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Jitka Ulrichova - One of the best experts on this subject based on the ideXlab platform.
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Identification of Human Sulfotransferases Active towards Silymarin Flavonolignans and Taxifolin.
Metabolites, 2020Co-Authors: Jiří Vrba, Pavel Kosina, Barbora Papoušková, Kateřina Valentova, Kateřina Lněničková, Jitka UlrichovaAbstract:Natural phenolic compounds are known to be metabolized by phase II metabolic reactions. In this study, we examined the in vitro sulfation of the main constituents of silymarin, an herbal remedy produced from the fruits of the milk thistle. The study focused on major flavonolignan constituents, including silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin, as well as the flavonoid taxifolin. Using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS), individual Flavonolignans and taxifolin were found to be sulfated by human liver and human intestinal cytosols. Moreover, experiments with recombinant enzymes revealed that human sulfotransferases (SULTs) 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C4, and 1E1 catalyzed the sulfation of all of the tested compounds, with the exception of silydianin, which was not sulfated by SULT1B1 and SULT1C4. The sulfation products detected were monosulfates, of which some of the major ones were identified as silybin A 20-O-sulfate, silybin B 20-O-sulfate, and isosilybin A 20-O-sulfate. Further, we also observed the sulfation of the tested compounds when they were tested in the silymarin mixture. Sulfates of Flavonolignans and of taxifolin were produced by incubating silymarin with all of the above SULT enzymes, with human liver and intestinal cytosols, and also with human hepatocytes, even though the spectrum and amount of the sulfates varied among the metabolic models. Considering our results and the expression patterns of human sulfotransferases in metabolic tissues, we conclude that Flavonolignans and taxifolin can potentially undergo both intestinal and hepatic sulfation, and that SULTs 1A1, 1A3, 1B1, and 1E1 could be involved in the biotransformation of the constituents of silymarin.
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Biotransformation of Silymarin Flavonolignans by Human Fecal Microbiota
Metabolites, 2020Co-Authors: Kateřina Valentova, Jitka Ulrichova, Pavel Kosina, Barbora Papoušková, Lucie Petraskova, Jaroslav Havlik, Jose Diogenes Jaimes, Kristýna Káňová, Vladimír KřenAbstract:Flavonolignans occur typically in Silybum marianum (milk thistle) fruit extract, silymarin, which contains silybin, isosilybin, silychristin, silydianin, and their 2,3-dehydroderivatives, together with other minor flavonoids and a polymeric phenolic fraction. Biotransformation of individual silymarin components by human microbiota was studied ex vivo, using batch incubations inoculated by fecal slurry. Samples at selected time points were analyzed by ultrahigh-performance liquid chromatography equipped with mass spectrometry. The initial experiment using a concentration of 200 mg/L showed that Flavonolignans are resistant to the metabolic action of intestinal microbiota. At the lower concentration of 10 mg/L, biotransformation of Flavonolignans was much slower than that of taxifolin, which was completely degraded after 16 h. While silybin, isosilybin, and 2,3-dehydrosilybin underwent mostly demethylation, silychristin was predominantly reduced. Silydianin, 2,3-dehydrosilychristin and 2,3-dehydrosilydianin were reduced, as well, and decarbonylation and cysteine conjugation proceeded. No low-molecular-weight phenolic metabolites were detected for any of the compounds tested. Strong inter-individual differences in the biotransformation profile were observed among the four fecal-material donors. In conclusion, the Flavonolignans, especially at higher (pharmacological) doses, are relatively resistant to biotransformation by gut microbiota, which, however, depends strongly on the individual structures of these isomeric compounds, but also on the stool donor.
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Identification of UDP-glucuronosyltransferases involved in the metabolism of silymarin Flavonolignans.
Journal of pharmaceutical and biomedical analysis, 2019Co-Authors: Jiří Vrba, Pavel Kosina, Barbora Papoušková, Kateřina Lněničková, Vladimír Křen, Jitka UlrichovaAbstract:Abstract Silybum marianum (milk thistle) is a medicinal plant used for producing the hepatoprotective remedy silymarin. Its main bioactive constituents, including silybin and related Flavonolignans, can be metabolized directly by phase II conjugation reactions. This study was designed to identify UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of six silymarin Flavonolignans, namely silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. UHPLC-MS analyses showed that all of the tested compounds, both individually and in silymarin, were glucuronidated by human liver microsomes, and that glucuronidation was the main metabolic transformation in human hepatocytes. Further, each compound was glucuronidated by multiple recombinant human UGT enzymes. UGTs 1A1, 1A3, 1A8 and 1A9 were able to conjugate all of the tested Flavonolignans, and some of them were also metabolized by UGTs 1A6, 1A7, 1A10, 2B7 and 2B15. In contrast, no glucuronides were produced by UGTs 1A4, 2B4, 2B10 and 2B17. With silymarin, we found that UGT1A1 and, to a lesser extent UGT1A9, were primarily responsible for the glucuronidation of the flavonolignan constituents. It is concluded that the metabolism of silymarin Flavonolignans may involve multiple UGT enzymes, of which UGT1A1 appears to play the major role in the glucuronidation. These results may be relevant for future research on the metabolism of Flavonolignans in humans.
