Identification of UDP-glucuronosyltransferases involved in the metabolism of silymarin flavonolignans.

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.

Tyrosinase inhibitory study of flavonolignans from the seeds of Silybum marianum (Milk thistle).

Anti-melanogenesis effects of silymarin from milk thistle have been reported recently, but detailed tyrosinase inhibition properties of individual components have not been investigated. This study purported to substantiate tyrosinase inhibition and its mechanism based on a single metabolite. The responsible components for tyrosinase inhibition of target source were found out as flavonolignans which consist of isosilybin A (1), isosilybin B (2), silydianin (3), 2,3-dihydrosilychristin (4), silychristin A (5), silychristin B (6) and silybin (7), respectively. The isolated flavonolignans (1-7) inhibited both monophenolase (IC50 = 1.7-7.6 µM) and diphenolase (IC50 = 12.1-44.9 µM) of tyrosinase significantly. Their inhibitions were 10-fold effective in comparison with their mother skeletons (8-10). Inhibitory functions were also proved by HPLC analysis using N-acetyl-l-tyrosine as substrate. The predominant formation of Emet·I was confirmed from a long prolongation of lag time and a decrease of the static state activity of the enzyme. All tested compounds had a significant binding affinity to tyrosinase with KSV values of 0.06-0.27 × 104 L·mol-1, which are well correlated with IC50s. In kinetic study, all flavonolignan (1-7) were mixed type I (KI < KIS) inhibitors, whereas their mother skeletons (8-10) were competitive ones. The UPLC-ESI-TOF/MS analysis showed that the isolated inhibitors are the most abundant metabolites in the target plant.

Chemotaxonomic and biosynthetic relationships between flavonolignans produced by Silybum marianum populations.

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 1H nuclear magnetic resonance spectroscopy (qHNMR). These correlations can be interpreted as evidence for the involvement of a flavonoid radical in the biosynthesis of the flavonolignans in S. marianum. The predominance of silybins A & B over isosilybin A & B in the silybin-rich samples is discussed in light of the relative stabilities of their respective radical flavonoid biosynthetic intermediates.

A new approach to the rapid separation of isomeric compounds in a Silybum marianum extract using UHPLC core-shell column with F5 stationary phase.

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-1 flow rate and 50°C temperature was used for the separation. The detection wavelength was set at 288nm. Under optimal chromatographic conditions, good linearity with a correlation coefficient of R2 >0.999 for all compounds was achieved. The available commercial samples of herbal teas and food supplements were extracted with methanol using an ultrasonic bath. After dilution with water and centrifugation, a 2µL sample of the filtered supernatant was directly injected into the UHPLC system. The use of a pentafluorophenyl stationary phase with methanol as the organic component of the mobile phase showed new ways to effectively separate isomeric compounds in herbal extracts, which could not be done with the conventional C18 stationary phase.

Curcumin, Silybin Phytosome(®) and α-R-Lipoic Acid Mitigate Chronic Hepatitis in Rat by Inhibiting Oxidative Stress and Inflammatory Cytokines Production.

Chronic hepatitis is recognized as a worldwide health problem that gradually progresses towards cirrhosis and hepatocellular carcinoma. Despite the large number of experiments using animal models for allergic hepatitis, it is still difficult to produce a picture of chronic hepatitis. Therefore, this study was conducted to introduce an animal model approximating to the mechanism of chronicity in human hepatitis. The study also aimed to examine the hepatoprotective effects of curcumin, silybin phytosome(®) and α-R-lipoic acid against thioacetamide (TAA)-induced chronic hepatitis in rat model. TAA was administered intraperitoneally at a dose of 200 mg/kg three times weekly for 4 weeks. At the end of this period, a group of rats was killed to assess the development of chronic hepatitis in comparison with their respective control group. TAA administration was then discontinued, and the remaining animals were subsequently allocated into four groups. Group 1 was left untreated, whereas groups 2-4 were allowed to receive daily oral doses of curcumin, silybin phytosome(®) or α-R-lipoic acid, respectively, for 7 weeks. Increases in hepatic levels of malondialdehyde associated with TAA administration were inhibited in groups receiving supplements. Furthermore, glutathione depletion, collagen deposition, macrophage activation and nuclear factor κappa-B expression as well as tumour necrosis factor-α and interleukin-6 levels were significantly decreased in response to supplements administration. Serological analysis of liver function and liver histopathological examination reinforced the results. The above evidence collectively indicates that the antioxidant and anti-inflammatory activities of curcumin, silybin phytosome(®) and α-R-lipoic acid may confer therapeutic efficacy against chronic hepatitis.

The effects of milk thistle (Silybum marianum) on human cytochrome P450 activity.

Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities.

Modulatory effects of curcumin, silybin-phytosome and alpha-R-lipoic acid against thioacetamide-induced liver cirrhosis in rats.

