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

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.

• Keywords
• antiradical activity, density functional theory, lipid peroxidation, metabolites, molecular dynamics, quercetin, sulfates, sulfotransferase,
• MeSH
• Antioxidants MeSH
• Arylsulfotransferase metabolism   MeSH
• Desulfitobacterium enzymology   MeSH
• Quercetin analogs & derivatives   analysis   chemical synthesis   metabolism   MeSH
• Sulfates analysis   chemical synthesis   metabolism   MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antioxidants MeSH
• Arylsulfotransferase MeSH
• quercetin 3'-sulfate MeSH Browser
• Quercetin MeSH
• Sulfates MeSH

Isoquercitrin, (IQ, quercetin-3-O-β-d-glucopyranoside) is known for strong chemoprotectant activities. Acylation of flavonoid glucosides with carboxylic acids containing an aromatic ring brings entirely new properties to these compounds. Here, we describe the chemical and enzymatic synthesis of a series of IQ derivatives at the C-6″. IQ benzoate, phenylacetate, phenylpropanoate and cinnamate were prepared from respective vinyl esters using Novozym 435 (Lipase B from Candida antarctica immobilized on acrylic resin). The enzymatic procedure gave no products with "hydroxyaromatic" acids, their vinyl esters nor with their benzyl-protected forms. A chemical protection/deprotection method using Steglich reaction yielded IQ 4-hydroxybenzoate, vanillate and gallate. In case of p-coumaric, caffeic, and ferulic acid, the deprotection lead to the saturation of the double bonds at the phenylpropanoic moiety and yielded 4-hydroxy-, 3,4-dihydroxy- and 3-methoxy-4-hydroxy-phenylpropanoates. Reducing capacity of the cinnamate, gallate and 4-hydroxyphenylpropanoate towards Folin-Ciocalteau reagent was significantly lower than that of IQ, while other derivatives displayed slightly better or comparable capacity. Compared to isoquercitrin, most derivatives were less active in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, but they showed significantly better 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid, ABTS) scavenging activity and were substantially more active in the inhibition of tert-butylhydroperoxide induced lipid peroxidation of rat liver microsomes. The most active compounds were the hydroxyphenylpropanoates.

• Keywords
• DPPH, Novozym 435, antioxidant activity, aromatic acid, cinnamic acid, gallic acid, isoquercitrin, lipase, lipoperoxidation,
• MeSH
• Esters * chemistry   MeSH
• Mass Spectrometry MeSH
• Molecular Structure MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Lipid Peroxidation drug effects   MeSH
• Quercetin analogs & derivatives   analysis   chemical synthesis   pharmacology   MeSH
• Free Radical Scavengers analysis   chemical synthesis   pharmacology   MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Esters * MeSH
• isoquercitrin MeSH Browser
• Quercetin MeSH
• Free Radical Scavengers MeSH