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.

• Klíčová slova
• Desulfitobacterium hafniense, HPLC analysis, aryl sulfotransferase, flavonoids, phenolic acid, polyphenols, sulfates,
• MeSH
• fenoly analýza   MeSH
• flavonoidy analýza   MeSH
• flavonolignany * MeSH
• sírany * MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fenoly MeSH
• flavonoidy MeSH
• flavonolignany * MeSH
• sírany * MeSH

This review focuses on the specific biological effects of optically pure silymarin flavo-nolignans, mainly silybins A and B, isosilybins A and B, silychristins A and B, and their 2,3-dehydro derivatives. The chirality of these flavonolignans is also discussed in terms of their analysis, preparative separation and chemical reactions. We demonstrated the specific activities of the respective diastereomers of flavonolignans and also the enantiomers of their 2,3-dehydro derivatives in the 3D anisotropic systems typically represented by biological systems. In vivo, silymarin flavonolignans do not act as redox antioxidants, but they play a role as specific ligands of biological targets, according to the "lock-and-key" concept. Estrogenic, antidiabetic, anticancer, antiviral, and antiparasitic effects have been demonstrated in optically pure flavonolignans. Potential application of pure flavonolignans has also been shown in cardiovascular and neurological diseases. Inhibition of drug-metabolizing enzymes and modulation of multidrug resistance activity by these compounds are discussed in detail. The future of "silymarin applications" lies in the use of optically pure components that can be applied directly or used as valuable lead structures, and in the exploration of their true molecular effects.

• Klíčová slova
• Silybum marianum, chirality, dehydroflavonolignan, diastereomer, flavonoid, flavonolignan, isosilybin, milk thistle, silibinin, silybin, silychristin, silydianin, silymarin,
• MeSH
• antiinfekční látky chemie   farmakologie   MeSH
• antioxidancia chemie   farmakologie   MeSH
• fytogenní protinádorové látky chemie   farmakologie   MeSH
• lidé MeSH
• silibinin chemie   farmakologie   MeSH
• stereoizomerie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antiinfekční látky MeSH
• antioxidancia MeSH
• fytogenní protinádorové látky MeSH
• silibinin MeSH

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.

• Klíčová slova
• dihydroquercetin, isosilybin, metabolism, silybin, silychristin, silydianin, sulfation,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• Silybum marianum, activity, biotransformation, metabolites, sulfate, sulfotransferase,
• MeSH
• antioxidancia chemie   MeSH
• flavonolignany chemie   MeSH
• hmotnostní spektrometrie MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární struktura MeSH
• ostropestřec mariánský chemie   MeSH
• ovoce chemie   MeSH
• potravní doplňky MeSH
• rostliny chemie   ultrastruktura   MeSH
• scavengery volných radikálů chemie   MeSH
• sírany chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia MeSH
• flavonolignany MeSH
• scavengery volných radikálů MeSH
• sírany MeSH

Sulfated quercetin derivatives are important authentic standards for metabolic studies. Quercetin-3'-O-sulfate, quercetin-4'-O-sulfate, and quercetin-3-O-sulfate as well as quercetin-di-O-sulfate mixture (quercetin-7,3'-di-O-sulfate, quercetin-7,4'-di-O-sulfate, and quercetin-3',4'-di-O-sulfate) were synthetized by arylsulfotransferase from Desulfitobacterium hafniense. Purified monosulfates and disulfates were fully characterized using MS and NMR and tested for 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, Folin-Ciocalteau reduction (FCR), ferric reducing antioxidant power (FRAP), and anti-lipoperoxidant activities in rat liver microsomes damaged by tert-butylhydroperoxide. Although, as expected, the sulfated metabolites were usually less active than quercetin, they remained still effective antiradical and reducing agents. Quercetin-3'-O-sulfate was more efficient than quercetin-4'-O-sulfate in DPPH and FCR assays. In contrast, quercetin-4'-O-sulfate was the best ferric reductant and lipoperoxidation inhibitor. The capacity to scavenge ABTS+• and DMPD was comparable for all substances, except for disulfates, which were the most efficient. Quantum calculations and molecular dynamics simulations on membrane models supported rationalization of free radical scavenging and lipid peroxidation inhibition. These results clearly showed that individual metabolites of food bioactives can markedly differ in their biological activity. Therefore, a systematic and thorough investigation of all bioavailable metabolites with respect to native compounds is needed when evaluating food health benefits.

• Klíčová slova
• antiradical activity, density functional theory, lipid peroxidation, metabolites, molecular dynamics, quercetin, sulfates, sulfotransferase,
• MeSH
• antioxidancia MeSH
• arylsulfotransferasa metabolismus   MeSH
• Desulfitobacterium enzymologie   MeSH
• quercetin analogy a deriváty   analýza   chemická syntéza   metabolismus   MeSH
• sírany analýza   chemická syntéza   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia MeSH
• arylsulfotransferasa MeSH
• quercetin 3'-sulfate MeSH Prohlížeč
• quercetin MeSH
• sírany MeSH