Selectively Halogenated Flavonolignans-Preparation and Antibacterial Activity.

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.

Bacterial Aryl Sulfotransferases in Selective and Sustainable Sulfation of Biologically Active Compounds using Novel Sulfate Donors.

Regioselective sulfation of bioactive compounds is a vital and scarcely studied topic in enzyme-catalyzed transformations and metabolomics. The major bottleneck of enzymatic sulfation consists in finding suitable sulfate donors. In this regard, 3'-phosphoadenosine 5'-phosphosulfate (PAPS)-independent aryl sulfotransferases using aromatic sulfate donors are a favored choice due to their cost-effectiveness. This work presents a unique study of five sulfate donors differing in their leaving group pKa values with a new His-tagged construct of aryl sulfotransferase from Desulfitobacterium hafniense (DhAST-tag). DhAST-tag was purified to homogeneity and biochemically characterized. Two new donors (3-nitrophenyl sulfate and 2-nitrophenyl sulfate) were synthesized. The kinetic parameters of these and other commercial sulfates (4-nitrophenyl, 4-methylumbelliferyl, and phenyl) revealed large differences with respect to the structure of the leaving group. These donors were screened for the sulfation of selected flavonoids (myricetin, chrysin) and phenolic acids (gallate, 3,4-dihydroxyphenylacetate). The donor impact on the sulfation regioselectivity and yield was assessed. The obtained regioselectively sulfated compounds are authentic human metabolites required as standards in clinical trials.

Sulfated Phenolic Substances: Preparation and Optimized HPLC Analysis.

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.

Interaction of silymarin components and their sulfate metabolites with human serum albumin and cytochrome P450 (2C9, 2C19, 2D6, and 3A4) enzymes.

Silymarin is a mixture of flavonolignans isolated from the fruit of milk thistle (Silybum marianum (L.) Gaertner). Milk thistle extract is the active ingredient of several medications and dietary supplements to treat liver injury/diseases. After the oral administration, flavonolignans are extensively biotransformed, resulting in the formation of sulfate and/or glucuronide metabolites. Previous studies demonstrated that silymarin components form stable complexes with serum albumin and can inhibit certain cytochrome P450 (CYP) enzymes. Nevertheless, in most of these investigations, silybin was tested; while no or only limited information is available regarding other silymarin components and metabolites. In this study, the interactions of five silymarin components (silybin A, silybin B, isosilybin A, silychristin, and 2,3-dehydrosilychristin) and their sulfate metabolites were examined with human serum albumin and CYP (2C9, 2C19, 2D6, and 3A4) enzymes. Our results demonstrate that each compound tested forms stable complexes with albumin, and certain silymarin components/metabolites can inhibit CYP enzymes. Most of the sulfate conjugates were less potent inhibitors of CYP enzymes, but 2,3-dehydrosilychristin-19-O-sulfate showed the strongest inhibitory effect on CYP3A4. Based on these observations, the simultaneous administration of high dose silymarin with medications should be carefully considered, because milk thistle flavonolignans and/or their sulfate metabolites may interfere with drug therapy.

Sulfated Metabolites of Luteolin, Myricetin, and Ampelopsin: Chemoenzymatic Preparation and Biophysical Properties.

Authentic standards of food flavonoids are important for human metabolic studies. Their isolation from biological materials is impracticable; however, they can be prepared in vitro. Twelve sulfated metabolites of luteolin, myricetin, and ampelopsin were obtained with arylsulfotransferase from Desulfitobacterium hafniense and fully characterized by high-performance liquid chromatography, MS, and NMR. The compounds were tested for their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), and N,N-dimethyl-p-phenylenediamine radicals, to reduce ferric ions and Folin-Ciocalteu reagent, and to inhibit tert-butyl hydroperoxide-induced lipid peroxidation of rat liver microsomes. The activity differed considerably even between monosulfate isomers. The parent compounds and myricetin-3'-O-sulfate were the most active while other compounds displayed significantly lower activity, particularly luteolin sulfates. No mutagenic activity of the parent compounds and their main metabolites was observed; only myricetin showed minor pro-mutagenicity. The prepared sulfated metabolites are now available as authentic standards for future in vitro and in vivo metabolic studies.

Multidrug Resistance Modulation Activity of Silybin Derivatives and Their Anti-inflammatory Potential.

