silychristin and silidianin

Silybum marianum (milk thistle) is a medicinal plant used for producing the hepatoprotective remedy silymarin. Its main bioactive constituents, including silybin and related flavonolignans, can be metabolized directly by phase II conjugation reactions. This study was designed to identify UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of six silymarin flavonolignans, namely silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. UHPLC-MS analyses showed that all of the tested compounds, both individually and in silymarin, were glucuronidated by human liver microsomes, and that glucuronidation was the main metabolic transformation in human hepatocytes. Further, each compound was glucuronidated by multiple recombinant human UGT enzymes. UGTs 1A1, 1A3, 1A8 and 1A9 were able to conjugate all of the tested flavonolignans, and some of them were also metabolized by UGTs 1A6, 1A7, 1A10, 2B7 and 2B15. In contrast, no glucuronides were produced by UGTs 1A4, 2B4, 2B10 and 2B17. With silymarin, we found that UGT1A1 and, to a lesser extent UGT1A9, were primarily responsible for the glucuronidation of the flavonolignan constituents. It is concluded that the metabolism of silymarin flavonolignans may involve multiple UGT enzymes, of which UGT1A1 appears to play the major role in the glucuronidation. These results may be relevant for future research on the metabolism of flavonolignans in humans.

Topics: Adult; Cells, Cultured; Flavonolignans; Glucuronides; Glucuronosyltransferase; Hepatocytes; Humans; Male; Microsomes, Liver; Silybin; Silybum marianum; Silymarin

Topics: Animals; Cytochrome P-450 Enzyme System; Glutathione; Humans; Hydroxylation; Isomerism; Microsomes, Liver; Oxidation-Reduction; Rats; Silybin; Silymarin; Tandem Mass Spectrometry

The exposure of naked unprotected skin to solar radiation may result in numerous acute and chronic undesirable effects. Evidence suggests that silymarin, a standardized extract from Silybum marianum (L.) Gaertn. seeds, and its major component silybin suppress UVB-induced skin damage. Here, we aimed to investigate the UVA-protective effects of silymarin's less abundant flavonolignans, specifically isosilybin (ISB), silychristin (SC), silydianin (SD), and 2,3-dehydrosilybin (DHSB). Normal human dermal fibroblasts (NHDF) pre-treated for 1 h with flavonolignans were then exposed to UVA light using a solar simulator. Their effects on reactive oxygen species (ROS), carbonylated proteins and glutathione (GSH) level, caspase-3 activity, single-strand breaks' (SSBs) formation and protein level of matrix metalloproteinase-1 (MMP-1), heme oxygenase-1 (HO-1), and heat shock protein (HSP70) were evaluated. The most pronounced preventative potential was found for DHSB, a minor component of silymarin, and SC, the second most abundant flavonolignan in silymarin. They had significant effects on most of the studied parameters. Meanwhile, a photoprotective effect of SC was mostly found at double the concentration of DHSB. ISB and SD protected against GSH depletion, the generation of ROS, carbonylated proteins and SSBs, and caspase-3 activation, but had no significant effect on MMP-1, HO-1, or HSP70. In summary, DHSB and to a lesser extent other silymarin flavonolignans are potent UVA-protective compounds. However, due to the in vitro phototoxic potential of DHSB published elsewhere, further studies are needed to exclude phototoxicity for humans as well as to confirm our results on human skin ex vivo and in vivo.

Topics: Caspase 3; Cells, Cultured; Cytoprotection; DNA Breaks, Single-Stranded; Fibroblasts; Glutathione; Heme Oxygenase-1; HSP70 Heat-Shock Proteins; Humans; Matrix Metalloproteinase 1; Protein Carbonylation; Reactive Oxygen Species; Silymarin; Skin; Sunscreening Agents; Ultraviolet Rays

