The 2-aminoethyl diphenylborinate-based fluorescent method identifies quercetin and luteolin metabolites as substrates of Organic anion transporting polypeptides, OATP1B1 and OATP2B1.

Organic anion transporting polypeptides (OATPs), OATP1B1 and OATP2B1 are membrane proteins mediating the cellular uptake of chemically diverse organic compounds. OATP1B1 is exclusively expressed in hepatocytes and plays a key role in hepatic detoxification. The ubiquitously expressed OATP2B1 promotes the intestinal absorption of orally administered drugs. Flavonoids are widely found in foods and beverages, and many of them can inhibit OATP function, resulting in food-drug interactions. In our previous work, we have shown that not only luteolin (LUT) and quercetin (Q), but also some of their metabolites can inhibit OATP1B1 and OATP2B1 activity. However, data about the potential direct transport of these flavonoids by OATPs have been incomplete. Hence, in the current study, we developed a simple, fluorescence-based method for the measurement of intracellular flavonoid levels. The method applies a cell-permeable small molecule (2-aminoethyl diphenylborinate, 2-APB), that, upon forming a complex with flavonoids, results in their fluorescence enhancement. This way the direct uptake of LUT and Q, and also their metabolites' could be investigated both by confocal microscopy and in a fluorescence plate reader in living cells. With this approach we identified quercetin-3'-O-sulfate, luteolin-3'-O-glucuronide, luteolin-7-O-glucuronide and luteolin-3'-O-sulfate as substrates of both OATP1B1 and OATP2B1. Our results highlight that OATP1B1 and OATP2B1 can be key participants in the transmembrane movement of LUT and Q conjugates with otherwise low cell permeability. In addition, the novel method developed in this study can be a good completion to existing fluorescence-based assays to investigate OATP function.

Flavonoids as Aglycones in Retaining Glycosidase-Catalyzed Reactions: Prospects for Green Chemistry.

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.

Inhibition of xanthine oxidase-catalyzed xanthine and 6-mercaptopurine oxidation by luteolin, naringenin, myricetin, ampelopsin and their conjugated metabolites.

Luteolin, naringenin, myricetin, and ampelopsin are abundant flavonoids in nature, and several dietary supplements also contain them at very high doses. After the peroral intake, flavonoids go through extensive presystemic biotransformation; therefore, typically their sulfate/glucuronic acid conjugates reach high concentrations in the circulation. Xanthine oxidase (XO) enzyme is involved in uric acid production, and it also takes part in the elimination of certain drugs (e.g., 6-mercaptopurine). The inhibitory effects of flavonoid aglycones on XO have been widely studied; however, only limited data are available regarding their sulfate and glucuronic acid conjugates. In this study, we examined the impacts of luteolin, naringenin, myricetin, ampelopsin, and their sulfate/glucuronide derivatives on XO-catalyzed xanthine and 6-mercaptopurine oxidations employing in vitro enzyme incubation assays and molecular modeling studies. Our major results/conclusions are the following: (1) Sulfate metabolites were stronger while glucuronic acid derivatives were weaker inhibitors of XO compared to the parent flavonoids. (2) Naringenin, ampelopsin, and their metabolites were weak inhibitors of the enzyme. (3) Luteolin, myricetin, and their sulfates were highly potent inhibitors of XO, and the glucuronides of luteolin showed moderate inhibitory impacts. (4) Conjugated metabolites of luteolin and myricetin can be involved in the inhibitory effects of these flavonoids on XO enzyme.

Biotransformation of Natural Products and Phytochemicals: Metabolites, Their Preparation, and Properties.

The term "biotransformation" refers to the process by which various compounds are biocatalyzed and enzymatically modified, as well as the metabolic changes that occur in organisms as a result of exposure to xenobiotics [...].

Combination of plant phenolics and isoquinolinium alkaloids protects gingival fibroblast and improves post-extraction healing after lower third molar extraction.

