Anti-Cancer Efficacy of Silybin Derivatives - A Structure-Activity Relationship

Silybin or silibinin, a flavonolignan isolated from Milk thistle seeds, is one of the popular dietary supplements and has been extensively studied for its antioxidant, hepatoprotective and anti-cancer properties. We have envisioned that potency of silybin could be further enhanced through suitable modification/s in its chemical structure. Accordingly, here, we synthesized and characterized a series of silybin derivatives namely 2,3-dehydrosilybin (DHS), 7-O-methylsilybin (7OM), 7-O-galloylsilybin (7OG), 7,23-disulphatesilybin (DSS), 7-O-palmitoylsilybin (7OP), and 23-O-palmitoylsilybin (23OP); and compared their anti-cancer efficacy using human bladder cancer HTB9, colon cancer HCT116 and prostate carcinoma PC3 cells. In all the 3 cell lines, DHS, 7OM and 7OG demonstrated better growth inhibitory effects and compared to silybin, while other silybin derivatives showed lesser or no efficacy. Next, we prepared the optical isomers (A and B) of silybin, DHS, 7OM and 7OG, and compared their anti-cancer efficacy. Isomers of these three silybin derivatives also showed better efficacy compared with respective silybin isomers, but in each, there was no clear cut silybin A versus B isomer activity preference. Further studies in HTB cells found that DHS, 7OM and 7OG exert better apoptotic activity than silibinin. Clonogenic assays in HTB9 cells further confirmed that both the racemic mixtures as well as pure optical isomers of DHS, 7OM and 7OG were more effective than silybin. Overall, these results clearly suggest that the anti-cancer efficacy of silybin could be significantly enhanced through structural modifications, and identify strong anti-cancer efficacy of silybin derivatives, namely DHS, 7OM, and 7OG, signifying that their efficacy and toxicity should be evaluated in relevant pre-clinical cancer models in rodents.     

Alkaloids from Portulaca oleracea L

Five alkaloids (oleraceins A, B, C, D and E) were isolated from Portulaca oleracea L., and their structures determined by spectroscopic methods as 5-hydroxy-1-p-coumaric acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside, 5-hydroxy-1-ferulic acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside, 5-hydroxy-1-(p-coumaric acyl-7'-O-beta-D-glucopyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside, 5-hydroxy-1-(ferulic acyl-7'-O-beta-D-glucopyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-beta-D-glucopyranoside and 8,9-dihydroxy-1,5,6,10b-tetrahydro-2H-pyrrolo[2,1-a]isoquinolin-3-one, respectively.     

Preparative chromatographic purification of diastereomers of silybin and their quantification in human plasma by liquid chromatography-tandem mass spectrometry

A novel preparative HPLC method separating silybin has been developed to meet the need for both silybin A and silybin B standard. After the preparation of silybin A and silybin B standard, a simple, sensitive, selective and reproducible liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with negative electrospray ionization (ESI) was developed for the quantification of silybin A and silybin B in human plasma. Following rapid sample preparation, silybin A, silybin B and naringin (internal standard, ISTD) were separated on a Zorbax Eclipse XDB-C18 column, using methanol-water containing 0.1% formic acid (48:52, v/v) as the mobile phase. The mass spectrometer was operated in selected reaction monitoring (SRM) mode using the transition m/z 481.1-->300.9 for both silybin A and silybin B and m/z 579.2-->271.1 for naringin, respectively. Linear calibration curves were obtained in the concentration range of 2-5000ng/ml with a lower limit of quantitation (LLOQ) of 2ng/ml for both silybin A and silybin B, respectively. The intra- and inter-day precision values were below 7.5% and accuracy was within +/-4.9% at all three quality control (QC) levels, for both silybin A and silybin B, respectively. This method was successfully applied to the stereospecific analysis of silybin in plasma samples from a pharmacokinetic study of silybin A and silybin B in 22 healthy male Chinese volunteers after a single oral dose of silybin-phosphatidylcholine complex (equivalent to 280mg silybin, including 133mg silybin A and 147mg silybin B).     

Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation—the Phase II conjugation reaction—of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3′-phosphoadenosine-5′-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.     