The primary biological function of platelets is to form hemostatic thrombi that prevent blood loss and maintain vascular integrity. These multi-responding cells are activated by different endogenous, physiological agonists due to the vast number of receptors present on the surface of the platelets. Collagen represents up to 40% of the total protein presented in the vessel wall and is the major activator of the platelets' response after tissue injury, and is the only matrix protein which supports both platelet adhesion and complete activation. The aim of our study was to determine the effects of three major flavonolignans (silybin, silychristin and silydianin) on collagen-induced blood platelets' activation, adhesion, aggregation and secretion of PF-4. We observed that depending on the dose, silychristin and silybin have anti-platelet properties observed as inhibition of collagen-induced activation (formation of blood platelet aggregates and microparticles, as well as decreased expression of P-selectin and activation of integrin α

Topics: Blood Coagulation; Blood Platelets; Cell Adhesion; Collagen; Flavonolignans; Hemostatics; Humans; Platelet Activation; Platelet Aggregation; Platelet Factor 4; Platelet Glycoprotein GPIIb-IIIa Complex; Silybin; Silymarin

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet activation.

Topics: Adenosine Diphosphate; Blood Platelets; Cell Adhesion Molecules; Fibrinogen; Flavonolignans; Humans; Microfilament Proteins; Phosphoproteins; Phosphorylation; Platelet Activation; Platelet Aggregation; Platelet-Rich Plasma; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y12; Signal Transduction; Silybin; Silymarin

Flavonolignans constitute an important class of plant secondary metabolites formed by oxidative coupling of one flavonoid and one phenylpropanoid moiety. The standardized flavonolignan-rich extract prepared from the fruits of Silybum marianum is known as silymarin and has long been used medicinally, prominently as an antihepatotoxic and as a chemopreventive agent. Principal component analysis of the variation in flavonolignan content in S. marianum samples collected from different locations in Egypt revealed biosynthetic relationships between the flavonolignans. Silybin A, silybin B, and silychristin are positively correlated as are silydianin, isosilychristin, and isosilybin B. The detection of silyamandin in the extracts of S. marianum correlates with isosilychristin and silydianin content. The positive correlation between silydianin, isosilychristin, and silyamandin was demonstrated using quantitative

Topics: Egypt; Flavonolignans; Fruit; Molecular Structure; Secondary Metabolism; Silybin; Silybum marianum; Silymarin

Silybum marianum (milk thistle) is a medicinal plant used for the treatment of various liver disorders. This study examined whether the main flavonolignans from S. marianum (i.e. silybin, silychristin, silydianin) and their 2,3-dehydro derivatives (i.e. 2,3-dehydrosilybin, 2,3-dehydrosilychristin, 2,3-dehydrosilydianin) activate the Nrf2 pathway, which regulates the expression of genes encoding many cytoprotective enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1). After 48h of exposure, 2,3-dehydrosilydianin at concentrations of 25μM and higher significantly elevated the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. In contrast, other tested compounds at non-cytotoxic concentrations had a mild or negligible effect on the NQO1 activity. Using a luciferase reporter assay, 2,3-dehydrosilydianin was found to significantly activate transcription via the antioxidant response element in stably transfected human AREc32 reporter cells. Moreover, 2,3-dehydrosilydianin caused the accumulation of Nrf2 and significantly induced the expression of the Nqo1 gene at both the mRNA and protein levels in Hepa1c1c7 cells. We found that 2,3-dehydrosilydianin also increased to some extent the expression of other Nrf2 target genes, namely of the heme oxygenase-1 gene (Hmox1) and the glutamate-cysteine ligase modifier subunit gene (Gclm). We conclude that 2,3-dehydrosilydianin activates Nrf2 and induces Nrf2-mediated gene expression in Hepa1c1c7 cells.