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A pilot study of the UVA-photoprotective potential of dehydrosilybin, isosilybin, silychristin, and silydianin on human dermal fibroblasts
Archives of Dermatological Research, 2019Co-Authors: Alena Rajnochová Svobodová, David Biedermann, Jitka Ulrichova, Eva Gabrielová, Bohumil Zálešák, Jitka VostalovaAbstract:The exposure of naked unprotected skin to solar radiation may result in numerous acute and chronic undesirable effects. Evidence suggests that silymarin, a standardized extract from Silybum marianum (L.) Gaertn. seeds, and its major component silybin suppress UVB-induced skin damage. Here, we aimed to investigate the UVA-protective effects of silymarinʼs less abundant Flavonolignans, specifically isosilybin (ISB), silychristin (SC), silydianin (SD), and 2,3-dehydrosilybin (DHSB). Normal human dermal fibroblasts (NHDF) pre-treated for 1 h with Flavonolignans were then exposed to UVA light using a solar simulator. Their effects on reactive oxygen species (ROS), carbonylated proteins and glutathione (GSH) level, caspase-3 activity, single-strand breaks’ (SSBs) formation and protein level of matrix metalloproteinase-1 (MMP-1), heme oxygenase-1 (HO-1), and heat shock protein (HSP70) were evaluated. The most pronounced preventative potential was found for DHSB, a minor component of silymarin, and SC, the second most abundant flavonolignan in silymarin. They had significant effects on most of the studied parameters. Meanwhile, a photoprotective effect of SC was mostly found at double the concentration of DHSB. ISB and SD protected against GSH depletion, the generation of ROS, carbonylated proteins and SSBs, and caspase-3 activation, but had no significant effect on MMP-1, HO-1, or HSP70. In summary, DHSB and to a lesser extent other silymarin Flavonolignans are potent UVA-protective compounds. However, due to the in vitro phototoxic potential of DHSB published elsewhere, further studies are needed to exclude phototoxicity for humans as well as to confirm our results on human skin ex vivo and in vivo.
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Skin Protective Activity of Silymarin and its Flavonolignans.
Molecules, 2019Co-Authors: Jitka Vostalova, Eva Tinková, David Biedermann, Jitka Ulrichova, Pavel Kosina, Alena Rajnochova SvobodovaAbstract:Silybum marianum (L.) is a medicinal plant traditionally used in treatment of liver disorders. In last decades, silymarin (SM), a standardized extract from S. marianum seeds has been studied for its dermatological application, namely for UVB-protective properties. However, information on SM and its polyphenols effect on activity of enzymes participating in the (photo)aging process is limited. Therefore, evaluation of SM and its Flavonolignans potential to inhibit collagenase, elastase, and hyaluronidase in tube tests was the goal of this study. The antioxidant and UV screening properties of SM and its Flavonolignans silybin, isosilybin, silydianin, silychristin and 2,3-dehydrosilybin (DHSB) were also evaluated by a DPPH assay and spectrophotometrical measurement. DHSB showed the highest ability to scavenge DPPH radical and also revealed the highest UVA protection factor (PF-UVA) that corresponds with its absorption spectrum. SM and studied Flavonolignans were found to exhibit anti-collagenase and anti-elastase activity. The most potent flavonolignan was DHSB. None of studied Flavonolignans or SM showed anti-hyaluronidase activity. Our results suggest that SM and its Flavonolignans may be useful agents for skin protection against the harmful effects of full-spectrum solar radiation including slowing down skin (photo)aging.
Nicholas H Oberlies - One of the best experts on this subject based on the ideXlab platform.
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Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides
Drug metabolism and disposition: the biological fate of chemicals, 2015Co-Authors: Brandon T Gufford, Nicholas H Oberlies, Tyler N. Graf, Noemi D. Paguigan, Mary F PaineAbstract:Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multimilligram quantities of glucuronides of select constituents (Flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDP glucuronic acid) compared with typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three monoglucuronides, respectively. Large-scale synthesis (40 mg of starting material) generated three glucuronides of silybin A: silybin A-7-O-β-d-glucuronide (15.7 mg), silybin A-5-O-β-d-glucuronide (1.6 mg), and silybin A-4´´-O-β-d-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action.
