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.

Deoxypodophyllotoxin Induces ROS-Mediated Apoptosis by Modulating the PI3K/AKT and p38 MAPK-Dependent Signaling in Oral Squamous Cell Carcinoma.

Deoxypodophyllotoxin (DPT), a naturally occurring flavonolignan, possesses several pharmacological properties, including anticancer property. However, the mechanisms underlying DPT mode of action in oral squamous cell carcinoma (OSCC) remain unknown. This study aimed to investigate the anticancer effects of DPT on OSCC and the underlying mechanisms. Results of the MTT assay revealed that DPT significantly reduced the cell viability in a time- and dose-dependent manner. Flow cytometry analysis revealed that DPT induces apoptosis in OSCC cells in a dose-dependent manner. Moreover, DPT enhanced the production of mitochondrial reactive oxygen species (ROS) in OSCC cells. Mechanistically, DPT induced apoptosis in OSCC cells by suppressing the PI3K/AKT signaling pathway while activating the p38 MAPK signaling to regulate the expression of apoptotic proteins. Treatment with SC79, an AKT activator, reversed the effects of DPT on AKT signaling in OSCC cells. Taken together, these results provide the basis for the use of DPT in combination with conventional chemotherapy for the treatment of oral cancer.

Natural flavonolignans as potential therapeutic agents against common diseases.

OBJECTIVES: Plant-derived flavonolignans had been demonstrated to have various biological functions. They are an important class of natural products combined by a flavonoid unit and a phenylpropanoid unit. KEY FINDINGS: From the literature survey, 88 constituents from natural resources were identified. Different derivatives of flavonolignans were listed, fused phenylpropanoid unit with dioxane ring, or cyclic ether, or simple ether side chain, or lactone, and so on. Besides, the pharmacological effects of flavonolignans were summarized as well. It has a wide range of anti-tumour, antioxidant, anti-microorganic and anti-inflammatory effects. SUMMARY: This review had provided a full-scale profile of flavonolignans on its plant sources, phytochemistry and pharmacology, and also proposed some issues and perspectives which may be of concern in the future. It was greatly anticipated that the commercialization of the flavonolignans would lead to uplift the financial abilities of communities attending the growing of the flavonolignans and the relevant and potential production becoming an international herbal and pharmaceutical commodity.

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.

Structural Modification of Sylibin to Derivatives of Sylibin/Hydnocarpin D/Silandrin, and Evaluation of their Cytotoxicity against Cancer Cells.

BACKGROUND: Flavonolignans like silybin, hydnocarpin, and siliandrin are a group of natural compounds combining the structural moieties of flavonoid and phenylpropanoid (lignan). Hydnocarpin and silandrin have been less explored because of their trace occurrence in nature. OBJECTIVE: The present study aimed at chemical conversion of silybin to hydnocarpin and siliandrin. Another objective was to synthesize a series of amide derivatives and biologically evaluate them with regard to their anti-cancer effects. METHODS: In order to selectively convert silybin to 23-iodo silybin, 23-iodo hydnocarpin D and 23- iodo isosilandrin, the ratio of Ph3P, imidazole and molecular iodine was meticulously adjusted. These three iodide compounds were converted into amide compounds by chemical transformation. MTT method was applied to evaluate their anti-cancer potency. The binding affinity to related proteins was calculated by molecular docking. RESULTS: A total of 45 new amido-derivatives were synthesized and structurally characterized by NMR and HRMS. Some of them showed moderate to good antiproliferative potency against cancer cells. The activity of compound 10j was further testified by colony formation assay and molecular docking. CONCLUSION: The synthesis of 23-iodo silybin, 23-iodo hydnocarpin D and 23-iodo isosilandrin from silybin was successfully accomplished by one simple iodination reaction. Some of the amide derivatives of sylibin/hydnocarpin D /silandrin exhibited a remarkable inhibitory effect of proliferation on cancer cells compared to silybin. These results would pave the way for further investigation on the derivatives of flavonolignans for the treatment of cancer.

Inhibitory effect of hydnocarpin D on T-cell acute lymphoblastic leukemia via induction of autophagy-dependent ferroptosis.

Hydnocarpin D (HD) is a bioactive flavonolignan compound that possesses promising anti-tumor activity, although the mechanism is not fully understood. Using T cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat and Molt-4 as model system, we found that HD suppressed T-ALL proliferation in vitro, via induction of cell cycle arrest and subsequent apoptosis. Furthermore, HD increased the LC3-II levels and the formation of autophagolysosome vacuoles, both of which are markers for autophagy. The inhibition of autophagy by either knockdown of ATG5/7 or pre-treatment of 3-MA partially rescued HD-induced apoptosis, thus suggesting that autophagy enhanced the efficacy of HD. Interestingly, this cytotoxic autophagy triggered ferroptosis, as evidenced by the accumulation of lipid ROS and decrease of GSH and GPX4, while inhibition of autophagy impeded ferroptotic cell death. Our study suggests that HD triggers multiple cell death processes and is an interesting compound that should be evaluated in future preclinical studies.