Topics: Animals; Cell Line, Tumor; Gene Expression; Glutamate-Cysteine Ligase; Heme Oxygenase-1; Humans; Membrane Proteins; Mice; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Silybin; Silybum marianum; Silymarin; Up-Regulation

Arachidonic acid metabolism by cyclooxygenase (COX) is a major pathway for blood platelets' activation, which is associated with pro-thrombotic platelet activity and the production of pro-inflammatory mediators. Inhibition of COX activity is one of the major means of anti-platelet pharmacotherapy preventing arterial thrombosis and reducing the incidence of cardiovascular events. Recent studies have presented that a silymarin (standardized extract of Milk thistle (Silybum marianum)) can inhibit the COX pathway. Accordingly, the aim of our study was to determine the effects of three major flavonolignans (silybin, silychristin and silydianin) on COX pathway activity in blood platelets.. We determined the effect of flavonolignans on arachidonic acid induced blood platelet aggregation, COX pathway metabolites formation, as well as COX activity in platelets. Additionally, we analysed the potential mechanism of this interaction using the bioinformatic ligand docking method.. We observed that tested compounds decrease the platelet aggregation level, both thromboxane A. The results obtained from this study clearly present the potential of flavonolignans as novel antiplatelet and anti-inflammatory agents.

Topics: Anti-Inflammatory Agents; Arachidonic Acid; Binding, Competitive; Blood Platelets; Flavonolignans; Humans; Inflammation Mediators; Malondialdehyde; Molecular Docking Simulation; Plant Extracts; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin-Endoperoxide Synthases; Silybin; Silybum marianum; Silymarin; Thromboxane A2

Interleukin-1 beta (IL-1β)-the most potent pro-inflammatory is responsible for a broad spectrum of immune and inflammatory responses, it induces T-cell and B-cell activation and consequently the synthesis of other pro-inflammatory cytokines (such as IFN-γ and TNF). IL-1β induces the formation of blood platelet-leukocyte aggregates (PLAs), which suggests that IL-1β significantly affects the cross-talk between blood platelets and the immune response system, leading to coronary thrombosis. The aim of our study is to investigate the effect of flavonolignans (silybin, silychristin and silydianin) on the IL-1β-induced interaction between platelets and leukocytes, as well as on the expression and the secretion of pro-inflammatory factors. Whole blood samples were pre-incubated with commercially available flavonolignans (silybin, silychristin and silydianin) in a concentration range of 10-100 µM (30 min, 37 °C). Next, samples were activated by IL-1β for 1 h. Blood platelet-leukocyte aggregates were detected by using the double-labeled flow cytometry (CD61/CD45). The level of produced cytokines was estimated via the ELISA immunoenzymatic method. IFN-γ and TNF gene expression was evaluated using Real Time PCR with TaqMan arrays. We observed that in a dose-dependent manner, silybin and silychristin inhibit the IL-1β-induced formation of blood platelet-leukocyte aggregates in whole blood samples, as well as the production of pro-inflammatory cytokines-IL-2, TNF, INF-α, and INF-γ. Additionally, these two flavonolignans abolished the IL-1β-induced expression of mRNA for IFN-γ and TNF. Our current results demonstrate that flavonolignans can be novel compounds used in the prevention of cardiovascular diseases with dual-use action as antiplatelet and anti-inflammatory agents.

Topics: Anti-Inflammatory Agents; Blood Platelets; Cells, Cultured; Cytokines; Flavonolignans; Humans; Interleukin-1beta; Leukocytes; Platelet Aggregation Inhibitors; Silybin; Silymarin

Flavonolignans are the main components of silymarin, which represents 1.5-3% of the dry fruit weight of Milk thistle (

Topics: A549 Cells; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Blood Platelets; Cell Survival; DNA Copy Number Variations; DNA Damage; DNA, Mitochondrial; Dose-Response Relationship, Drug; Flavonolignans; Fruit; Humans; Leukocytes, Mononuclear; Plant Extracts; Reactive Oxygen Species; Silybin; Silybum marianum; Silymarin

In this paper, a new ultra-high performance liquid chromatography (UHPLC) method using a core-shell column with a pentafluorophenyl stationary phase for separation of seven active compounds of a Silybum marianum extract was developed and validated. Silymarin, an extract of Silybum marianum, is known for its abilities to protect the liver from toxic substances, hepatitis therapy, and anti-tumour activity. Silymarin is currently being widely used in commercial preparations and herbal teas. Separation of seven compounds contained in the Silybum marianum extract (taxifolin, silychristin, silydianin, silybin A, silybin B, isosilybin A, isosilybin B) and other substances occurring in real samples was performed on the Kinetex 1.7μ F5 100A (150×2.1mm), 1.7μm particle size core-shell column, with a mobile phase methanol/100mM phosphate buffer pH 2.0 according to the gradient program. A mobile phase 0.35mLmin