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Flavonolignans from Aspergillus iizukae, a Fungal Endophyte of Milk Thistle (Silybum marianum)
Journal of natural products, 2014Co-Authors: Tamam El-elimat, Huzefa A. Raja, Tyler N. Graf, Stanley H. Faeth, Nadja B. Cech, Nicholas H OberliesAbstract:Silybin A (1), silybin B (2), and isosilybin A (3), three of the seven Flavonolignans that constitute silymarin, an extract of the fruits of milk thistle (Silybum marianum), were detected for the first time from a fungal endophyte, Aspergillus iizukae, isolated from the surface-sterilized leaves of S. marianum. The Flavonolignans were identified using a UPLC-PDA-HRMS-MS/MS method by matching retention times, HRMS, and MS/MS data with authentic reference compounds. Attenuation of flavonolignan production was observed following successive subculturing of the original flavonolignan-producing culture, as is often the case with endophytes that produce plant-based secondary metabolites. However, production of 1 and 2 resumed when attenuated spores were harvested from cultures grown on a medium to which autoclaved leaves of S. marianum were added. The cycle of attenuation followed by resumed biosynthesis of these Flavonolignans was replicated in triplicate.
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semisynthesis cytotoxicity antiviral activity and drug interaction liability of 7 o methylated analogues of Flavonolignans from milk thistle
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Hanan S Althagafy, Tyler N. Graf, Arlene A Sycordero, Brandon T Gufford, Mary F Paine, Jessica Wagoner, Stephen J Polyak, Mitchell P Croatt, Nicholas H OberliesAbstract:Silymarin, an extract of the seeds of milk thistle (Silybum marianum), is used as an herbal remedy, particularly for hepatoprotection. The main chemical constituents in silymarin are seven Flavonolignans. Recent studies explored the non-selective methylation of one flavonolignan, silybin B, and then tested those analogues for cytotoxicity and inhibition of both cytochrome P450 (CYP) 2C9 activity in human liver microsomes and hepatitis C virus infection in a human hepatoma (Huh7.5.1) cell line. In general, enhanced bioactivity was observed with the analogues. To further probe the biological consequences of methylation of the seven major Flavonolignans, a series of 7-O-methylFlavonolignans were generated. Optimization of the reaction conditions permitted selective methylation at the phenol in the 7-position in the presence of each metabolite’s 4–5 other phenolic and/or alcoholic positions without the use of protecting groups. These 7-O-methylated analogues, in parallel with the corresponding parent compounds, were evaluated for cytotoxicity against Huh7.5.1 cells; in all cases the monomethylated analogues were more cytotoxic than the parent compounds. Moreover, parent compounds that were relatively non-toxic and inactive or weak inhibitors of hepatitis C virus infection had enhanced cytotoxicity and anti-HCV activity upon 7-O-methylation. Also, the compounds were tested for inhibition of major drug metabolizing enzymes (CYP2C9, CYP3A4/5, UDP-glucuronsyltransferases) in pooled human liver or intestinal microsomes. Methylation of Flavonolignans differentially modified inhibitory potency, with compounds demonstrating both increased and decreased potency depending upon the compound tested and the enzyme system investigated. In total, these data indicated that monomethylation modulates the cytotoxic, antiviral, and drug interaction potential of silymarin Flavonolignans.
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Interaction of silymarin Flavonolignans with organic anion-transporting polypeptides.
Drug Metabolism and Disposition, 2013Co-Authors: Kathleen Köck, Roy L. Hawke, Nicholas H Oberlies, Kim L. R. BrouwerAbstract:Organic anion-transporting polypeptides (OATPs) are multispecific transporters mediating the uptake of endogenous compounds and xenobiotics in tissues that are important for drug absorption and elimination, including the intestine and liver. Silymarin is a popular herbal supplement often used by patients with chronic liver disease; higher oral doses than those customarily used (140 mg three times/day) are being evaluated clinically. The present study examined the effect of silymarin Flavonolignans on OATP1B1-, OATP1B3-, and OATP2B1-mediated transport in cell lines stably expressing these transporters and in human hepatocytes. In overexpressing cell lines, OATP1B1- and OATP1B3-mediated estradiol-17β-glucuronide uptake and OATP2B1-mediated estrone-3-sulfate uptake were inhibited by most of the silymarin Flavonolignans investigated. OATP1B1-, OATP1B3-, and OATP2B1-mediated substrate transport was inhibited efficiently by silymarin (IC50 values of 1.3, 2.2 and 0.3 µM, respectively), silybin A (IC50 values of 9.7, 2.7 and 4.5 µM, respectively), silybin B (IC50 values of 8.5, 5.0 and 0.8 µM, respectively), and silychristin (IC50 values of 9.0, 36.4, and 3.6 µM, respectively). Furthermore, silymarin, silybin A, and silybin B (100 µM) significantly inhibited OATP-mediated estradiol-17β-glucuronide and rosuvastatin uptake into human hepatocytes. Calculation of the maximal unbound portal vein concentrations/IC50 values indicated a low risk for silymarin-drug interactions in hepatic uptake with a customary silymarin dose. The extent of silymarin-drug interactions depends on OATP isoform specificity and concentrations of Flavonolignans at the site of drug transport. Higher than customary doses of silymarin, or formulations with improved bioavailability, may increase the risk of flavonolignan interactions with OATP substrates in patients.
