The preparation of pure metabolites of bioactive compounds, particularly (poly)phenols, is essential for the accurate determination of their pharmacological profiles in vivo. Since the extraction of these metabolites from biological material is tedious and impractical, they can be synthesized enzymatically in vitro by bacterial PAPS-independent aryl sulfotransferases (ASTs). However, only a few ASTs have been studied and used for (poly)phenol sulfation. This study introduces new fully characterized recombinant ASTs selected according to their similarity to the previously characterized ASTs. These enzymes, produced in Escherichia coli, were purified, biochemically characterized, and screened for the sulfation of nine flavonoids and two phenolic acids using p-nitrophenyl sulfate. All tested compounds were proved to be substrates for the new ASTs, with kaempferol and luteolin being the best converted acceptors. ASTs from Desulfofalx alkaliphile (DalAST) and Campylobacter fetus (CfAST) showed the highest efficiency in the sulfation of tested polyphenols. To demonstrate the efficiency of the present sulfation approach, a series of new authentic metabolite standards, regioisomers of kaempferol sulfate, were enzymatically produced, isolated, and structurally characterized.

• Keywords
• aryl sulfotransferase, enzymatic sulfation, kaempferol sulfate, metabolite, polyphenol,
• MeSH
• Arylsulfotransferase * metabolism   chemistry   genetics   MeSH
• Bacterial Proteins metabolism   chemistry   genetics   MeSH
• Biocatalysis MeSH
• Escherichia coli metabolism   genetics   enzymology   MeSH
• Polyphenols * metabolism   chemistry   MeSH
• Sulfates metabolism   chemistry   MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arylsulfotransferase * MeSH
• Bacterial Proteins MeSH
• Polyphenols * MeSH
• Sulfates MeSH

A library of previously unknown halogenated derivatives of flavonolignans (silybins A and B, 2,3-dehydrosilybin, silychristin A, and 2,3-dehydrosilychristin A) was prepared. The effect of halogenation on the biological activity of flavonolignans was investigated. Halogenated derivatives had a significant effect on bacteria. All prepared derivatives inhibited the AI-2 type of bacterial communication (quorum sensing) at concentrations below 10 µM. All prepared compounds also inhibited the adhesion of bacteria (Staphyloccocus aureus and Pseudomonas aeruginosa) to the surface, preventing biofilm formation. These two effects indicate that the halogenated derivatives are promising antibacterial agents. Moreover, these derivatives acted synergistically with antibiotics and reduced the viability of antibiotic-resistant S. aureus. Some flavonolignans were able to reverse the resistant phenotype to a sensitive one, implying that they modulate antibiotic resistance.

• Keywords
• bacteria, biological activity, flavonoids, flavonolignans, halogenation, multidrug resistance,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacteria MeSH
• Biofilms MeSH
• Methicillin-Resistant Staphylococcus aureus * MeSH
• Pseudomonas aeruginosa MeSH
• Quorum Sensing MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH

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

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.

• Keywords
• dihydroquercetin, isosilybin, metabolism, silybin, silychristin, silydianin, sulfation,
• Publication type
• Journal Article 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.

• Keywords
• Silybum marianum, activity, biotransformation, metabolites, sulfate, sulfotransferase,
• MeSH
• Antioxidants chemistry   MeSH
• Flavonolignans chemistry   MeSH
• Mass Spectrometry MeSH
• Magnetic Resonance Spectroscopy MeSH
• Molecular Structure MeSH
• Silybum marianum chemistry   MeSH
• Fruit chemistry   MeSH
• Dietary Supplements MeSH
• Plants chemistry   ultrastructure   MeSH
• Free Radical Scavengers chemistry   MeSH
• Sulfates chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antioxidants MeSH
• Flavonolignans MeSH
• Free Radical Scavengers MeSH
• Sulfates MeSH

The natural flavonoid quercetin is known to activate the transcription factor Nrf2, which regulates the expression of cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, a novel semisynthetic flavonoid 7-O-galloylquercetin (or quercetin-7-gallate, 3) was prepared by direct galloylation of quercetin, and its effect on the Nrf2 pathway was examined. A luciferase reporter assay showed that 7-O-galloylquercetin, like quercetin, significantly activated transcription via the antioxidant response element in a stably transfected human AREc32 reporter cell line. In addition, 7-O-galloylquercetin caused the accumulation of Nrf2 and induced the expression of HO-1 at both the mRNA and protein levels in murine macrophage RAW264.7 cells. The induction of HO-1 by 7-O-galloylquercetin was significantly suppressed by N-acetyl-l-cysteine and SB203580, indicating the involvement of reactive oxygen species and p38 mitogen-activated protein kinase activity, respectively. HPLC/MS analyses also showed that 7-O-galloylquercetin was not degalloylated to quercetin, but it was conjugated with glucuronic acid and/or methylated in RAW264.7 cells. Furthermore, 7-O-galloylquercetin was found to increase the protein levels of Nrf2 and HO-1, and also the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. Taken together, we conclude that 7-O-galloylquercetin increases Nrf2 activity and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells.

• Keywords
• Heme oxygenase-1, Metabolism, Methyl gallate, Nrf2, Quercetin, Quercetin-7-gallate,
• MeSH
• Biotransformation drug effects   MeSH
• Enzyme Induction drug effects   MeSH
• NF-E2-Related Factor 2 metabolism   MeSH
• Heme Oxygenase-1 biosynthesis   MeSH
• Mass Spectrometry MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Metabolome drug effects   MeSH
• p38 Mitogen-Activated Protein Kinases metabolism   MeSH
• Mice MeSH
• NAD(P)H Dehydrogenase (Quinone) metabolism   MeSH
• Cell Line, Tumor MeSH
• Quercetin chemical synthesis   chemistry   pharmacology   MeSH
• RAW 264.7 Cells MeSH
• Reactive Oxygen Species metabolism   MeSH
• Gene Expression Regulation drug effects   MeSH
• Genes, Reporter MeSH
• Cell Survival drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NF-E2-Related Factor 2 MeSH
• Heme Oxygenase-1 MeSH
• Protein Kinase Inhibitors MeSH
• p38 Mitogen-Activated Protein Kinases MeSH
• NAD(P)H Dehydrogenase (Quinone) MeSH
• Quercetin MeSH
• Reactive Oxygen Species MeSH