Topics: Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Dietary Supplements; Isomerism; Plant Extracts; Silybin; Silybum marianum; Silymarin; Teas, Herbal

The protective constituents of silymarin, an extract from Silybum marianum fruits, have been extensively studied in terms of their antioxidant and hepatoprotective activities. Here, we explore the electron-donor properties of the major silymarin flavonolignans. Silybin (SB), silychristin (SCH), silydianin (SD) and their respective 2,3-dehydroderivatives (DHSB, DHSCH and DHSD) were oxidized electrochemically and their antiradical/antioxidant properties were investigated. Namely, Folin-Ciocalteau reduction, DPPH and ABTS(+) radical scavenging, inhibition of microsomal lipid peroxidation and cytoprotective effects against tert-butyl hydroperoxide-induced damage to a human hepatocellular carcinoma HepG2 cell line were evaluated. Due to the presence of the highly reactive C3-OH group and the C-2,3 double bond (ring C) allowing electron delocalization across the whole structure in the 2,3-dehydroderivatives, these compounds are much more easily oxidized than the corresponding flavonolignans SB, SCH and SD. This finding was unequivocally confirmed not only by experimental approaches, but also by density functional theory (DFT) calculations. The hierarchy in terms of ability to undergo electrochemical oxidation (DHSCH~DHSD>DHSB>>SCH/SD>SB) was consistent with their antiradical activities, mainly DPPH scavenging, as well as in vitro cytoprotection of HepG2 cells. The results are discussed in the context of the antioxidant vs. prooxidant activities of flavonolignans and molecular interactions in complex biological systems.

Topics: Animals; Antioxidants; Cytoprotection; Electron Spin Resonance Spectroscopy; Flavonolignans; Hep G2 Cells; Humans; Male; Rats; Silybin; Silymarin; Structure-Activity Relationship

Mature fruits collected from different milk thistle (Silybum marianum (L.) Gaertn.) plants, grown in various habitats in Europe, were analysed for silymarin content and variation in component composition. Two different German and Polish cultivars each, as well as fruits from Hungary and Bulgaria have been compared with respect to their ratio of flavonolignan regioisomers. Besides differences in total silymarin content (0.8%-4.9%), three distinct chemotypical variations in fruit flavonolignan regioisomer composition in the cultivars have been observed. Although the differences in the diastereomer ratios of silybin A/B and isosilybin A/B were not significant, they never appeared in a 1:1 ratio. In vitro cultures have been established from seedlings of three typical chemotypes for further insights into flavonolignan content and composition in suspension cultures and the release of these specialized compounds to the extracellular space. The differences in the three Silybum marianum chemotypes were also observed in the composition of the intracellular silymarin of suspension-cultured cells. Silymarin components released to the cell culture medium, however, showed a highly differing composition with only low amounts of silychristin and silydianin. Assays with crude protein extracts prepared from suspension cells or habituated medium of these three chemotypes did not result in differences in silymarin content or composition. In in vitro assays the formation of the regioisomers silydianin and silychristin were strongly influenced by the taxifolin:coniferyl alcohol concentration ratio.