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Angiopreventive Efficacy of Pure Flavonolignans from Milk Thistle Extract against Prostate Cancer: Targeting VEGF-VEGFR Signaling
PloS one, 2012Co-Authors: Gagan Deep, Nicholas H Oberlies, Subhash Chander Gangar, Subapriya Rajamanickam, Komal Raina, Chapla Agarwal, Rajesh AgarwalAbstract:The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric Flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these Flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these Flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These Flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure Flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.
David Biedermann - One of the best experts on this subject based on the ideXlab platform.
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A pilot study of the UVA-photoprotective potential of dehydrosilybin, isosilybin, silychristin, and silydianin on human dermal fibroblasts
Archives of Dermatological Research, 2019Co-Authors: Alena Rajnochová Svobodová, David Biedermann, Jitka Ulrichova, Eva Gabrielová, Bohumil Zálešák, Jitka VostalovaAbstract:The exposure of naked unprotected skin to solar radiation may result in numerous acute and chronic undesirable effects. Evidence suggests that silymarin, a standardized extract from Silybum marianum (L.) Gaertn. seeds, and its major component silybin suppress UVB-induced skin damage. Here, we aimed to investigate the UVA-protective effects of silymarinʼs less abundant Flavonolignans, specifically isosilybin (ISB), silychristin (SC), silydianin (SD), and 2,3-dehydrosilybin (DHSB). Normal human dermal fibroblasts (NHDF) pre-treated for 1 h with Flavonolignans were then exposed to UVA light using a solar simulator. Their effects on reactive oxygen species (ROS), carbonylated proteins and glutathione (GSH) level, caspase-3 activity, single-strand breaks’ (SSBs) formation and protein level of matrix metalloproteinase-1 (MMP-1), heme oxygenase-1 (HO-1), and heat shock protein (HSP70) were evaluated. The most pronounced preventative potential was found for DHSB, a minor component of silymarin, and SC, the second most abundant flavonolignan in silymarin. They had significant effects on most of the studied parameters. Meanwhile, a photoprotective effect of SC was mostly found at double the concentration of DHSB. ISB and SD protected against GSH depletion, the generation of ROS, carbonylated proteins and SSBs, and caspase-3 activation, but had no significant effect on MMP-1, HO-1, or HSP70. In summary, DHSB and to a lesser extent other silymarin Flavonolignans are potent UVA-protective compounds. However, due to the in vitro phototoxic potential of DHSB published elsewhere, further studies are needed to exclude phototoxicity for humans as well as to confirm our results on human skin ex vivo and in vivo.
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Preparation of Retinoyl-Flavonolignan Hybrids and Their Antioxidant Properties.
Antioxidants (Basel Switzerland), 2019Co-Authors: Christopher S Chambers, David Biedermann, Kateřina Valentova, Marek Kuzma, Lucie Petraskova, Jitka Viktorova, Vladimír KřenAbstract:Antioxidants protect the structural and functional components in organisms against oxidative stress. Most antioxidants are of plant origin as the plants are permanently exposed to oxidative stress (UV radiation, photosynthetic reactions). Both carotenoids and flavonoids are prominent antioxidant and anti-radical agents often occurring together in the plant tissues and acting in lipophilic and hydrophilic milieu, respectively. They are complementary in their anti-radical activity. This study describes the synthesis of a series of hybrid ester conjugates of retinoic acid with various Flavonolignans, such as silybin, 2,3-dehydrosilybin and isosilybin. Antioxidant/anti-radical activities and bio-physical properties of novel covalent carotenoid-flavonoid hybrids, as well as various mixtures of the respective parent components, were investigated. Retinoyl conjugates with silybin—which is the most important flavonolignan in silymarin complex—(and its pure diastereomers) displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than both the parent compounds and their equimolar mixtures.
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2,3-Dehydroderivatives of Silymarin Flavonolignans: Prospective Natural Compounds for the Prevention of Chronic Diseases
Proceedings, 2019Co-Authors: Kateřina Valentova, David Biedermann, Vladimír KřenAbstract:Silybum marianum fruit extract silymarin displays various biological activities, which are attributed mostly to its major component silybin. However, silymarin contain several other isomeric Flavonolignans (isosilybin, silychristin, silydianin) and their oxidation products, the 2,3-dehydroFlavonolignans (2,3-dehydrosilybin, 2,3-dehydrosilychristin, 2,3-dehydrosilydianin). The latter compounds were found to be 1-2 orders of magnitude more efficient radical scavengers, reducing, chelating, cytoprotective, anti-aging, anti-cancer and anti-angiogenic agents than the parent Flavonolignans. Although 2,3-dehydroFlavonolignans occur in silymarin as minorities, they seem to be responsible for the majority of the biological activity and therefore have potential for the prevention of chronic diseases.
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Skin Protective Activity of Silymarin and its Flavonolignans.