Structurally Diverse Flavonolignans with Immunosuppressive and Neuroprotective Activities from the Fruits of Hippophae rhamnoides L.

The fruit of Hippophae rhamnoides L. has been used for centuries in Europe and Asia as a food with high nutritional and medicinal values. In this study, a bioactivity-guided phytochemical investigation of H. rhamnoides L. has resulted in four new dimethylallylated flavonolignans (1-4), four new isopropylpentenone-flavonolignan heterodimers (5-8), two new geranylated flavonolignans (9 and 10), and 14 known flavonolignan derivatives (11-24); they were elucidated by their spectrometric and spectroscopic methods, including HR-ESI-MS, NMR, IR, and UV from the fruit of H. rhamnoides L. for the first time. Among them, compounds 2, 5, 6, 20, and 21 showed potent immunosuppressive activities with IC50 values from 19.42 ± 3.91 to 48.05 ± 12.56 µM. Meanwhile, compounds 1, 4, 11, 12, and 13 showed moderate neuroprotective activities, which increased the cell survival rate from 50.30 ± 4.24% for the model group to 71.63 ± 3.04%, 70.02 ± 4.13%, 61.53 ± 5.93%, 61.08 ± 3.58%, and 65.68 ± 4.88% at 10 µM, respectively. The hypothetical biogenetic pathway and preliminary structure-activity relationship were found and discussed scientifically.

Anti-Alzheimer's flavanolignans from Ceiba pentandra aerial parts.

Four flavanolignans, ceibapentains A (1) and B (2) and cinchonains Ia (3) and Ib (4), were isolated for the first time from an ethyl acetate extract of Ceiba pentandra (L) (Bombacaceae) aerial parts. The ceibapentains A (1) and B (2) are new compounds and their structures, including the absolute configurations, were determined by HRESIMS, 1D and 2D NMR, and electronic circular dichroism analyses, then compared with reported data. Compounds 1-4 were tested for their anti-Alzheimer's activity via an assessment of their inhibitory effect on amyloid ß42 aggregation using a thioflavin T assay. The results revealed that cinchonain Ia (3) showed a higher inhibitory effect (91%) than the standard curcumin (70%). Compounds 1, 2, and 4 exhibited moderate activity, with inhibition ratios of 43%, 47%, and 58%, respectively. A molecular docking study on the binding mode of 3 and curcumin with an amyloid ß1-40 peptide fibril structure indicated a high affinity of cinchonain 1a (3) towards amyloid ß1-40 peptide, in agreement with the experimental results.

Flavonolignans: One Step Further in the Broad-Spectrum Approach of Cancer.

BACKGROUND: The small chemical class of flavonolignans encompasses unique hybrid molecules with versatile biological activities. Their anticancer effects have received considerable attention, and a large body of supporting evidence has accumulated. Moreover, their ability to interact with proteins involved in drug resistance, and to enhance the effects of conventional chemotherapeutics in decreasing cell viability make them influential partners in addressing cancer. OBJECTIVE: The review provides an outline of the various ways in which flavonolignans advance the combat against cancer. While the main focus falls on flavonolignans from milk thistle, attention is drawn to the yet, underexplored potential of less known flavonolignan subgroups derived from isoflavonoids and aurones. METHODS: Proceeding from the presentation of natural flavonolignan subtypes and their occurrence, the present work reviews these compounds with regard to their molecular targets in cancer, anti-angiogenetic effects, synergistic efficacy in conjunction with anticancer agents, reversal of drug resistance, and importance in overcoming the side effects of anticancer therapy. Recent advances in the endeavor to improve flavonolignan bioavailability in cancer are also presented. CONCLUSIONS: Significant progress has been achieved in detailing the molecular mechanisms of silybin and its congeners in experimental models of cancer. The availability of novel formulations with improved bioavailability, and data from phase I clinical trials in cancer patients provide an encouraging basis for more extensive trials aimed at evaluating the benefits of Silybum flavonolignans in cancer management. On the other hand, further research on the antitumor efficacy of iso-flavonolignans and other subtypes of flavonolignans should be pursued.

Samin-derived flavonolignans, a new series of antidiabetic agents having dual inhibition against α-glucosidase and free radicals.

A series of novel flavonolignans were synthesized by the reaction between a lignan named samin (1) and a range of flavonoids. This simple and rapid approach allowed direct assembly of these two bulky motifs in good yields without the formation of byproducts. Upon evaluation of antidiabetic activity of the synthesized products, epicatechinosamin (ß-2g) was the most active α-glucosidase inhibitor toward maltase and sucrase. The kinetic study indicated that ß-2 g inhibited the enzymes in a mixed manner of competitive and noncompetitive inhibition.