Topics: Bulgaria; Cells, Cultured; Europe; Flavonols; Fruit; Lignin; Quercetin; Silybin; Silybum marianum; Silymarin; Stereoisomerism

According to the results obtained in this study, drought stress can enhance the accumulation of silymarin in milk thistle seeds. Moreover, under drought stress, the share of silybin increased which possess the greatest degree of biological activity among the silymarin components. Silymarin, an isomeric mixture of flavonolignans found in milk thistle (Silybum marianum (L.) Gaertn) seeds, has been used for its hepatoprotective effects for more than 2,000 years. Biosynthesis and accumulation of active substances like silymarin in plant tissues highly interacts with the environmental conditions. Effects of moderate and severe drought stress (based on soil moisture depletion) on silymarin content and composition in milk thistle seeds were evaluated in a field study. Averaged across treatments, milk thistle seeds contained 19.3 g kg(-1) silymarin. Drought stress enhanced silymarin accumulation in milk thistle seeds. Plants grown under moderate and severe drought stress treatments contained 4 and 17 % greater silymarin than those grown in well-watered condition, respectively. Greater content of sylimarin in stressed plants was attributed to more contents of silybin, isosilybin and silychristin, while silydianin content was lower under drought condition. According to the results obtained in this study, drought stress enhanced accumulation of silymarin in milk thistle seeds and improved its quality by increasing the share of silybin, which possess the greatest degree of biological activity among the silymarin components.

Topics: Antioxidants; Droughts; Seeds; Silybin; Silybum marianum; Silymarin

The optimal conditions of supercritical carbon dioxide (SC-CO2) (160-220 bars, 40-80 °C) technology combined with co-solvent (ethanol), to recover oil, flavonolignans (silychristin, silydianin and silybinin) and fatty acids from milk thistle seeds, to be used as food additives and/or nutraceuticals, were studied. Moreover, the antioxidant and cytotoxic activities of the SC-CO2 oil seeds extracts were evaluated in Caco-2 carcinoma cells. Pressure and temperature had a significant effect on oil and flavonolignans recovery, although there was not observed a clear trend. SC-CO2 with co-solvent extraction at 220 bars, 40 °C was the optimum treatment to recover oil (30.8%) and flavonolignans from milk thistle seeds. Moreover, linoleic (47.64-66.70%), and oleic (19.68-24.83%) acids were the predominant fatty acids in the oil extracts recovered from milk thistle under SC-CO2. In addition, SC-CO2 extract showed a high antioxidant activity determined by DPPH and ABTS tests. Cytotoxic activities of silychristin, silydianin and silybinin and the obtained SC-CO2 extract (220 bars, 40 °C) were evaluated against Caco-2 cells. The SC-CO2 extract inhibited the proliferation of Caco-2 cells in a dose-responsive manner and induced the highest percentage of mortality of Caco-2 cells (from 43 to 71% for concentrations from 10 up to 100 μg/ml of SC-CO2 oil seeds).

Topics: Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Antioxidants; Caco-2 Cells; Carcinoma; Cell Survival; Chromatography, Supercritical Fluid; Dietary Supplements; Fatty Acids; Flavonolignans; Food Additives; Humans; Plant Oils; Seeds; Silybin; Silybum marianum; Silymarin; Tunisia

Milk thistle (Silybum marianum) extracts, one of the most widely used dietary supplements, contain a mixture of six major flavonolignans (silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin) and other components. However, the pharmacokinetics of the free individual flavonolignans have been only partially investigated in humans. Furthermore, antioxidant effects of the extract, which may underlie the basis of many therapeutic effects, have not been thoroughly assessed. The present study evaluated the pharmacokinetics of the six major flavonolignans in healthy volunteers receiving single doses of either one (175 mg), two (350 mg), or three (525 mg) milk thistle capsule(s) on three separate study visits. Additionally, the steady-state pharmacokinetic parameters were determined after the subjects were administered one capsule three times daily for 28 consecutive days. Our results demonstrated that all six flavonolignans were rapidly absorbed and eliminated. In order of abundance, the exposure to free flavonolignans was greatest for silybin A followed by silybin B, isosilybin B, isosilybin A, silychristin, and silydianin. The systemic exposure to these compounds appeared linear and dose proportional. The disposition of flavonolignans was stereoselective, as evidenced by the apparent clearance of silybin B, which was significantly greater than silybin A, whereas the apparent clearance of isosilybin B was significantly lower than isosilybin A. The concentrations of urinary 8-epi-prostaglandin F2α, a commonly used biomarker of oxidative status in humans, were considerably decreased in study subjects after a 28-day exposure to the extract (1.3 ± 0.9 versus 0.8 ± 0.9 ng/mg creatinine) but failed to reach statistical significance (P = 0.076).