Molecules, 2019Co-Authors: Jitka Vostalova, Eva Tinková, David Biedermann, Jitka Ulrichova, Pavel Kosina, Alena Rajnochova SvobodovaAbstract:Silybum marianum (L.) is a medicinal plant traditionally used in treatment of liver disorders. In last decades, silymarin (SM), a standardized extract from S. marianum seeds has been studied for its dermatological application, namely for UVB-protective properties. However, information on SM and its polyphenols effect on activity of enzymes participating in the (photo)aging process is limited. Therefore, evaluation of SM and its Flavonolignans potential to inhibit collagenase, elastase, and hyaluronidase in tube tests was the goal of this study. The antioxidant and UV screening properties of SM and its Flavonolignans silybin, isosilybin, silydianin, silychristin and 2,3-dehydrosilybin (DHSB) were also evaluated by a DPPH assay and spectrophotometrical measurement. DHSB showed the highest ability to scavenge DPPH radical and also revealed the highest UVA protection factor (PF-UVA) that corresponds with its absorption spectrum. SM and studied Flavonolignans were found to exhibit anti-collagenase and anti-elastase activity. The most potent flavonolignan was DHSB. None of studied Flavonolignans or SM showed anti-hyaluronidase activity. Our results suggest that SM and its Flavonolignans may be useful agents for skin protection against the harmful effects of full-spectrum solar radiation including slowing down skin (photo)aging.
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Sulfated Metabolites of Flavonolignans and 2,3-DehydroFlavonolignans: Preparation and Properties.
International journal of molecular sciences, 2018Co-Authors: Kateřina Valentova, David Biedermann, Lenka Roubalová, Lucie Petraskova, Kateřina Purchartová, Lenka Rydlová, Alena Křenková, Helena Pelantová, Veronika Holečková-moravcová, Eva TesařováAbstract:Silymarin, an extract from milk thistle (Silybum marianum) fruits, is consumed in various food supplements. The metabolism of silymarin Flavonolignans in mammals is complex, the exact structure of their metabolites still remains partly unclear and standards are not commercially available. This work is focused on the preparation of sulfated metabolites of silymarin Flavonolignans. Sulfated Flavonolignans were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate as a sulfate donor and characterized by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR). Their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging; ferric (FRAP) and Folin–Ciocalteu reagent (FCR) reducing activity; anti-lipoperoxidant potential; and effect on the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway were examined. Pure silybin A 20-O-sulfate, silybin B 20-O-sulfate, 2,3-dehydrosilybin-20-O-sulfate, 2,3-dehydrosilybin-7,20-di-O-sulfate, silychristin-19-O-sulfate, 2,3-dehydrosilychristin-19-O-sulfate, and silydianin-19-O-sulfate were prepared and fully characterized. Sulfated 2,3-dehydroderivatives were more active in FCR and FRAP assays than the parent compounds, and remaining sulfates were less active chemoprotectants. The sulfated Flavonolignans obtained can be now used as authentic standards for in vivo metabolic experiments and for further research on their biological activity.
Vladimír Křen - One of the best experts on this subject based on the ideXlab platform.
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Biotransformation of Silymarin Flavonolignans by Human Fecal Microbiota
Metabolites, 2020Co-Authors: Kateřina Valentova, Jitka Ulrichova, Pavel Kosina, Barbora Papoušková, Lucie Petraskova, Jaroslav Havlik, Jose Diogenes Jaimes, Kristýna Káňová, Vladimír KřenAbstract:Flavonolignans occur typically in Silybum marianum (milk thistle) fruit extract, silymarin, which contains silybin, isosilybin, silychristin, silydianin, and their 2,3-dehydroderivatives, together with other minor flavonoids and a polymeric phenolic fraction. Biotransformation of individual silymarin components by human microbiota was studied ex vivo, using batch incubations inoculated by fecal slurry. Samples at selected time points were analyzed by ultrahigh-performance liquid chromatography equipped with mass spectrometry. The initial experiment using a concentration of 200 mg/L showed that Flavonolignans are resistant to the metabolic action of intestinal microbiota. At the lower concentration of 10 mg/L, biotransformation of Flavonolignans was much slower than that of taxifolin, which was completely degraded after 16 h. While silybin, isosilybin, and 2,3-dehydrosilybin underwent mostly demethylation, silychristin was predominantly reduced. Silydianin, 2,3-dehydrosilychristin and 2,3-dehydrosilydianin were reduced, as well, and decarbonylation and cysteine conjugation proceeded. No low-molecular-weight phenolic metabolites were detected for any of the compounds tested. Strong inter-individual differences in the biotransformation profile were observed among the four fecal-material donors. In conclusion, the Flavonolignans, especially at higher (pharmacological) doses, are relatively resistant to biotransformation by gut microbiota, which, however, depends strongly on the individual structures of these isomeric compounds, but also on the stool donor.
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Identification of UDP-glucuronosyltransferases involved in the metabolism of silymarin Flavonolignans.