Antimicrobial constituents of Scrophularia deserti

A study of the chemistry and antibacterial activity of Scrophularia deserti led to the isolation of eight compounds, including the metabolite 3(zeta)-hydroxy-octadeca-4(E),6(Z)-dienoic acid (1). The known compounds ajugoside (2), scropolioside B (3), 6-O-alpha-L-rhamnopyranosylcatalpol (4), buddlejoside A(8) (5), scrospioside A (6), laterioside (7) and 3R-1-octan-3-yl-3-O-beta-D-glucopyranoside (8) were also isolated. Compounds 1-3 exhibited moderate antibacterial activity against strains of multidrug and methicillin-resistant Staphylococcus aureus (MRSA) and a panel of rapidly growing mycobacteria with minimum inhibitory concentration (MIC) values ranging from 32 to 128 microg/ml.     

Iridoids from Globularia dumulosa

Two new iridoids, 10-O-benzoylglobularigenin (1) and dumuloside (2) were isolated from the aerial parts of Globularia dumulosa together with seven known iridoid glucosides, davisioside (3), aucubin (4), melampyroside (5), catalpol (6), 10-O-benzoylcatalpol (7), alpinoside (8) and deacetylalpinoside (9). Three phenylethanoid glycosides, verbascoside, decaffeoyl-verbascoside, leucosceptoside A and three flavone glucosides, pectolinarigenin 7-O-beta-D-glucopyranoside, nepetin 7-O-B-D-glucopyranoside, demethoxycentaureidin 7-O-beta-D-glucopyranoside were also isolated and characterized. The structure elucidation of the isolated compounds was performed by spectroscopic (UV, IR, HR-MALDIMS, 1D- and 2D NMR) methods.     

Phenylethanoid glycosides from Phlomis integrifolia Hub.-Mor

Two new phenylethanoid glycosides integrifoliosides A (2) and B (3), along with a known phenylethanoid glycoside alyssonoside (1) and a flavone glucoside chrysoeriol-7-O-beta-D-glucopyranoside (4) were isolated from the aerial parts of Phlomis integrifolia. The structures of the new compounds were identified as 3,4-dihydroxy-beta-phenylethoxy-O-beta-D-apiofuranosyl-(1 --> 4)-alpha-L-rhamnopyranosyl-(1 --> 3)-4-O-feruloyl-beta-D-glucopyranoside (2) and 3-hydroxy-4-methoxy-beta-phenylethoxy-O-beta-D-apiofuranosyl-(1 --> 4)-alpha-L-rhamnopyranosyl-(1 --> 3)-4-O-feruloyl-beta-D-glucopyranoside (3), on the basis of spectroscopic (UV, IR, 1D- and 2D-NMR, and HR-FABMS) methods.     

Two flavonol glycosides, hexandrasides C and D, from the underground parts of Vancouveria hexandra.

In addition to four known glycosides, icariin, epimedin B, epimedosides A and E, two new glycosides of a flavonol with a gamma,gamma-dimethylallyl group were isolated from the underground parts of Vancouveria hexandra. The structures were determined to be des-O-methyl-anhydroicaritin 3-O-beta-D-xylopyranosyl(1----2)-alpha-L-rhamnopyranoside 7-O-beta-D-glucopyranosyl(1----2)-beta-D-glucopyranoside and anhydroicaritin 3-O-alpha-L-rhamnopyranosyl(1----3)-alpha-L-rhamnopyranoside 7-O-beta-D-glucopyranoside by means of spectral analysis.     

6-hydroxyflavonoids as alpha-glucosidase inhibitors from marjoram (Origanum majorana) leaves

A methanol extract of marjoram leaves strongly inhibited rat intestinal alpha-glucosidase. Five 6-hydroxyflavonoids, 6-hydroxyapigenin (scutellarein; IC50 for sucrose hydrolysis by rat intestinal alpha-glucosidase, 12 microM), 6-hydroxyapigenin-7-O-beta-D-glucopyranoside (> 500 microM), 6-hydroxyluteolin-7-O-beta-D-glucopyranoside (300 microM), 6-hydroxyapigenin-7-O-(6-O-feruloyl)-beta-D-glucopyranoside (>500 microM), and 6-hydroxyluteolin-7-O-(6-O-feruloyl)-beta-D-glucopyranoside (> 500 microM), were isolated as active principles and related compounds. The two feruloylglucosides are novel compounds.     