Silybin is considered to be the main biologically active component of silymarin. Its oxidized derivative 2,3-dehydrosilybin typically occurs in silymarin in small, but non-negligible amounts (up to 3%). Here, we investigated in detail complex biological activities of silybin and 2,3-dehydrosilybin optical isomers. Antioxidant activities of pure stereomers A and B of silybin and 2,3-dehydrosilybin, as well as their racemic mixtures, were investigated by using oxygen radical absorption capacity (ORAC) and cellular antioxidant activity (CAA) assay. All substances efficiently reduced nitric oxide production and cytokines (TNF-α, IL-6) release in a dose-dependent manner. Multidrug resistance (MDR) modulating potential was evaluated as inhibition of P-glycoprotein (P-gp) ATPase activity and regulation of ATP-binding cassette (ABC) protein expression. All the tested compounds showed strong dose-dependent inhibition of P-gp pump. Moreover, 2,3-dehydrosilybin A (30 µM) displayed the strongest sensitization of doxorubicin-resistant ovarian carcinoma. Despite these significant effects, silybin B was the only compound acting directly upon P-gp in vitro and also downregulating the expression of respective MDR genes. This compound altered the expression of P-glycoprotein (P-gp, ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2). 2,3-Dehydrosilybin AB exhibited the most effective inhibition of acetylcholinesterase activity. We can clearly postulate that silybin derivatives could serve well as modulators of a cancer drug-resistant phenotype.

Biotransformation of Silymarin Flavonolignans by Human Fecal Microbiota.

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.

Simple and Rapid HPLC Separation and Quantification of Flavonoid, Flavonolignans, and 2,3-Dehydroflavonolignans in Silymarin.

Herbal preparations from Silybum marianum have been used since the fourth century BC in liver disease treatment and against numerous other pathologies. Consumption of silymarin containing drugs and food supplements continues to increase. Precise, fast, reliable, and complex determination of all components of silymarin preparations is paramount for assessing its pharmacological quality. We present here simple and fast HPLC-DAD and LC-MS analytical methods for the determination and quantification of all known silymarin components, including 2,3-dehydroflavonolignans that has not been achieved so far. The first method, using a common C18 column, allows baseline separation of previously inseparable silychristin A, B, isosilychristin, and silydianin. Moreover, this method allowed detection of three so far unknown silymarin components. In addition, the first analytical separation of enantiomers of 2,3-dehydrosilybin was achieved using a Lux 3µ Cellulose-4 chiral column, providing even more accurate description of silymarin composition. 2,3-Dehydroflavonolignans were isolated for the first time from silymarin using preparative chromatography on C18 and ASAHIPAK columns, and 2,3-dehydrosilychristin and 2,3-dehydrosilybin were for the first time conclusively confirmed by HPLC, MS, and NMR to be silymarin components. Using the optimized analytical methods, six various silymarin preparations were analyzed showing substantial differences in the composition.

Antioxidant, Anti-Inflammatory, and Multidrug Resistance Modulation Activity of Silychristin Derivatives.

Silychristin A is the second most abundant compound of silymarin. Silymarin complex was previously described as an antioxidant with multidrug resistance modulation activity. Here, the results of a classical biochemical antioxidant assay (ORAC) were compared with a cellular assay evaluating the antioxidant capacity of pure silychristin A and its derivatives (anhydrosilychristin, isosilychristin and 2,3-dehydrosilychristin A). All the tested compounds acted as antioxidants within the cells, but 2,3-dehydro- and anhydro derivatives were almost twice as potent as the other tested compounds. Similar results were obtained in LPS-stimulated macrophages, where 2,3-dehydro- and anhydrosilychristin inhibited NO production nearly twice as efficiently as silychristin A. The inhibition of P-glycoprotein (P-gp) was determined in vitro, and the respective sensitization of doxorubicin-resistant ovarian carcinoma overproducing P-gp was detected. Despite the fact that the inhibition of P-gp was demonstrated in a concentration-dependent manner for each tested compound, the sensitization of the resistant cell line was observed predominantly for silychristin A and 2,3-dehydrosilychristin A. However, anhydrosilychristin and isosilychristin affected the expression of both the P-gp (ABCB1) and ABCG2 genes. This is the first report showing that silychristin A and its 2,3-dehydro-derivative modulate multidrug resistance by the direct inhibition of P-gp, in contrast to anhydrosilychristin and isosilychristin modulating multidrug resistance by downregulating the expression of the dominant transmembrane efflux pumps.

Chemoenzymatic Preparation and Biophysical Properties of Sulfated Quercetin Metabolites.

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.