Topics: Adult; Antioxidants; Dinoprost; F2-Isoprostanes; Female; Flavonolignans; Healthy Volunteers; Humans; Male; Silybin; Silybum marianum; Silymarin; Young Adult

Silibinin (SB), silydianin (SD), and silychristin (SC) are components of silymarin. These compounds can be used to protect the skin from oxidative stress induced by ultraviolet (UV) irradiation and treat it. To this end, the absorption of silymarin constituents via the skin was examined in the present report.. Transport of SB, SD, and SC under the same thermodynamic activity through and into the skin and the effects of pH were studied in vitro using a Franz diffusion assembly.. The lipophilicity increased in the order of SC

Topics: Administration, Cutaneous; Animals; Antioxidants; Diffusion Chambers, Culture; Female; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; In Vitro Techniques; Mice; Mice, Nude; Permeability; Silybin; Silymarin; Skin Absorption; Skin Irritancy Tests; Thermodynamics; Time Factors

Silymarin is one of the most potent antioxidant so far developed from plant sources used as hepatoprotectants. Influence of different concentrations (0, 1, 2, 4, 6 and 8mg/50ml culture) and exposure time (24, 48, 72, 96 and 120h) of salicylic acid on lipoxygenase activity, linoleic acid content, growth and production of silymarin in hairy root cultures of S. marianum were investigated. Detection and identification of flavonolignans was carried out by high performance liquid chromatograph method. Salicylic acid enhanced silymarin production (1.89mgg(-1) DW). The optimal feeding condition was the addition of salicylic acid (6 mg/50 ml culture) after 24h in which the silymarin content was 2.42 times higher than the control (0.78mgg(-1) DW). The content of silybin, isosilybin, silychristin, silydianin and taxifolin were 0.703, 0.017, 0.289, 0.02 and 0.863mgg(-1) DW respectively in these samples, while in non-treated hairy roots were 0.027, 0.046, 0.23, 0.022 and 0.453 respectively. Lipoxygenase activity also affected by elicitation. lipoxygenase activity increased 24h after treatment by approximately 1.57- fold (0.21 Delta OD(234)/mgproteinmin(-1)). Upon elicitation with salicylic acid, linoleic acid content of hairy roots (38.26mgg(-1) DW) were also elevated after 24h, in which the linoleic acid content was 2.37 times higher than the control (16.1mgg(-1) DW). It is feasible that elicitation with salicylic acid regulates the jasmonate pathway, which in turn mediates the elicitor-induced accumulation of silymarin.

Topics: Antioxidants; Cell Culture Techniques; Flavonolignans; Linoleic Acid; Lipoxygenase; Plant Roots; Quercetin; Salicylic Acid; Silybin; Silybum marianum; Silymarin

Prostate cancer (PCa) is the leading cause of cancer-related deaths in men; urgent measures are warranted to lower this deadly malignancy. Silymarin is a known cancer chemopreventive agent, but the relative anticancer efficacy of its constituents is still unknown. Here, we compared the efficacy of 7 pure flavonolignan compounds isolated from silymarin, namely silybin A, silybin B, isosilybin A, isosilybin B, silydianin, isosilydianin, silychristin and isosilychristin, in advanced human PCa PC3 cells. Silybin A, silybin B, isosilybin A, isosilybin B, silibinin and silymarin strongly inhibited the colony formation by PC3 cells (p < 0.001), while silydianin, silychristin and isosilychristin had marginal effect (p < 0.05). Using cell growth and death assays, we identified isosilybin B as the most effective isomer. FACS analysis for cell cycle also showed that silybin A, silybin B, isosilybin A, isosilybin B, silibinin and silymarin treatment resulted in strong cell cycle arrest in PC3 cells after 72 hr of treatment, while the effect of silydianin, silychristin and isosilychristin was marginal (if any). Western blot analysis also showed the differential effect of these compounds on the levels of cell cycle regulators-cyclins (D, E, A and B), CDKs (Cdk2, 4 and Cdc2), CDKIs (p21 and p27) and other cell cycle regulators (Skp2, Cdc25A, B, C and Chk2). This study provided further evidence for differential anticancer potential among each silymarin constituent, which would have potential implications in devising better formulations of silymarin against prostate and other cancers.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; Blotting, Western; cdc25 Phosphatases; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor Proteins; Cyclin-Dependent Kinases; Cyclins; Drug Screening Assays, Antitumor; Flavonolignans; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isomerism; Male; Prostatic Neoplasms; Protective Agents; Silybin; Silymarin; Time Factors