AIMS: The effect of polyphenolic fraction of Lonicera caerulea (PFLC) and alkaloid fraction of Macleaya cordata (AFMC) mix on the production of inflammatory mediators in human gingival fibroblasts pretreated with lipopolysaccharide (LPS) was investigated. In addition, protective effects of mucoadhesive paste containing combination of PFLC and AFMC (0.05% and 0.01%, respectively; n=15, Group A) and placebo (n=15, Group B) were evaluated in patients after surgical extraction of lower third molars. METHODS: Gingival fibroblasts were pre-treated with LPS (10 µg/mL; 24 h) and PFLC/AFMC (25/0.25; 50/0.25; 100/0.25; 25/0.5; 50/0.5; 100/0.5 µg/mL) in serum-free medium was applied for 4 h. Then the interleukin-6 (IL-6), reactive oxygen species (ROS) generation, level of intracellular glutathione (GSH) and expression of cyclooxygenase-2 (COX-2) were evaluated. The study was a 6-day, single-center, randomized, double-blind and placebo-controlled trial consisting of two parallel treatment arms. A modified Oral health impact profile questionnaire including both general oral condition and extraction related questions, was used to evaluate the oral condition and other changes before (day 0) and on the days 1, 3 and 6 after surgical extraction. RESULTS AND CONCLUSION: The combination of PFLC with AFMC caused a reduction of ROS generation, reduced IL-6 production and suppressed the expression of COX-2. In group A the paste treatment contributed to improvement of oral health-related quality of life. Topical application of PFLC and AFMC into the extraction wound improved post-extraction site wound healing probably by antioxidant and anti-inflammatory mechanisms.

Interaction of luteolin, naringenin, and their sulfate and glucuronide conjugates with human serum albumin, cytochrome P450 (CYP2C9, CYP2C19, and CYP3A4) enzymes and organic anion transporting polypeptide (OATP1B1 and OATP2B1) transporters.

Luteolin and naringenin are flavonoids found in various foods/beverages and present in certain dietary supplements. After a high intake of these flavonoids, their sulfate and glucuronide conjugates reach micromolar concentrations in the bloodstream. Some pharmacokinetic interactions of luteolin and naringenin have been investigated in previous studies; however, only limited data are available in regard to their metabolites. In this study, we aimed to investigate the interactions of the sulfate and glucuronic acid conjugates of luteolin and naringenin with human serum albumin, cytochrome P450 (CYP2C9, 2C19, and 3A4) enzymes, and organic anion transporting polypeptide (OATP1B1 and OATP2B1) transporters. Our main findings are as follows: (1) Sulfate conjugates formed more stable complexes with albumin than the parent flavonoids. (2) Luteolin and naringenin conjugates showed no or only weak inhibitory action on the CYP enzymes examined. (3) Certain conjugates of luteolin and naringenin are potent inhibitors of OATP1B1 and/or OATP2B1 enzymes. (4) Conjugated metabolites of luteolin and naringenin may play an important role in the pharmacokinetic interactions of these flavonoids.

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.

Sulfation of Phenolic Acids: Chemoenzymatic vs. Chemical Synthesis.

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.

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 Flavonoids with Zinc and Zinc-Containing Enzymes.

The current chelation therapy has several drawbacks, including lack of selectivity, which could lead to trace metal depletion. Consequently, the proper function of metalloenzymes can be disrupted. Flavonoids possess chelating properties and hence interfere with the homeostasis of essential metals. We focused on zinc, an important trace metal required for the function of many enzymes and transcription factors. After making an initial evaluation of the Zn2+-chelating properties of a series of flavonoids, the effect of these compounds on various zinc-containing enzymes was also investigated. We performed enzyme inhibition assays spectrophotometrically using yeast and equine alcohol dehydrogenases and bovine glutamate dehydrogenase. Nine of the 21 flavonoids tested were capable of chelating Zn2+. Baicalein and 3-hydroxyflavone were the most potent Zn2+ chelators under slightly acidic and neutral pH conditions. This chelation was also confirmed by the ability to reverse Zn2+-induced enzymatic inhibition of bovine glutamate dehydrogenase. Although some flavonoids were also able to inhibit zinc-containing alcohol dehydrogenases, this inhibition was likely not caused by Zn2+ chelation. Luteolin was a relatively potent inhibitor of these enzymes regardless of the presence of Zn2+. Docking studies confirmed the binding of active flavonoids to equine alcohol dehydrogenase without any significant interaction with the catalytic zinc.

Silybin and its congeners: from traditional medicine to molecular effects.

Covering: 2015 up to 2022 (Feb)Silymarin, an extract of milk thistle (Silybum marianum) fruits, has been used in various medicinal applications since ancient times. A major component of silymarin is the flavonolignan silybin and its relatives isosilybin, silychristin, silydianin, 2,3-dehydrosilybin, and some others. Except for silydianin, they occur in nature as two stereomers. This review focuses on recent developments in chemistry, biosynthesis, modern advanced analytical methods, and transformations of flavonolignans specifically reflecting their chirality. Recently described chemotypes of S. marianum, but also the newest findings regarding the pharmacokinetics, hepatoprotective, antiviral, neuroprotective, and cardioprotective activity, modulation of endocrine functions, modulation of multidrug resistance, and safety of flavonolignans are discussed. A growing number of studies show that the respective diastereomers of flavonolignans have significantly different activities in anisotropic biological systems. Moreover, it is now clear that flavonolignans do not act as antioxidants in vivo, but as specific ligands of biological targets and therefore their chirality is crucial. Many controversies often arise, mainly due to the non-standard composition of this phytopreparation, the use of various undefined mixtures, the misattribution of silymarin vs. silybin, and also the failure to consider the chemistry of the respective components of silymarin.

