Silibinin suppresses TGFß2-induced lens epithelial cell migration and epithelial-mesenchymal transition.

Growth factor-induced migration of lens epithelial cell (LEC) toward the posterior of lens capsule bag and their epithelial-mesenchymal transition (EMT) is the key process involved in the pathogenesis of posterior capsular opacification (PCO). Silibinin, a natural flavonolignan, confers therapeutic effects to different cells by regulation of signalling pathways; however, its role in the prevention of migration and EMT of LECs is yet to be analysed. In this study, the inhibitory capabilities of silibinin on migration and EMT were analysed in response to TGFß2 stimulation in HLE B-3 cells. The anti-migratory effect of silibinin was analysed using wound healing assay. Transcriptional and translational expression of genes related to LEC migration, EMT, and transcription factors related to EMT were studied by quantitative real-time PCR and Western blotting. Immunofluorescence analysis was utilized to study the localization of fibronectin. Silibinin reduced the viability of LECs in a concentration-dependent manner and inhibited the wound healing capacity of LECs induced by TGFß2. Silibinin also suppressed alteration in the EMT-related markers such as cytoskeletal proteins, cell adhesion markers, extracellular matrix molecules, and transcription factors. Analysis of downstream signalling revealed that treatment with silibinin decreased phosphorylated Akt (Ser473, Thr308), PDK1 (Ser241), PTEN (Ser380), c-Raf (Ser259), and GSK3ß (Ser9) in TGFß-stimulated cells. The effect of silibinin treatment on phosphorylated Akt resembled that of the PI3K inhibitor LY294002. Our results suggest that silibinin can suppress LEC migration and EMT, which involves the inactivation of the PI3K-Akt signalling pathway. Silibinin might be a good candidate for PCO prevention; however, functional evaluation of silibinin using in vivo models is a pre-requisite.

Hepatic organic anion transporting polypeptides mediate disposition of milk thistle flavonolignans and pharmacokinetic silymarin-drug interactions.

Silybum marianum (L.) Gaertn. (Asteraceae), commonly known as milk thistle, is a botanical natural product used to self-treat multiple diseases such as Type 2 diabetes mellitus and nonalcoholic steatohepatitis (NASH). An extract from milk thistle seeds (achenes), termed silymarin, is comprised primarily of several flavonolignans. Systemic concentrations of these flavonolignans can influence the potential biologic effects of silymarin and the risk for pharmacokinetic silymarin-drug interactions. The aims of this research were to determine the roles of organic anion transporting polypeptides (OATPs/Oatps) in silymarin flavonolignan disposition and in pharmacokinetic silymarin-drug interactions. The seven major flavonolignans from silymarin were determined to be substrates for OATP1B1, OATP1B3, and OATP2B1. Sprague Dawley rats were fed either a control diet or a NASH-inducing diet and administered pitavastatin (OATP/Oatp probe substrate), followed by silymarin via oral gavage. Decreased protein expression of Oatp1b2 and Oatp1a4 in NASH animals increased flavonolignan area under the plasma concentration-time curve (AUC) and maximum plasma concentration. The combination of silymarin inhibition of Oatps and NASH-associated decrease in Oatp expression caused an additive increase in plasma pitavastatin AUC in the animals. These data indicate that OATPs/Oatps contribute to flavonolignan cellular uptake and mediate the interaction between silymarin and NASH on pitavastatin systemic exposure.

Simulated Gastrointestinal Biotransformation of Chlorogenic Acid, Flavonoids, Flavonolignans and Triterpenoid Saponins in Cecropia obtusifolia Leaf Extract.

It is well known that biotransformation processes in the human body are crucial to form potentially bioactive metabolites from particular classes of natural products. However, little research has been conducted concerning the bioavailability of polyphenols, especially in the colon. The gastrointestinal stability and colonic biotransformation of the crude extract of the leaves of Cecropia obtusifolia, rich in flavone C-glycosides, was investigated under in vitro conditions, and the processing and interpretation of results were facilitated by using an automated machine learning model. This investigation revealed that flavone C-glycosides and flavonolignans from C. obtusifolia were stable throughout their passage in the simulated gastrointestinal tract including the colon phase. On the other hand, the colon bacteria extensively metabolized chlorogenic acid, flavonol, and triterpenoid O-glycosides. This investigation revealed that the colonic microbiota has an important role in the biotransformation of some chemical constituents of this extract.

Metabolism, Transport and Drug-Drug Interactions of Silymarin.

Silymarin, the extract of milk thistle, and its major active flavonolignan silybin, are common products widely used in the phytotherapy of liver diseases. They also have promising effects in protecting the pancreas, kidney, myocardium, and the central nervous system. However, inconsistent results are noted in the different clinical studies due to the low bioavailability of silymarin. Extensive studies were conducted to explore the metabolism and transport of silymarin/silybin as well as the impact of its consumption on the pharmacokinetics of other clinical drugs. Here, we aimed to summarize and highlight the current knowledge of the metabolism and transport of silymarin. It was concluded that the major efflux transporters of silybin are multidrug resistance-associated protein (MRP2) and breast cancer resistance protein (BCRP) based on results from the transporter-overexpressing cell lines and MRP2-deficient (TR-) rats. Nevertheless, compounds that inhibit the efflux transporters MRP2 and BCRP can enhance the absorption and activity of silybin. Although silymarin does inhibit certain drug-metabolizing enzymes and drug transporters, such effects are unlikely to manifest in clinical settings. Overall, silymarin is a safe and well-tolerated phytomedicine.