Journal of pharmaceutical and biomedical analysis, 2019Co-Authors: Jiří Vrba, Pavel Kosina, Barbora Papoušková, Kateřina Lněničková, Vladimír Křen, Jitka UlrichovaAbstract:Abstract Silybum marianum (milk thistle) is a medicinal plant used for producing the hepatoprotective remedy silymarin. Its main bioactive constituents, including silybin and related Flavonolignans, can be metabolized directly by phase II conjugation reactions. This study was designed to identify UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of six silymarin Flavonolignans, namely silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. UHPLC-MS analyses showed that all of the tested compounds, both individually and in silymarin, were glucuronidated by human liver microsomes, and that glucuronidation was the main metabolic transformation in human hepatocytes. Further, each compound was glucuronidated by multiple recombinant human UGT enzymes. UGTs 1A1, 1A3, 1A8 and 1A9 were able to conjugate all of the tested Flavonolignans, and some of them were also metabolized by UGTs 1A6, 1A7, 1A10, 2B7 and 2B15. In contrast, no glucuronides were produced by UGTs 1A4, 2B4, 2B10 and 2B17. With silymarin, we found that UGT1A1 and, to a lesser extent UGT1A9, were primarily responsible for the glucuronidation of the flavonolignan constituents. It is concluded that the metabolism of silymarin Flavonolignans may involve multiple UGT enzymes, of which UGT1A1 appears to play the major role in the glucuronidation. These results may be relevant for future research on the metabolism of Flavonolignans in humans.
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Preparation of Retinoyl-Flavonolignan Hybrids and Their Antioxidant Properties.
Antioxidants (Basel Switzerland), 2019Co-Authors: Christopher S Chambers, David Biedermann, Kateřina Valentova, Marek Kuzma, Lucie Petraskova, Jitka Viktorova, Vladimír KřenAbstract:Antioxidants protect the structural and functional components in organisms against oxidative stress. Most antioxidants are of plant origin as the plants are permanently exposed to oxidative stress (UV radiation, photosynthetic reactions). Both carotenoids and flavonoids are prominent antioxidant and anti-radical agents often occurring together in the plant tissues and acting in lipophilic and hydrophilic milieu, respectively. They are complementary in their anti-radical activity. This study describes the synthesis of a series of hybrid ester conjugates of retinoic acid with various Flavonolignans, such as silybin, 2,3-dehydrosilybin and isosilybin. Antioxidant/anti-radical activities and bio-physical properties of novel covalent carotenoid-flavonoid hybrids, as well as various mixtures of the respective parent components, were investigated. Retinoyl conjugates with silybin—which is the most important flavonolignan in silymarin complex—(and its pure diastereomers) displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than both the parent compounds and their equimolar mixtures.
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2,3-Dehydroderivatives of Silymarin Flavonolignans: Prospective Natural Compounds for the Prevention of Chronic Diseases
Proceedings, 2019Co-Authors: Kateřina Valentova, David Biedermann, Vladimír KřenAbstract:Silybum marianum fruit extract silymarin displays various biological activities, which are attributed mostly to its major component silybin. However, silymarin contain several other isomeric Flavonolignans (isosilybin, silychristin, silydianin) and their oxidation products, the 2,3-dehydroFlavonolignans (2,3-dehydrosilybin, 2,3-dehydrosilychristin, 2,3-dehydrosilydianin). The latter compounds were found to be 1-2 orders of magnitude more efficient radical scavengers, reducing, chelating, cytoprotective, anti-aging, anti-cancer and anti-angiogenic agents than the parent Flavonolignans. Although 2,3-dehydroFlavonolignans occur in silymarin as minorities, they seem to be responsible for the majority of the biological activity and therefore have potential for the prevention of chronic diseases.
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Metabolism of Flavonolignans in human hepatocytes
Journal of pharmaceutical and biomedical analysis, 2018Co-Authors: Jiří Vrba, David Biedermann, Jitka Ulrichova, Barbora Papoušková, Martina Zatloukalova, Kateřina Valentova, Vladimír Křen, Lenka Roubalová, Jan VacekAbstract:Abstract This study examined the in vitro biotransformation of eight structurally related Flavonolignans, namely silybin, 2,3-dehydrosilybin, silychristin, 2,3-dehydrosilychristin, silydianin, 2,3-dehydrosilydianin, isosilybin A and isosilybin B. The metabolic transformations were performed using primary cultures of human hepatocytes and recombinant human cytochromes P450 (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4). The metabolites produced were analyzed by ultra-performance liquid chromatography coupled with tandem mass spectrometry. We found that each of the tested compounds was metabolized in vitro by one or more CYP enzymes, which catalyzed O-demethylation, hydroxylation, hydrogenation and dehydrogenation reactions. In human hepatocytes, silybin, 2,3-dehydrosilybin, silychristin, 2,3-dehydrosilychristin, and isosilybins A and B were directly conjugated by sulfation or glucuronidation. Moreover, isosilybin A was also converted to a methyl derivative, while isosilybin B was hydroxylated and methylated. Silydianin and 2,3-dehydrosilydianin were found to undergo hydrogenation and/or glucuronidation. In addition, 2,3-dehydrosilydianin was found to be metabolically the least stable flavonolignan in human hepatocytes, and its main metabolite was a cleavage product corresponding to a loss of CO. We conclude that the hepatic biotransformation of Flavonolignans primarily involves the phase II conjugation reactions, however in some cases the phase I reactions may also occur. These results are highly relevant for research focused on flavonolignan metabolism and pharmacology.