Flavonolignans from silymarin modulate antibiotic resistance and virulence in Staphylococcus aureus.

Antibiotic resistance is currently a serious health problem. Since the discovery of new antibiotics no longer seems to be a sufficient tool in the fight against multidrug-resistant infections, adjuvant (combination) therapy is gaining in importance as well as reducing bacterial virulence. Silymarin is a complex of flavonoids and flavonolignans known for its broad spectrum of biological activities, including its ability to modulate drug resistance in cancer. This work aimed to test eleven, optically pure silymarin flavonolignans for their ability to reverse the multidrug resistance phenotype of Staphylococcus aureus and reduce its virulence. Silybin A, 2,3-dehydrosilybin B, and 2,3-dehydrosilybin AB completely reversed antibiotic resistance at concentrations of 20 µM or less. Both 2,3-dehydrosilybin B and AB decreased the antibiotic-induced gene expression of representative efflux pumps belonging to the major facilitator (MFS), multidrug and toxic compound extrusion (MATE), and ATP-binding cassette (ABC) families. 2,3-Dehydrosilybin B also inhibited ethidium bromide accumulation and efflux in a clinical isolate whose NorA and MdeA overproduction was induced by antibiotics. Most of the tested flavonolignans reduced cell-to-cell communication on a tetrahydrofuran-borate (autoinducer-2) basis, with isosilychristin leading the way followed by 2,3-dehydrosilybin A and AB, which halved communication at 10 µM. Anhydrosilychristin was the only compound that reduced communication based on acyl-homoserine lactone (autoinducer 1), with an IC50 of 4.8 µM. Except for isosilychristin and anhydrosilychristin, all of the flavonolignans inhibited S. aureus surface colonization, with 2,3-dehydrosilybin A being the most active (IC50 10.6 µM). In conclusion, the selected flavonolignans, particularly derivatives of 2,3-dehydrosilybin B, 2,3-dehydrosilybin AB, and silybin A are non-toxic modulators of S. aureus multidrug resistance and can decrease the virulence of the bacterium, which deserves further detailed research.

Preparation of Synthetic and Natural Derivatives of Flavonoids Using Suzuki-Miyaura Cross-Coupling Reaction.

Herein, we report the use of the Suzuki-Miyaura cross-coupling reaction for the preparation of a library of synthetic derivatives of flavonoids for biological activity assays. We have investigated the reactivity of halogenated flavonoids with aryl boronates and with boronyl flavonoids. This reaction was used to prepare new synthetic derivatives of flavonoids substituted at C-8 with aryl, heteroaryl, alkyl, and boronate substituents. The formation of flavonoid boronate enabled a cross-coupling reaction with halogenated flavones yielding biflavonoids connected at C-8. This method was used for the preparation of natural compounds including C-8 prenylated compounds, such as sinoflavonoid NB. Flavonoid boronates were used for the preparation of rare C-8 hydroxyflavonoids (natural flavonoids gossypetin and hypolaetin). A series of previously unknown derivatives of quercetin and luteolin were prepared and fully characterized.

Common Cosmetic Compounds Can Reduce Air Pollution-Induced Oxidative Stress and Pro-Inflammatory Response in the Skin.

INTRODUCTION: Constantly increasing air pollution (AP) poses a concern affecting not only our health but also our skin. A typical manifestation of the skin damage induced by AP is its premature aging, irritation, skin barrier impairment, pigmentation disorders, and development or exacerbation of various skin diseases. For these reasons, it is crucial to protect the skin from the negative effects of AP. In this study, we evaluated the ability of some compounds commonly used in dermatological or cosmetic preparations with various biological activities to reduce AP-induced skin damage. METHODS: We established a new experimental model using porcine skin explants exposed to cigarette smoke (CS) in which we determined the level of reactive oxygen species (ROS) in the stratum corneum, skin barrier lipids peroxidation, and gene expression of the pro-inflammatory cytokine interleukin 6 in the epidermis. Then, we tested several polysaccharides and their derivatives such as sodium hyaluronate (SH) of different molecular weight (MW, 1.6 MDa, 300 kDa, 15 kDa, 5 kDa), yeast glucomannan, schizophyllan, and carboxymethyl ß-glucan, then vitamin C derivative sodium ascorbyl phosphate, niacinamide, and D-panthenol for their ability to prevent CS-induced skin damage. For the evaluation and comparison of their mechanism of action, film-forming effect was determined by TEWL and gloss measurements and the antioxidant properties were assessed by DPPH assay. RESULTS: In the skin samples exposed to CS, we observed significant negative changes such as the presence of large amount of ROS in the stratum corneum, high level of skin barrier lipids peroxidation and upregulated IL6 gene expression. Pretreatment of the skin samples with all the tested substances significantly prevented CS-induced skin damage. The most effective were high MW SH probably due to its best film-forming effect and sodium ascorbyl phosphate with the best antioxidant properties. CONCLUSION: AP leads to a significant skin damage which can be effectively prevented using some conventional cosmetic and dermatological ingredients with various mechanisms of action.