Ecdysteroid glycosides from Sida rhombifolia L

Seven ecdysteroids, including the three new compounds 1-3, were isolated from Sida rhombifolia L. Their structures and configurations were determined by extensive spectroscopic techniques in combination with chemical derivatization. The four known compounds--ecdysone (4), 20-hydroxyecdysone (5), 2-deoxy-20-hydroxyecdysone-3-O-beta-D-glucopyranoside (6), and 20-hydroxyecdysone-3-O-beta-D-glucopyranoside (7)--are reported for the first time from this plant.     

Isoflavonoid glycosides and rotenoids from Pongamia pinnata leaves

Chromatographic separation of a 70% aqueous methanol extract (AME) of Pongamia pinnata (Linn.) Pierre (Leguminosae) leaves has led to the isolation of two new isoflavonoid diglycosides, 4'-O-methyl-genistein 7-O-beta-D-rutinoside (2) and 2',5'-dimethoxy-genistein 7-O-beta-D-apiofuranosyl-(1"'-->6")-O-beta-D-glucopyranoside (6), and a new rotenoid, 12a-hydroxy-alpha-toxicarol (5), together with nine known metabolites, vecinin-2 (1), kaempferol 3-O-beta-D-rutinoside (3), rutin (4), vitexin (7), isoquercitrin (8), kaempferol 3-O-beta-D-glucopyranoside (9), 11,12a-dihydroxy-munduserone (10), kaempferol (11), and quercetin (12). Their structures were elucidated on the basis of chemical and spectroscopic analyses.     

Effect of silybin on phorbol myristate actetate-induced protein kinase C translocation, NADPH oxidase activity and apoptosis in human neutrophils.

Mechanism of the action of silybin (1) and its derivatives (2-4), possessing different lipid solubility in PMA-stimulated neutrophils was evaluated. Silybin (1) inhibited the calcium, phosphatidylserine- and diacylglycerol-dependent protein kinase C translocation and the NADPH oxidase activity in PMA-stimulated neutrophils and resulted in decreased apoptosis. Furthermore, silybin (1) inhibited xanthine oxidase activity and hem-mediated oxidative degradation of low-density lipoprotein, as well. Its derivatives (2-4), possessing different lipid-solubility, affected all the studied parameters. The lipid solubility of silybin (1) was enhanced by methylation (5'7'4'trimethylsilybin: 2), whereas a decrease in lipid-solubility by acetylation of compound 2 (5',7,'4"-trimethylsilybin-acetate: 3) or all the hydroxyl groups of silybin (peracetyl-silybin: 4) attenuated the antioxidant capacity by decreasing the inhibition in PKC translocation and NADPH oxidase activation. All the derivatives of silybin (2-4) showed no inhibition in cell free systems; e.g. did not alter the xanthine oxidase activity and the hem-mediated oxidative degradation of LDL. In conclusion, the antioxidant activity of (1) might be due to its ability to inhibit PKC translocation and NADPH oxidase activation in PMA-stimulated neutrophils. The increase of lipid solubility of silybin (1) supports its penetration through cell membrane and enhances its inhibitory effects. This structural modification of (1) might have pharmacological consequences.     

Anthraquinones from the stem bark of Stereospermum zenkeri with antimicrobial activity

Two anthraquinones, zenkequinones A and B were isolated from the stem bark of Stereospermum zenkeri together with known sterequinone-F, p-coumaric acid, sitosterol-3-O-beta-D-glucopyranoside and 3beta-hydroxyolean-12-en-28-O-beta-D-glucopyranoside. Their structures were established by spectroscopic methods. The antimicrobial activity of the isolated compounds was evaluated against six multiresistant strains of pathogens. Zenkequinone B showed the best antibacterial activity (MIC 9.50 microg/ml) against gram-negative Pseudomonas aeruginosa.     