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Salicylic acid increases Flavonolignans accumulation in the fruits of hydroponically cultured Silybum marianum
Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society, 2020Co-Authors: Hayam S. Ahmed, Sameh Abouzid, Abeer Moawad, Asmaa I. OwisAbstract:Abstract Silybum marianum (L.) Gaertn. (Asteraceae) was hydroponically cultured using a nutrient film technique system. Silibinin, isosilibinin and silychristin were detected in the fruits of the cultured plants. The effect of salicylic acid on the improvement of Flavonolignans production by the fruits of the hydroponically cultured S. marianum was investigated. Salicylic acid was added to the nutrient solution at different concentrations (100, 200 and 400 µM) and the mature fruits of the plant were collected five days after elicitor addition. The fruits were then analyzed for their total Flavonolignans contents and individual components using quantitative proton nuclear magnetic resonance spectroscopy (qHNMR) and high-performance liquid chromatography (HPLC). The results showed that elicitation with salicylic acid at 200 µM for five days increased production of total Flavonolignans (1.7-fold by qHNMR and 1.6-fold by HPLC) higher than the control cultures and (1.4-fold by qHNMR and 1.1-fold by HPLC) higher than the cultivated plants. Silychristin was the major flavonolignan produced by the cultured plant. Elicitation by 200 µM salicylic acid increased silychristin production (1.6-fold by qHNMR and HPLC) higher than the control cultures and (1.3-fold by qHNMR and 1.0-fold by HPLC) higher than the cultivated plants. The present study provides a chance to improve secondary metabolite yield, serves as a useful tool for studying the biosynthesis of these medicinally valuable compounds and its regulation in plant and spots more light on hydroponic system as an important agricultural technique.
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Linear regression analysis of silychristin A, silybin A and silybin B contents in Silybum marianum
2018Co-Authors: Sameh Abouzid, Hayam S. Ahmed, Asmaa I. Owis, Shao-nong Chen, Amandine Nachtergael, James B. Mcalpine, Abd-el Mageed Abd El A. Mageed, Abeer S. Moawad, Brent J. Friesen, Guido F. PauliAbstract:Quantitative correlations between the contents of the Flavonolignans silychristin A and silybins A/B provide biosynthetic clues that support a pathway in which one mesomeric form of a taxifolin radical is undergoing an oxidative coupling with a coniferyl alcohol radical. The flavonolignan content and patterns reported in the literature for 53 samples, representing populations of the Silybum marianum plant growing in different parts of the world, were subject to a meta-analysis. Linear regression analyses were carried out on these data sets, and a mathematical model was derived that predicts the content of silychristin A relative to the metabolomic pattern of its congeners. The validity of the model was verified by applying it to test samples. This approach could potentially become a tool to enhance the understanding of both the relative composition of the silymarin complex and the biosynthetic pathways that underlie its formation.
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Chemotaxonomic and biosynthetic relationships between Flavonolignans produced by Silybum marianum populations
Fitoterapia, 2017Co-Authors: Sameh Abouzid, Hayam S. Ahmed, Abeer Moawad, Asmaa I. Owis, Shao-nong Chen, Amandine Nachtergael, James B. Mcalpine, J. Brent Friesen, Guido F. PauliAbstract:Flavonolignans constitute an important class of plant secondary metabolites formed by oxidative coupling of one flavonoid and one phenylpropanoid moiety. The standardized flavonolignan-rich extract prepared from the fruits of Silybum marianum is known as silymarin and has long been used medicinally, prominently as an antihepatotoxic and as a chemopreventive agent. Principal component analysis of the variation in flavonolignan content in S. marianum samples collected from different locations in Egypt revealed biosynthetic relationships between the Flavonolignans. Silybin A, silybin B, and silychristin are positively correlated as are silydianin, isosilychristin, and isosilybin B. The detection of silyamandin in the extracts of S. marianum correlates with isosilychristin and silydianin content. The positive correlation between silydianin, isosilychristin, and silyamandin was demonstrated using quantitative 1H nuclear magnetic resonance spectroscopy (qHNMR). These correlations can be interpreted as evidence for the involvement of a flavonoid radical in the biosynthesis of the Flavonolignans in S. marianum. The predominance of silybins A & B over isosilybin A & B in the silybin-rich samples is discussed in light of the relative stabilities of their respective radical flavonoid biosynthetic intermediates.