Novel flavonolignan hybrid antioxidants: From enzymatic preparation to molecular rationalization.

A series of antioxidants was designed and synthesized based on conjugation of the hepatoprotective flavonolignan silybin with l-ascorbic acid, trolox alcohol or tyrosol via a C12 aliphatic linker. These hybrid molecules were prepared from 12-vinyl dodecanedioate-23-O-silybin using the enzymatic regioselective acylation procedure with Novozym 435 (lipase B) or with lipase PS. Voltammetric analyses showed that the silybin-ascorbic acid conjugate exhibited excellent electron donating ability, in comparison to the other conjugates. Free radical scavenging, antioxidant activities and cytoprotective action were evaluated. The silybin-ascorbic acid hybrid exhibited the best activities (IC50 = 30.2 µM) in terms of lipid peroxidation inhibition. The promising protective action of the conjugate against lipid peroxidation can be attributed to modulated electron transfer abilities of both the silybin and ascorbate moieties, but also to the hydrophobic C12 linker facilitating membrane insertion. This was supported experimentally and theoretically by density functional theory (DFT) and molecular dynamics (MD) calculations. The results presented here can be used in the further development of novel multipotent antioxidants and cytoprotective agents, in particular for substances acting at an aqueous/lipid interface.

Flavonolignans inhibit ADP induced blood platelets activation and aggregation in whole blood.

Flavonolignans are a group of active chemical compounds presented in the silymarin - a standardized extract obtained from fruits and seeds of Milk thistle (Silybum marianum L. Gaernt.). Since the 70s of the last century, flavonolignans have been regarded to the official medicine as a substances having hepatoprotective properties. However many researches performed in recent years have demonstrated that flavonolignans posses many other healthy properties including modulation of variety cell-signaling pathways. The aim of our study was to examine the effects of three major flavonolignans (silybin, silychristin and silydianin) on ADP-induced blood platelet activation using the flow cytometry analysis as well as determine the mechanism of this interaction by bioinformatic ligand docking method. We observed that all tested flavonolignans in dose-dependent manner inhibit formation of blood platelet aggregates and microparticles as well as decrease expression of P-selectin and activation of integrin αIIbß3. Our computer-generated models confirm the flow cytometry analysis. We observed that all tested flavonolignans have conformations which are able to bind to the extracellular domain of P2Y12 receptor and probably block interaction with ADP. Our studies may help in the development of a new potential anti-platelet agent, which might be an alternative to the current using drugs.

The Potential of Flavonolignans in Prostate Cancer Management.

BACKGROUND: The generic name "flavonolignan" was created in 1968 for a relatively small class of naturally occurring hybrid molecules biogenetically originated from ubiquitous flavonoids and lignans (phenylpropanoids). The first group of flavonolignans was extracted from Silybum marianum that has long been used for hepatoprotection. Recently, the medicinal merit of flavonolignans has been extended to the prostate cancer management. METHODS: Systematic interpretation and summarization of the relevant literature. RESULTS: Over forty naturally occurring flavonolignans have so far been obtained from various plants. Certain flavonolignans have been demonstrated by in vitro cell-based and in vivo animal-based experiments, and human clinical studies i) to possess effective chemopreventive function against various tumor promoters; ii) to show the anti-angiogenic efficacy; iii) to have potential in treating prostate cancer; iv) to sensitize prostate tumors to chemotherapeutic agents through down-regulation of P-glycoprotein and other mechanisms; and v) to be used by prostate cancer patients to protect or treat the hepatotoxicity caused by several chemotherapies. Certain flavonolignans can synergize with well-established chemotherapeutic agents for prostate cancer. CONCLUSION: This review provides a systematic and in-depth overview of the promise and potential of flavonolignans in prostate cancer management, which covers their chemopreventive effect, chemotherapeutic treatment, mechanisms of actions, synthetic derivatives, structure-activity relationships, and the difference in inhibiting prostate cancer cell proliferation between certain flavonoligans and their respective flavonoid counterpart. This summarization aims to provide valuable insights into further and rational development of flavonolignans for prostate cancer management by interpreting the data reported in the literature.

Supercritical CO2 extraction of oil, fatty acids and flavonolignans from milk thistle seeds: Evaluation of their antioxidant and cytotoxic activities in Caco-2 cells.

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).

Biotransformation on the flavonolignan constituents of Silybi Fructus by an intestinal bacterial strain Eubacterium limosum ZL-II.

Eubacterium limosum ZL-II is an anaerobic bacterium with demethylated activity, which was isolated from human intestinal bacteria in our previous work. In this study, the flavonolignan constituents of Silybi Fructus were biotransformed by E. limosum(1) ZL-II, producing four new transformation products - demethylisosilybin B (T1), demethylisosilybin A (T2), demethylsilybin B (T3) and demethylsilybin A (T4), among which T1 and T2 were new compounds. Their chemical structures were identified by ESI-TOF/MS, (1)H NMR, (13)C NMR, HMBC and CD spectroscopic data. The bioassay results showed that the transformation products T1-T4 exhibited significant inhibitory activities on Alzheimer's amyloid-ß 42 (Aß42(2)) aggregation with IC50 values at 7.49 µM-10.46 µM, which were comparable with that of the positive control (epigallocatechin gallate, EGCG(3), at 9.01 µM) and much lower than those of their parent compounds (at not less than 145.10 µM). The method of biotransformation by E. limosum ZL-II explored a way to develop the new and active lead compounds in Alzheimer's disease from Silybi Fructus. However, the transformation products T1-T4 exhibited decreased inhibitory activities against human tumor cell lines comparing with their parent compounds.