The Effect of Silymarin Flavonolignans and Their Sulfated Conjugates on Platelet Aggregation and Blood Vessels Ex Vivo.

Silymarin is a traditional drug and food supplement employed for numerous liver disorders. The available studies indicate that its activities may be broader, in particular due to claimed benefits in some cardiovascular diseases, but the contributions of individual silymarin components are unclear. Therefore, we tested silymarin flavonolignans as pure diastereomers as well as their sulfated metabolites for potential vasorelaxant and antiplatelet effects in isolated rat aorta and in human blood, respectively. Eleven compounds from a panel of 17 tested exhibited a vasorelaxant effect, with half maximal effective concentrations (EC50) ranging from 20 to 100 µM, and some substances retained certain activity even in the range of hundreds of nM. Stereomers A were generally more potent as vasorelaxants than stereomers B. Interestingly, the most active compound was a metabolite-silychristin-19-O-sulfate. Although initial experiments showed that silybin, 2,3-dehydrosilybin, and 2,3-dehydrosilychristin were able to substantially block platelet aggregation, their effects were rapidly abolished with decreasing concentration, and were negligible at concentrations ≤100 µM. In conclusion, metabolites of silymarin flavonolignans seem to have biologically relevant vasodilatory properties, but the effect of silymarin components on platelets is low or negligible.

Exploring Mitochondria-Mediated Intrinsic Apoptosis by New Phytochemical Entities: An Explicit Observation of Cytochrome c Dynamics on Lung and Melanoma Cancer Cells.

Hydnocarpin (Hy) is a flavonoid isolated and purified from the seeds of Hydnocarpus wightiana Blume. Herein, we have developed a built-in semi-synthetic modification on Hy by one-pot multi-component reaction and a [3 + 2] cycloaddition strategy to append five membered isoxazole and isoxazolone as new phytochemical entities (NPCEs). Two selected NPCEs viz Hy-ISO-VIII and Hy-ISO-G from the library of 20 newly synthesized derivatives after in vitro screening unveiled promising cytotoxicity and induced caspase-mediated apoptosis against the human lung and melanoma cancer cells which were well supported by virtual screening based on ligand binding affinity and molecular dynamic simulations. As a new insight, we introduced surface-enhanced Raman spectroscopy to identify the chemo-marker molecular fingerprint to confirm the cellular uptake, cytochrome c release, and DNA fragmentation in a label-free manner. The present findings throw up a surfeit of seminal reasons behind the semi-synthetic modification of Hy, stepping forward to cancer chemotherapy.

Antiviral Activities of Silymarin and Derivatives.

Silymarin flavonolignans are well-known agents that typically possess antioxidative, anti-inflammatory, and hepatoprotective functions. Recent studies have also documented the antiviral activities of silymarin and its derivatives against several viruses, including the flaviviruses (hepatitis C virus and dengue virus), togaviruses (Chikungunya virus and Mayaro virus), influenza virus, human immunodeficiency virus, and hepatitis B virus. This review will describe some of the latest preclinical and clinical studies detailing the antiviral profiles of silymarin and its derivatives, and discuss their relevance for antiviral drug development.

The protective effects of vernicilignan A, a new flavonolignan isolated from Toxicodendron vernicifluum on SH-SY5Y cells against oxidative stress-induced injury.

In this study, a new flavonolignan vernicilignan A was isolated from Toxicodendron vernicifluum. The neuroprotective effects of this compound against H2O2 induced cell injury in SH-SY5Y cells were evaluated by MTT assay and LDH release assay. Vernicilignan A dose-dependently attenuated the cell injury and LDH release induced by H2O2 in SH-SY5Y cells. Further study indicated that vernicilignan A reduced cell apoptosis caused by H2O2 treatment via regulation of some apoptotic related proteins including Bax, Bcl-2, caspase 3 and caspase 9. Also, vernicilignan A increase the cell viability of H2O2 treated cells via the activation of Akt and GSK3ß. Base on the findings, vernicilignan A exhibited neuroprotective effects through the activation of PI3K/Akt signaling and inhibition of mitochondria apoptosis pathway. Vernicilignan A might be a promising therapeutic agent for oxidative stress induced neurodegenerative diseases.

1,4-Benzodioxane Lignans: An Efficient, Asymmetric Synthesis of Flavonolignans and Study of Neolignan Cytotoxicity and Antiviral Profiles.