A sensitive method for the simultaneous quantitation of six active constituents in commercial silymarin standardized extracts was developed based on liquid chromatography (LC) in combination with mass spectrometry (MS). The six main active constituents, namely, silydianin, silychristin, diastereoisomers of silybin (silybin A and B), and diastereoisomers of isosilybin (isosilybin A and B) were completely separated and quantified by LC/MS. Silymarin obtained from Sigma-Aldrich Co. was evaluated and used as standard reference material for the six individual constituents in comparing the relative content of silymarin and the relative ratio of each constituent in commercial standardized silymarin extracts, respectively. Significant variation was found between different commercial silymarin sources. As a result, this method has proven useful in evaluating and quantifying the six active constituents in commercial milk thistle extracts. The calibration curves were over the range from 0.25 to 100 microg/mL for silychristin and silydianin, and from 0.10 to 100 microg/mL for silybin A, silybin B, isosilybin A and isosilybin B, respectively (r(2)> or =0.9958). For all six active constituents, the overall intra-day precision values, based on the relative standard deviation replicate for four QC levels, ranged from 1.18% to 12.4% and accuracy ranged from 89.4% to 112%. This methodology could easily be incorporated into standardized testing to assess content uniformity including lot-to-lot variation as part of routine process controls as well as a means to describe cross-product variation among the exiting marketed formulations.

Topics: Chromatography, Liquid; Plant Extracts; Reproducibility of Results; Sensitivity and Specificity; Silybin; Silymarin; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Tandem Mass Spectrometry

Development of sustained delivery systems for herbal medicines was very difficult because of their complexity in composition. The concept of synchronized release from sustained release systems, which is characterized by release of multiple components in their original ratio that defines a herbal medicine, served as the basis for keeping the original pharmacological activity. In this study, erodible matrix systems based on glyceryl monostearate and polyethylene glycol 6000 or poloxamer 188 were prepared to perform strict control on synchronized release of the five active components of silymarin, i.e. taxifolin, silychrystin, silydianin, isosilybin and silybin. The matrix system was prepared by a melt fusion method. Synchronized release was achieved with high similarity factor f(2) values between each two of the five components. Erosion profiles of the matrix were in good correlation with release profiles of the five components, showing erosion-controlled release mechanisms. Through tuning some of the formulation variables, the system can be adjusted for synchronized and sustained release of silymarin for oral administration. In vitro hemolysis study indicated that the synchronized release samples showed a much better stabilizing effect on erythrocyte membrane.

Topics: Animals; Chemistry, Pharmaceutical; Delayed-Action Preparations; Drug Carriers; Drug Compounding; Flavonols; Glycerides; Hemolysis; In Vitro Techniques; Molecular Structure; Poloxamer; Polyethylene Glycols; Protective Agents; Quercetin; Rabbits; Silybin; Silymarin; Solubility; Technology, Pharmaceutical; Time Factors

A selective and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the characterization of silymarin in commercially available milk thistle extract. In this study, six main active constituents, including silydianin, silychristin, diastereomers of silybin (silybin A and B) and diastereomers of isosilybin (isosilybin A and B) in silymarin, were completely separated on a YMC ODS-AQ HPLC column using a gradient mobile phase system comprised of ammonium acetate and methanol/water/formic acid. Identification and characterization of the major constituents were based not only on the product ion scan, which provided unique fragmentation information of a selected molecular ion, but also on the specific fragmentation of multiple reaction monitoring (MRM) data, which confirmed the retention times of LC chromatographic peaks. The method was applied in the analysis of human plasma samples in the presence of silymarin and appeared to be suitable for the pharmacokinetic studies in which the discrimination of silymarin constituents is essential.

