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

Flavonoids and their glycosides are abundant in many plant-based foods. The (de)glycosylation of flavonoids by retaining glycoside hydrolases has recently attracted much interest in basic and applied research, including the possibility of altering the glycosylation pattern of flavonoids. Research in this area is driven by significant differences in physicochemical, organoleptic, and bioactive properties between flavonoid aglycones and their glycosylated counterparts. While many flavonoid glycosides are present in nature at low levels, some occur in substantial quantities, making them readily available low-cost glycosyl donors for transglycosylations. Retaining glycosidases can be used to synthesize natural and novel glycosides, which serve as standards for bioactivity experiments and analyses, using flavonoid glycosides as glycosyl donors. Engineered glycosidases also prove valuable for the synthesis of flavonoid glycosides using chemically synthesized activated glycosyl donors. This review outlines the bioactivities of flavonoids and their glycosides and highlights the applications of retaining glycosidases in the context of flavonoid glycosides, acting as substrates, products, or glycosyl donors in deglycosylation or transglycosylation reactions.

• Keywords
• Glucosidase, Glycoside hydrolase, Glycosyl donor, Glycosynthase, Hydrolysis, Rutinosidase, Transglycosylation,
• MeSH
• Flavonoids * chemistry   MeSH
• Glycoside Hydrolases * metabolism   MeSH
• Glycosides chemistry   MeSH
• Glycosylation MeSH
• Catalysis MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Flavonoids * MeSH
• Glycoside Hydrolases * MeSH
• Glycosides MeSH

Phenolic acids are known flavonoid metabolites, which typically undergo bioconjugation during phase II of biotransformation, forming sulfates, along with other conjugates. Sulfated derivatives of phenolic acids can be synthesized by two approaches: chemoenzymatically by 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferases or PAPS-independent aryl sulfotransferases such as those from Desulfitobacterium hafniense, or chemically using SO3 complexes. Both approaches were tested with six selected phenolic acids (2-hydroxyphenylacetic acid (2-HPA), 3-hydroxyphenylacetic acid (3-HPA), 4-hydroxyphenylacetic acid (4-HPA), 3,4-dihydroxyphenylacetic acid (DHPA), 3-(4-hydroxyphenyl)propionic acid (4-HPP), and 3,4-dihydroxyphenylpropionic acid (DHPP)) to create a library of sulfated metabolites of phenolic acids. The sulfates of 3-HPA, 4-HPA, 4-HPP, DHPA, and DHPP were all obtained by the methods of chemical synthesis. In contrast, the enzymatic sulfation of monohydroxyphenolic acids failed probably due to enzyme inhibition, whereas the same reaction was successful for dihydroxyphenolic acids (DHPA and DHPP). Special attention was also paid to the counterions of the sulfates, a topic often poorly reported in synthetic works. The products obtained will serve as authentic analytical standards in metabolic studies and to determine their biological activity.

• Keywords
• aryl sulfotransferase, biotransformation, flavonoid metabolites, phenolic acids, sulfation,
• MeSH
• Phosphoadenosine Phosphosulfate * chemistry   metabolism   MeSH
• Hydroxybenzoates MeSH
• Sulfates metabolism   MeSH
• Sulfotransferases * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphoadenosine Phosphosulfate * MeSH
• Hydroxybenzoates MeSH
• phenolic acid MeSH Browser
• Sulfates MeSH
• Sulfotransferases * MeSH

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.

• Keywords
• UHPLC–MS, biotransformation, flavonolignans, gut microbiota, inter-individual differences, metabolites, silymarin,
• Publication type
• Journal Article MeSH

Increased arterial stiffness is a degenerative vascular process, progressing with age that leads to a reduced capability of arteries to expand and contract in response to pressure changes. This progressive degeneration mainly affects the extracellular matrix of elastic arteries and causes loss of vascular elasticity. Recent studies point to significant interference of dietary polyphenols with mechanisms involved in the pathophysiology and progression of arterial stiffness. This review summarizes data from epidemiological and interventional studies on the effect of polyphenols on vascular stiffness as an illustration of current research and addresses possible etiological factors targeted by polyphenols, including pathways of vascular functionality, oxidative status, inflammation, glycation, and autophagy. Effects can either be inflicted directly by the dietary polyphenols or indirectly by metabolites originated from the host or microbial metabolic processes. The composition of the gut microbiome, therefore, determines the resulting metabolome and, as a consequence, the observed activity. On the other hand, polyphenols also influence the intestinal microbial composition, and therefore the metabolites available for interaction with relevant targets. As such, targeting the gut microbiome is another potential treatment option for arterial stiffness.

• Keywords
• aging, anti-inflammatory, antioxidant, arterial stiffness, autophagy, gut microbiome, polyphenol biotransformation, polyphenols,
• MeSH
• Food Analysis * MeSH
• Diet * MeSH
• Humans MeSH
• Polyphenols chemistry   pharmacology   MeSH
• Gastrointestinal Microbiome drug effects   MeSH
• Vascular Stiffness drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Polyphenols MeSH

Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.

• Keywords
• Aspergillus niger, quercetin, rutin, rutinose, rutinosidase, “solid-state biocatalysis”,
• MeSH
• Aspergillus niger enzymology   genetics   MeSH
• Biocatalysis * MeSH
• Disaccharides chemistry   metabolism   MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Glycoside Hydrolases genetics   metabolism   MeSH
• Pichia genetics   metabolism   MeSH
• Industrial Microbiology methods   MeSH
• Quercetin chemistry   metabolism   MeSH
• Rutin chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• beta-rutinosidase MeSH Browser
• Disaccharides MeSH
• Fungal Proteins MeSH
• Glycoside Hydrolases MeSH
• Quercetin MeSH
• Rutin MeSH
• rutinose MeSH Browser

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

Natural flavonoids, especially in their glycosylated forms, are the most abundant phenolic compounds found in plants, fruit, and vegetables. They exhibit a large variety of beneficial physiological effects, which makes them generally interesting in a broad spectrum of scientific areas. In this review, we focus on recent advances in the modifications of the glycosidic parts of various flavonoids employing glycosidases, covering both selective trimming of the sugar moieties and glycosylation of flavonoid aglycones by natural and mutant glycosidases. Glycosylation of flavonoids strongly enhances their water solubility and thus increases their bioavailability. Antioxidant and most biological activities are usually less pronounced in glycosides, but some specific bioactivities are enhanced. The presence of l-rhamnose (6-deoxy-α-l-mannopyranose) in rhamnosides, rutinosides (rutin, hesperidin) and neohesperidosides (naringin) plays an important role in properties of flavonoid glycosides, which can be considered as "pro-drugs". The natural hydrolytic activity of glycosidases is widely employed in biotechnological deglycosylation processes producing respective aglycones or partially deglycosylated flavonoids. Moreover, deglycosylation is quite commonly used in the food industry aiming at the improvement of sensoric properties of beverages such as debittering of citrus juices or enhancement of wine aromas. Therefore, natural and mutant glycosidases are excellent tools for modifications of flavonoid glycosides.

• Keywords
• catechin, enzymatic hydrolysis, hesperetin, icariin, naringenin, puerarin, quercetin, rhamnosidase, rutinosidase, transglycosylation,
• MeSH
• Flavonoids metabolism   MeSH
• Glycoside Hydrolases metabolism   MeSH
• Isoflavones metabolism   MeSH
• Catechin metabolism   MeSH
• Humans MeSH
• Quercetin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Flavonoids MeSH
• Glycoside Hydrolases MeSH
• Isoflavones MeSH
• Catechin MeSH
• puerarin MeSH Browser
• Quercetin MeSH