1,4-Benzodioxane lignans are a class of bioactive compounds that have received much attention through the years. Herein research pertaining to both 1,4-benzodioxane flavonolignans and 1,4-benzodioxane neolignans is presented. A novel synthesis of both traditional 1,4-benzodioxane flavonolignans and 3-deoxyflavonolignans is described. The antiviral and cytotoxic activities of 1,4-benzodioxane neolignans were then investigated; eusiderins A, B, G, and M, deallyl eusiderin A, and nitidanin, which contain the 1,4-benzodioxane motif but lack the chromanone motif found in the known antiviral flavonolignans, were tested. Notably, it was found that all eusiderin 1,4-benzodioxane neolignans exhibited greater antiviral activity than the potent and well-known silybin flavonolignans. While most modifications of the C-1' side chain did not significantly alter the cytotoxicity or antiviral activity, eusiderin M and nitidanin, which contain an allylic alcohol side chain, had lower cytotoxicity. All the eusiderins had similar antiviral activities, with eusiderin B having the best selectivity index. These results show that the chromanone moiety of the flavonolignans is not essential for bioactivity.

Silibinin to improve cancer therapeutic, as an apoptotic inducer, autophagy modulator, cell cycle inhibitor, and microRNAs regulator.

Silibinin is a natural plant polyphenol with high antioxidant and anticancer properties, which causes broad-spectrum efficacy against cancer, including cell cycle arrest and apoptosis in most cancer cell types. Silibinin, by modulating the apoptosis, cell cycle progression and autophagic pathways in various cellular and molecular routs might be used to design more effective anticancer strategies. Silibinin also regulates aberrant miRNAs expression linked to many aspects of cell biology in cancer. Maybe the most interesting aspect of silibinin is its ability to trigger multiple cellular signaling pathways to induce a particular biologic effect in various cell types. This review discusses investigations supporting the ability of silibinin to be as a natural modulator of involved cellular biological events in cancer progression. In this review, we introduce the salient features of silibinin therapy to optimize clinical outcomes for oncology patients. The goal of the treatments is to make it possible to eliminate the tumor with the minimum side effects and cure the patient in the early stage cancer. Therefore, plant extracts such as silibinin can be included in the treatments.

Identification of flavonolignans from Silybum marianum seeds as allosteric protein tyrosine phosphatase 1B inhibitors.

Protein tyrosine phosphatase 1B (PTP1B) is an attractive molecular target for anti-diabetes, anti-obesity, and anti-cancer drug development. From the seeds of Silybum marianum, nine flavonolignans, namely, silybins A, B (1, 2), isosilybins A, B (3, 4), silychristins A, B (5, 6), isosilychristin A (7), dehydrosilychristin A (8), and silydianin (11) were identified as a novel class of natural PTP1B inhibitors (IC50 1.3 7-23.87 µM). Analysis of structure-activity relationship suggested that the absolute configurations at C-7" and C-8" greatly affected the PTP1B inhibitory activity. Compounds 1-5 were demonstrated to be non-competitive inhibitors of PTP1B based on kinetic analyses. Molecular docking simulations resulted that 1-5 docked into the allosteric site, including α3, α6, and α7 helix of PTP1B. At a concentration inhibiting PTP1B completely, compounds 1-5 moderately inhibited VHR and SHP-2, and weakly inhibited TCPTP and SHP-1. These results suggested the potentiality of these PTP1B inhibitors as lead compounds for further drug developments.

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

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.

Regioselective synthesis of 7-O-esters of the flavonolignan silibinin and SARs lead to compounds with overadditive neuroprotective effects.

A series of neuroprotective hybrid compounds was synthesized by conjugation of the flavonolignan silibinin with natural phenolic acids, such as ferulic, cinnamic and syringic acid. Selective 7-O-esterfication without protection groups was achieved by applying the respective acyl chlorides. Sixteen compounds were obtained and SARs were established by evaluating antioxidative properties in the physicochemical FRAP assay, as well as in a cell-based neuroprotection assay using murine hippocampal HT-22 cells. Despite weak activities in the FRAP assay, esters of the α,ß-unsaturated acids showed pronounced overadditive effects at low concentrations greatly exceeding the effects of equimolar mixtures of silibinin and the respective acids in the neuroprotection assay. Cinnamic and ferulic acid esters (5a and 6a) also showed overadditive effects regarding inhibition of microglial activation, PC12 cell differentiation, in vitro ischemia as well as anti-aggregating abilities against Aß42 peptide and τ protein. Remarkably, the esters of ferulic acid with silybin A and silybin B (11a and 11b) showed a moderate but significant difference in both neuroprotection and in their anti-aggregating capacities. The results demonstrate that non-toxic natural antioxidants can be regioselectively connected as esters with medium-term stability exhibiting very pronounced overadditive effects in a portfolio of biological assays.

Flavonolignans reduce the response of blood platelet to collagen.

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 αIIbß3), aggregation, adhesion and secretion of PF-4. These effects highlight the potential of silybin and silychristin as supplementation to prevent primary and secondary thrombotic events wherein excessive blood platelet response to a physiological agonist is observed.