Suppression of up-regulated LXRα by silybin ameliorates experimental rheumatoid arthritis and abnormal lipid metabolism

BackgroundAs dysregulation of immunometabolism plays a key role in the immunological diseases, dyslipidemia frequently observed in rheumatoid arthritis (RA) patients (60%) is associated with the disease activity and has been considered as the potential target of anti-inflammatory strategy. However, targeting of metabolic events to develop novel anti-inflammatory therapeutics are far from clear as well as the mechanism of dyslipidemia in RA.PurposeTo explore the therapeutic potential and mechanisms of silybin again RA through the regulation of lipid metabolism.MethodsAdjuvant-induced arthritis (AIA) rat model was used to examine the effects of silybin on modulating dysregulated lipid metabolism and arthritis. Metabolomics, docking technology, and biochemical methods such as western blots, qRT-PCR, immunofluorescence staining were performed to understanding the underlying mechanisms. Moreover, knock-down of LXRα and LXRα agonist were used on LO2 cell lines to understand the action of silybin.ResultsWe are the first to demonstrate that silybin can ameliorate dyslipidemia and arthritis in AIA rats. Overexpression of LXRα and several key lipogenic enzymes regulated by LXRα, including lipoprotein lipase (LPL), cholesterol 7α and 27α hydroxylase (CYP7A, CYP27A), adipocyte fatty acid-binding protein (aP2/FABP4) and fatty acid translocase (CD36/FAT), were observed in AIA rats, which mostly accounted for dyslipidemia during arthritis development. Metabolomics, docking technology, and biochemical results indicated that anti-arthritis effects of silybin related to suppressing the up-regulated LXRα and abnormal lipid metabolism. Notably, activation of LXRα could potentiate cell inflammatory process induced by LPS through the regulation of NF-κB pathway, however, suppression of LXRα agonism by siRNA or silybin reduced the nuclear translocation of NF-κB as well as the induction of downstream cytokines, indicating LXRα agonism is the important factor for the arthritis development and could be a potential target.ConclusionThe up-regulation of LXRα can activate lipogenesis enzymes to worsen the inflammatory process in AIA rats as well as the development of dyslipidemia, therefore, rectifying lipid disorder via suppression of LXRα agonism pertains the capacity of drug target, which enables to discover and develop new drugs to treat rheumatoid arthritis with dyslipidaemia.     

Isolation and characterization of new limonoid glycosides from Citrus unshiu peels.

Three limonoid glycosides were isolated from Citrus unshiu peels, and their structures were determined based on MS and NMR spectroscopic data as nomilinic acid 17-O-beta-D-glucopyranoside (1), methyl nomilinate 17-O-beta-D-glucopyranoside (2), and obacunone 17-O-beta-D-glucopyranoside (3). In particular, the location of the sugar moiety was clearly determined by the B/E constant linked scan FABMS method. No limonoid glycosides obtained here were found to have antitumor activity in NCI-H292 and EL-4 cell lines.     

Enhanced bioactivity of silybin B methylation products

Flavonolignans from milk thistle (Silybum marianum) have been investigated for their cellular modulatory properties, including cancer chemoprevention and hepatoprotection, as an extract (silymarin), as partially purified mixtures (silibinin and isosilibinin), and as pure compounds (a series of seven isomers). One challenge with the use of these compounds in vivo is their relatively short half-life due to conjugation, particularly glucuronidation. In an attempt to generate analogues with improved in vivo properties, particularly reduced metabolic liability, a semi-synthetic series was prepared in which the hydroxy groups of silybin B were alkylated. A total of five methylated analogues of silybin B were synthesized using standard alkylation conditions (dimethyl sulfate and potassium carbonate in acetone), purified using preparative HPLC, and elucidated via spectroscopy and spectrometry. Of the five, one was monomethylated (3), one was dimethylated (4), two were trimethylated (2 and 6), and one was tetramethylated (5). The relative potency of all compounds was determined in a 72 h growth-inhibition assay against a panel of three prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a human hepatoma cell line (Huh7.5.1) and compared to natural silybin B. Compounds also were evaluated for inhibition of both cytochrome P450 2C9 (CYP2C9) activity in human liver microsomes and hepatitis C virus infection in Huh7.5.1 cells. The monomethyl and dimethyl analogues were shown to have enhanced activity in terms of cytotoxicity, CYP2C9 inhibitory potency, and antiviral activity (up to 6-fold increased potency) compared to the parent compound, silybin B. In total, these data suggested that methylation of flavonolignans can increase bioactivity.     

