mobile phase.

• Keywords
• Desulfitobacterium hafniense, HPLC analysis, aryl sulfotransferase, flavonoids, phenolic acid, polyphenols, sulfates,
• MeSH
• Phenols analysis   MeSH
• Flavonoids analysis   MeSH
• Flavonolignans * MeSH
• Sulfates * MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phenols MeSH
• Flavonoids MeSH
• Flavonolignans * MeSH
• Sulfates * MeSH

Milk thistle [Silybum marianum (L.) Gaertn.] achieved a significant increase in interest over the past few years from local and foreign pharmaceutical corporations. The silymarin complex of constituents extracted from milk thistle achenes provides compelling health benefits primarily thanks to antioxidant activities and hepatoprotective effects. However, consuming mycotoxin-contaminated plant material can cause immunosuppression and hepatotoxic problems. The aim of this study was to develop and validate a method for the determination of mycotoxin content in milk thistle. Fusarium toxins as T-2 and HT-2 toxins in grown milk thistle harvested from a breeding station in the Czech Republic during 2020-2021 were studied. The analysis of T-2 and HT-2 toxins was performed by UPLC-MS/MS after immunoaffinity columns EASI-EXTRACT® T-2 & HT-2 clean up. All analysed samples of milk thistle were contaminated with T-2 toxin and HT-2 toxin. The content of T-2 toxin in the samples from 2020 was in the range of 122.7-290.2 µg/kg and HT-2 toxin 157.0-319.0 µg/kg. In 2021, the content of T-2 toxin was in the range of 28.8-69.9 µg/kg and HT-2 toxin was 24.2-75.4 µg/kg. The results show that the climatic conditions of the year of harvesting have a highly statistically significant effect on the content of T-2 and HT-2 toxins in milk thistle.

• Keywords
• HT-2 toxin, T-2 toxin, UPLC-MS/MS, immunoaffinity column, milk thistle, validation method,
• MeSH
• Antioxidants MeSH
• Biological Products * MeSH
• Chromatography, Liquid MeSH
• Flavonoids MeSH
• Mycotoxins * MeSH
• Silybum marianum MeSH
• Seeds MeSH
• Silymarin * MeSH
• Plant Breeding MeSH
• T-2 Toxin * analogs & derivatives   MeSH
• Tandem Mass Spectrometry MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antioxidants MeSH
• Biological Products * MeSH
• Flavonoids MeSH
• HT-2 toxin MeSH Browser
• Mycotoxins * MeSH
• Silymarin * MeSH
• T-2 Toxin * MeSH

Milk thistle-based dietary supplements have become increasingly popular. The extract from milk thistle (Silybum marianum) is often used for the treatment of liver diseases because of the presence of its active component, silymarin. However, the co-occurrence of toxic mycotoxins in these preparations is quite frequent as well. The objective of this study was to investigate the changes in composition of liver lipidome and other clinical characteristics of experimental mice fed by a high-fat methionine-choline deficient diet inducing non-alcoholic fatty liver disease. The mice were exposed to (i) silymarin, (ii) mycotoxins (trichothecenes, enniatins, beauvericin, and altertoxins) and (iii) both silymarin and mycotoxins, and results were compared to the controls. The liver tissue extracts were analyzed by ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry. Using tools of univariate and multivariate statistical analysis, we were able to identify 48 lipid species from the classes of diacylglycerols, triacylglycerols, free fatty acids, fatty acid esters of hydroxy fatty acids and phospholipids clearly reflecting the dysregulation of lipid metabolism upon exposure to mycotoxin and/or silymarin.