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Silymarin Flavonolignans: Structure–Activity Relationship and Biosynthesis
Studies in natural products chemistry, 2013Co-Authors: Sameh Abouzid, Osama M. AhmedAbstract:Abstract Silymarin is a well-known hepatoprotective agent having an exceptional safety profile. It exerts its action by antioxidant, anti-inflammatory, immunomodulatory, antiproliferative, antifibrotic, and antiviral activities. Silymarin is composed of an isomeric mixture of seven Flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B, and silydianin and one flavonoid taxifolin. Silibinin, a mixture of silybin A and silybin B, is considered responsible for the hepatoprotective functions of silymarin. Purification of these compounds in gram scale allowed for testing the hepatoprotective effects of pure compounds in various assays. Some individual Flavonolignans showed stronger hepatoprotective functions than silymarin. Only isosilybin B showed high toxicity to human hepatoma cell line. Isosilybin A, taxifolin, and silibinin were the most effective hepatoprotectors. Isosilybin B showed the highest antiproliferative activity against human prostate carcinoma cell lines. Biosynthesis of silymarin Flavonolignans occurs by oxidative coupling between the phenylpropanoid coniferyl alcohol and the flavonoid taxifolin. Flavonoid biosynthesis involves phenylpropanoid and polyketide pathways. Plant tissue culture studies have largely contributed to our current understanding of silymarin biosynthesis. Production of silymarin in Silybum marianum cultures can be stimulated by treatment with elicitors such as yeast extract and methyl jasmonate. The components of phenylpropanoid pathway are not modified by elicitation of cell cultures of S. marianum with yeast extract or methyl jasmonate. This was concluded when the overall metabolic changes in elicitor-treated cultures were studied using nuclear magnetic spectroscopy. The flavonoid biosynthesis, not coniferyl alcohol biosynthesis, may be the candidate component of the signaling pathway involved in stimulation of flavonolignan biosynthesis with elicitors. Understanding the basic signaling components in the transduction of the elicitor signal to downstream responses such as silymarin production is mandatory for biotechnological exploitation of this valuable pharmaceutical raw material. Lipoxygenase involvement in the elicitor-induced accumulation of silymarin is well established. Inhibition of external and internal calcium fluxes significantly increases flavonolignan production. Activation of phospholipase D after elicitor treatment mediates silymarin secretion into the culture medium, indicating possible involvement of the enzyme in deposition of silymarin in the external cover of the fruits of S . marianum .
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silymarin Flavonolignans structure activity relationship and biosynthesis
Studies in natural products chemistry, 2013Co-Authors: Sameh Abouzid, Osama M. AhmedAbstract:Abstract Silymarin is a well-known hepatoprotective agent having an exceptional safety profile. It exerts its action by antioxidant, anti-inflammatory, immunomodulatory, antiproliferative, antifibrotic, and antiviral activities. Silymarin is composed of an isomeric mixture of seven Flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin A, silychristin B, and silydianin and one flavonoid taxifolin. Silibinin, a mixture of silybin A and silybin B, is considered responsible for the hepatoprotective functions of silymarin. Purification of these compounds in gram scale allowed for testing the hepatoprotective effects of pure compounds in various assays. Some individual Flavonolignans showed stronger hepatoprotective functions than silymarin. Only isosilybin B showed high toxicity to human hepatoma cell line. Isosilybin A, taxifolin, and silibinin were the most effective hepatoprotectors. Isosilybin B showed the highest antiproliferative activity against human prostate carcinoma cell lines. Biosynthesis of silymarin Flavonolignans occurs by oxidative coupling between the phenylpropanoid coniferyl alcohol and the flavonoid taxifolin. Flavonoid biosynthesis involves phenylpropanoid and polyketide pathways. Plant tissue culture studies have largely contributed to our current understanding of silymarin biosynthesis. Production of silymarin in Silybum marianum cultures can be stimulated by treatment with elicitors such as yeast extract and methyl jasmonate. The components of phenylpropanoid pathway are not modified by elicitation of cell cultures of S. marianum with yeast extract or methyl jasmonate. This was concluded when the overall metabolic changes in elicitor-treated cultures were studied using nuclear magnetic spectroscopy. The flavonoid biosynthesis, not coniferyl alcohol biosynthesis, may be the candidate component of the signaling pathway involved in stimulation of flavonolignan biosynthesis with elicitors. Understanding the basic signaling components in the transduction of the elicitor signal to downstream responses such as silymarin production is mandatory for biotechnological exploitation of this valuable pharmaceutical raw material. Lipoxygenase involvement in the elicitor-induced accumulation of silymarin is well established. Inhibition of external and internal calcium fluxes significantly increases flavonolignan production. Activation of phospholipase D after elicitor treatment mediates silymarin secretion into the culture medium, indicating possible involvement of the enzyme in deposition of silymarin in the external cover of the fruits of S . marianum .