Large-scale separation of silybin diastereoisomers using lipases

The flavonolignan silybin (1), isolated from the seeds of milk thistle (Silybum marianum), occurs in nature as an equimolar mixture of two diastereoisomers, silybin A and silybin B, that exhibit different biological activities. The preparative production of optically pure silybin A and B in a diastereoisomeric purity greater than 95% was accomplished using immobilized Candida antarctica lipase B (Novozym 435) in a combination of two reactions: regioselective acetylation of a natural silybin mixture (1) and subsequent stereoselective alcoholysis of the resulting 23-O-acetylsilybin (2). Several grams of the optically pure substances can be produced within one week using this new, robust and scalable process, which is selective, mild and high-yielding.     

Two chromone-secoiridoid glycosides and three indole alkaloid glycosides from Neonauclea sessilifolia

From the dried roots of Neonauclea sessilifolia, two new chromone-secoiridoid glycosides, sessilifoside and 7"-O-beta-D-glucopyranosylsessilifoside, and three novel indole alkaloid glycosides, neonaucleosides A, B, and C, were isolated along with the main known glycosides, 5-hydroxy-2-methylchromone-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, sweroside, loganin, grandifloroside, and quinovic acid 3 beta-O-beta-D-quinovopyranoside-28-O-beta-D-glucopyranoside. The structures of these new glycosides were determined by spectroscopic and chemical means. Neonaucleoside A and its C-3 epimer were prepared from secologanin and tryptamine.     

Glycosylation of hesperetin by plant cell cultures

The biotransformation of hesperetin by cultured cells of Ipomoea batatas and Eucalyptus perriniana was investigated. Three glycosides, hesperetin 3'-O-beta-D-glucopyranoside (33 microg/g fr. wt of cells), hesperetin 3',7-O-beta-D-diglucopyranoside (217 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(beta-D-glucopyranosyl)]-beta-d-glucopyranoside (beta-gentiobioside, 22 microg/g fr. wt of cells), together with three hitherto known glycosides, hesperetin 5-O-beta-d-glucopyranoside (23 microg/g fr. wt of cells), hesperetin 7-O-beta-D-glucopyranoside (57 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(alpha-L-rhamnopyranosyl)]-beta-D-glucopyranoside (beta-rutinoside, hesperidin, 13 microg/g fr. wt of cells), were isolated from cultured suspension cells of E. perriniana that had been treated with hesperetin. Oligosaccharide chains were regioselectively formed at the C-7 position of hesperetin to afford beta-gentiobioside and beta-rutinoside. On the other hand, cultured I. batatas cells converted hesperetin into hesperetin 3'-O-beta-D-glucopyranoside (60 microg/g fr. wt of cells), hesperetin 5-O-beta-D-glucopyranoside (23 microg/g fr. wt of cells), and hesperetin 7-O-beta-D-glucopyranoside (110 microg/g fr. wt of cells).     

Assessment of estrogenic activity of flavonoids from Mediterranean plants using an in vitro short-term test

Six isoflavones, daidzein (4',7,-dihydroxyisoflavone), genistein (4',5,7-trihydroxyisoflavone), genistin (genistein 7-O-beta-D-glucopyranoside), isoprunetin (4',7-dihydroxy, 5-metoxyisoflavone), isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside and four flavones, luteolin (3',4',5,7-tetrahydroxyflavone), luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C (4',5,7-trihydroxy,8-isoprenylflavone) were purified from Mediterranean plants (Genista morisii and Genista ephedroides) and their estrogenic activity was assessed by a yeast reporter gene assay (Saccharomyces cerevisiae RMY326 ER-ERE). Licoflavone C showed a powerful estrogenic activity at 10(-7) M (0.0338 microg/ml) and it was 47.45% than 10(-8) M 17beta-estradiol (0.00272 microg/ml). The estrogenicity of this flavone was found to be comparable to the activity showed by genistein at 10(-6) M (0.27 microg/ml). This study points out that a glucose substituent in flavones and isoflavones modulates the hormone-like activity in a different way. Isoflavone aglycones showed a more estrogenic activity than the corresponding glucosides. Conversely, the glucosidation made estrogenic the flavone luteolin and the position of substitution differently influenced the estrogenic activity of compounds.