Silymarin Dehydroflavonolignans Chelate Zinc and Partially Inhibit Alcohol Dehydrogenase.

Silymarin is known for its hepatoprotective effects. Although there is solid evidence for its protective effects against Amanita phalloides intoxication, only inconclusive data are available for alcoholic liver damage. Since silymarin flavonolignans have metal-chelating activity, we hypothesized that silymarin may influence alcoholic liver damage by inhibiting zinc-containing alcohol dehydrogenase (ADH). Therefore, we tested the zinc-chelating activity of pure silymarin flavonolignans and their effect on yeast and equine ADH. The most active compounds were also tested on bovine glutamate dehydrogenase, an enzyme blocked by zinc ions. Of the six flavonolignans tested, only 2,3-dehydroderivatives (2,3-dehydrosilybin and 2,3-dehydrosilychristin) significantly chelated zinc ions. Their effect on yeast ADH was modest but stronger than that of the clinically used ADH inhibitor fomepizole. In contrast, fomepizole strongly blocked mammalian (equine) ADH. 2,3-Dehydrosilybin at low micromolar concentrations also partially inhibited this enzyme. These results were confirmed by in silico docking of active dehydroflavonolignans with equine ADH. Glutamate dehydrogenase activity was decreased by zinc ions in a concentration-dependent manner, and this inhibition was abolished by a standard zinc chelating agent. In contrast, 2,3-dehydroflavonolignans blocked the enzyme both in the absence and presence of zinc ions. Therefore, 2,3-dehydrosilybin might have a biologically relevant inhibitory effect on ADH and glutamate dehydrogenase.

Dehydroflavonolignans from Silymarin Potentiate Transition Metal Toxicity In Vitro but Are Protective for Isolated Erythrocytes Ex Vivo.

2,3-Dehydrosilybin (DHS) was previously shown to chelate and reduce both copper and iron ions. In this study, similar experiments with 2,3-dehydrosilychristin (DHSCH) showed that this congener of DHS also chelates and reduces both metals. Statistical analysis pointed to some differences between both compounds: in general, DHS appeared to be a more potent iron and copper chelator, and a copper reducing agent under acidic conditions, while DHSCH was a more potent copper reducing agent under neutral conditions. In the next step, both DHS and DHSCH were tested for metal-based Fenton chemistry in vitro using HPLC with coulometric detection. Neither of these compounds were able to block the iron-based Fenton reaction and, in addition, they mostly intensified hydroxyl radical production. In the copper-based Fenton reaction, the effect of DHSCH was again prooxidant or neutral, while the effect of DHS was profoundly condition-dependent. DHS was even able to attenuate the reaction under some conditions. Interestingly, both compounds were strongly protective against the copper-triggered lysis of red blood cells, with DHSCH being more potent. The results from this study indicated that, notwithstanding the prooxidative effects of both dehydroflavonolignans, their in vivo effect could be protective.

Data sharing in PredRet for accurate prediction of retention time: Application to plant food bioactive compounds.

Prediction of retention times (RTs) is increasingly considered in untargeted metabolomics to complement MS/MS matching for annotation of unidentified peaks. We tested the performance of PredRet (http://predret.org/) to predict RTs for plant food bioactive metabolites in a data sharing initiative containing entry sets of 29-103 compounds (totalling 467 compounds, >30 families) across 24 chromatographic systems (CSs). Between 27 and 667 predictions were obtained with a median prediction error of 0.03-0.76 min and interval width of 0.33-8.78 min. An external validation test of eight CSs showed high prediction accuracy. RT prediction was dependent on shape and type of LC gradient, and number of commonly measured compounds. Our study highlights PredRet's accuracy and ability to transpose RT data acquired from one CS to another CS. We recommend extensive RT data sharing in PredRet by the community interested in plant food bioactive metabolites to achieve a powerful community-driven open-access tool for metabolomics annotation.

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.