Topics: Chromatography, High Pressure Liquid; Plant Extracts; Silybin; Silybum marianum; Silymarin; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet

Silybin and related flavonolignans form a major part of the Silybum marianum extract, silymarin, which has been used to treat liver diseases for hundreds of years. Although regarded as safe, many of the extract constituents remain thus far untested for their possible effects on liver biotransformation enzymes. Cytochromes P450 (CYP) are very important in this regard. We tested the effect of four flavonolignans: silybin, its hemisynthetic derivative dehydrosilybin, silydianin, and silycristin on three specific CYP activities: bufuralol 1'-hydroxylation (CYP2D6), p-nitrophenol hydroxylation (CYP2E1), and nifedipine oxidation (CYP3A4). All flavonolignans displayed dose-dependent inhibition of these activities with IC(50) values in the micromolar range. The inhibition was competitive or mixed as revealed by double reciprocal plots of kinetic experiments. However, the inhibition is not considered to be relevant for therapy because physiological concentrations of the individual flavonolignans do not exceed 0.5 microM. The data support the use of the extract as a dietary supplement.

Topics: Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Flavonoids; Humans; Inhibitory Concentration 50; Isomerism; Liver; Microsomes, Liver; Phytotherapy; Plant Extracts; Silybin; Silybum marianum; Silymarin

The mixture of flavonolignans [Legalon: silybin (2a), isosilybin (3), silydianin (4) and silychristin (5)] and derivatives of silybin (2b-d) were assessed for their inhibitory activity on the oxidative burst of PMA-stimulated human PMNLs. The inhibitory effect of flavonolignans on O(2)(-) release were compared with that of vitamin E (1). The flavonolignans tested exhibited the following order in inhibition of O(2)(-) release by PMA-stimulated PMNLs: 5,7,4"- trimethylsilybin (2c) approximately vitamin E (1) > Legalon >or= peracetylsilybin (2b) > silybin (2a) > peracetyl-5,7,4"-trimethylsilybin (2d). The flavonolignans inhibited not only the O(2)(-) release, but also the H(2)O(2) formation in PMA-stimulated PMNLs. The inhibitory capacity of flavonolignans on H(2)O(2) formation was similar to their inhibitory capacity on O(2)(-) release. These data suggest that the flavonolignans have antioxidant properties on the PMNL oxidative burst. The fact that the trimethyl derivative of silybin (2c) has a greater inhibitory effect than silybin itself suggests that the efficacy of the antioxidant properties is dependent on the lipophilicity of the molecules. This is underlined by the fact that peracetylation of all of the hydroxyl groups in silybin resulted in a total loss of the antioxidant activity of the molecule. In summary, flavonolignans inhibit the oxidative burst of PMNLs, and this inhibitory effect depends on the chemical structure of the flavonolignans.

Topics: Flavonoids; Humans; Hydrogen Peroxide; Neutrophils; Phytotherapy; Respiratory Burst; Silybum marianum; Silymarin; Structure-Activity Relationship; Superoxides; Vitamin E

Topics: Animals; Biological Availability; Cattle; Flavonoids; Protein Binding; Silymarin

Seedlings of Lepidium sativum L. contain 4 peroxidase isoenzymes. Their de-novo-synthesis is significantly depressed in the presence of 3.10(-3) moles/1 silybin, silydianin and silychristin in the incubation medium respectively.

Topics: Flavonoids; Isoenzymes; Isomerism; Peroxidases; Plants, Edible; Silymarin

Silybin and silychristin non-competitively inhibit the horseradish peroxidase in vitro. Silydianin on the other hand is a competitive inhibitor of the enzyme.

Topics: Flavonoids; Horseradish Peroxidase; Isomerism; Peroxidases; Silymarin