• Keywords
• lipidome, mass spectrometry, metabolome, mice liver, mycotoxins, silymarin,
• MeSH
• Liver * metabolism   drug effects   MeSH
• Lipidomics * MeSH
• Lipid Metabolism drug effects   MeSH
• Mycotoxins * toxicity   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Non-alcoholic Fatty Liver Disease * metabolism   drug therapy   chemically induced   MeSH
• Silymarin * pharmacology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Mycotoxins * MeSH
• Silymarin * MeSH

The consumption of herbal-based supplements, which are believed to have beneficial effects on human health with no side effects, has become popular around the world and this trend is still increasing. Silybum marianum (L.) Gaertn, commonly known as milk thistle (MT), is the most commonly studied herb associated with the treatment of liver diseases. The hepatoprotective effects of active substances in silymarin, with silybin being the main compound, have been demonstrated in many studies. However, MT can be affected by toxigenic micro-fungi and contaminated by mycotoxins with adverse effects. The beneficial effect of silymarin can thus be reduced or totally antagonized by mycotoxins. MT has proven to be affected by micro-fungi of the Fusarium and Alternaria genera, in particular, and their mycotoxins. Alternariol-methyl-ether (AME), alternariol (AOH), beauvericin (BEA), deoxynivalenol (DON), enniatin A (ENNA), enniatin A1 (ENNA1), enniatin B (ENNB), enniatin B1 (ENNB1), HT-2 toxin (HT-2), T-2 toxin (T-2), tentoxin (TEN), and zearalenone (ZEA) seem to be most significant in MT-based dietary supplements. This review focuses on summarizing cases of mycotoxins in MT to emphasize the need for strict monitoring and regulation, as mycotoxins in relation with MT-based dietary supplements are not covered by European Union legislation.

• Keywords
• food supplements, liver diseases, milk thistle, mycotoxins, silymarin,
• MeSH
• European Union MeSH
• Humans MeSH
• Mycotoxins adverse effects   analysis   MeSH
• Silybum marianum adverse effects   MeSH
• Dietary Supplements adverse effects   analysis   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Mycotoxins MeSH

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

• Keywords
• BRAF kinase, Smoothened, cytotoxicity, in silico methods, silybins,
• Publication type
• Journal Article MeSH

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.

• Keywords
• acute toxicity, cell protection, co-culture models, combined toxicity, genotoxicity, in silico prediction, silibinin,
• MeSH
• Cell Line MeSH
• Coculture Techniques MeSH
• Comet Assay MeSH
• Drug Interactions MeSH
• Humans MeSH
• Mycotoxins toxicity   MeSH
• Mice MeSH
• Protective Agents pharmacology   MeSH
• Silybum marianum chemistry   MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism   MeSH
• Computer Simulation MeSH
• Dietary Supplements MeSH
• Silybin pharmacology   MeSH
• Cell Survival drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mycotoxins MeSH
• Protective Agents MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 MeSH
• Silybin MeSH

Herbal preparations from Silybum marianum have been used since the fourth century BC in liver disease treatment and against numerous other pathologies. Consumption of silymarin containing drugs and food supplements continues to increase. Precise, fast, reliable, and complex determination of all components of silymarin preparations is paramount for assessing its pharmacological quality. We present here simple and fast HPLC-DAD and LC-MS analytical methods for the determination and quantification of all known silymarin components, including 2,3-dehydroflavonolignans that has not been achieved so far. The first method, using a common C18 column, allows baseline separation of previously inseparable silychristin A, B, isosilychristin, and silydianin. Moreover, this method allowed detection of three so far unknown silymarin components. In addition, the first analytical separation of enantiomers of 2,3-dehydrosilybin was achieved using a Lux 3μ Cellulose-4 chiral column, providing even more accurate description of silymarin composition. 2,3-Dehydroflavonolignans were isolated for the first time from silymarin using preparative chromatography on C18 and ASAHIPAK columns, and 2,3-dehydrosilychristin and 2,3-dehydrosilybin were for the first time conclusively confirmed by HPLC, MS, and NMR to be silymarin components. Using the optimized analytical methods, six various silymarin preparations were analyzed showing substantial differences in the composition.

• Keywords
• HPLC-MS separation, diastereoisomers, enantiomers, flavonolignans, milk thistle, quantification, silymarin,
• Publication type
• Journal Article MeSH

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.

• Keywords
• ABTS, DPPH, ORAC, U-HPLC-HRMS/MS, antioxidant activity, cellular antioxidant assay, milk thistle, silymarin,
• Publication type
• Journal Article MeSH