Liver cirrhosis is the final consequence of a progressive fibrotic process characterized by excessive collagen deposition and destruction of the normal liver architecture. This study aimed to investigate the protective effects of curcumin, silybin-phytosome and alpha-R-lipoic acid against thioacetamide-induced cirrhosis. Male rats were allocated into five groups of which one group received saline and served as normal control. Animals from groups 2-5 were treated with thioacetamide administered intraperitoneally at a dose of 200 mg/kg 3 times per week for 7 weeks. Group 2 was left untreated while groups from 3 to 5 were given a daily oral dose of curcumin, silybin-phytosome or alpha-R-lipoic acid simultaneously with thioacetamide. Increases in hepatic levels of malondialdehyde (MDA) and protein carbonyls (Pr Co) associated with thioacetamide administration were partially blocked in those groups receiving supplements. Glutathione (GSH) depletion, collagen deposition, matrix metalloproteinase-2 (MMP-2) activity, transforming growth factor-ß1 (TGF-ß1) level as well as α-smooth muscle actin (α-SMA) and heat shock protein-47 (HSP-47) gene expressions were also decreased in response to supplements administration. Serological analysis of liver function and histopathological examination reinforced the results. In conclusion, the present study highlights the antioxidant and the antifibrotic potentials of these supplements against chronic liver diseases caused by ongoing hepatic damage.

An assessment of pharmacokinetics and antioxidant activity of free silymarin flavonolignans in healthy volunteers: a dose escalation study.

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).

Identification of 23-(s)-2-amino-3-phenylpropanoyl-silybin as an antiviral agent for influenza A virus infection in vitro and in vivo.

It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection.

Milk thistle's active components silybin and isosilybin: novel inhibitors of PXR-mediated CYP3A4 induction.

Because cancer is often treated with combination therapy, unexpected pharmacological effects can occur because of drug-drug interactions. Several drugs are able to cause upregulation or downregulation of drug transporters or cytochrome P450 enzymes, particularly CYP3A4. Induction of CYP3A4 may result in decreased plasma levels and therapeutic efficacy of anticancer drugs. Since the pregnane X receptor (PXR) is one of the major transcriptional regulators of CYP3A4, PXR antagonists can possibly prevent CYP3A4 induction. Currently, a limited number of PXR antagonists are available. Some of these antagonists, such as sulphoraphane and coumestrol, belong to the so-called complementary and alternative medicines (CAM). Therefore, the aim was to determine the potential of selected CAM (ß-carotene, Echinacea purpurea, garlic, Ginkgo biloba, ginseng, grape seed, green tea, milk thistle, saw palmetto, valerian, St. John's Wort, and vitamins B6, B12, and C) to inhibit PXR-mediated CYP3A4 induction at the transcriptional level, using a reporter gene assay and a real-time polymerase chain reaction assay in LS180 colon adenocarcinoma cells. Furthermore, computational molecular docking and a LanthaScreen time-resolved fluorescence resonance energy transfer (TR-FRET) PXR competitive binding assay were performed to explore whether the inhibiting CAM components interact with PXR. The results demonstrated that milk thistle is a strong inhibitor of PXR-mediated CYP3A4 induction. The components of milk thistle responsible for this effect were identified as silybin and isosilybin. Furthermore, computational molecular docking revealed a strong interaction between both silybin and isosilybin and PXR, which was confirmed in the TR-FRET PXR assay. In conclusion, silybin and isosilybin might be suitable candidates to design potent PXR antagonists to prevent drug-drug interactions via CYP3A4 in cancer patients.

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.

Absorption and metabolism of milk thistle flavanolignans in humans.

This study evaluated the absorption and metabolism of milk thistle flavonolignans silychristin, silydianin, silybin and isosilybin isomers (all together known as silymarin) in humans. Fourteen volunteers consumed an extract of milk thistle and urine was collected up to 48 h after consumption. Thirty-one metabolites were identified in urine by means of HPLC-MS/MS, monoglucuronides being the most common excreted form, followed by sulphate-glucuronides and diglucuronides, respectively. The excretion of monoglucuronides peaked 2 h after consumption, whereas sulphate-glucuronide and diglucuronide excretion peaked at 8 h. The bioavailability of milk thistle flavanolignans was 0.45±0.28% (mean±SD). In conclusion, milk thistle flavonolignans are extensively modified after ingestion and recovered in urine as sulpho- and glucuronyl-conjugates, indicating a strong affinity for hepatic phase II enzymes. All future studies (in vitro and in vivo) dealing with the effects of milk thistle should start by considering the modification of its flavonolignans after ingestion by humans.

Angiopreventive efficacy of pure flavonolignans from milk thistle extract against prostate cancer: targeting VEGF-VEGFR signaling.

The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention.

Two flavonolignans from milk thistle (Silybum marianum) inhibit CYP2C9-mediated warfarin metabolism at clinically achievable concentrations.

Milk thistle (Silybum marianum) is a popular herbal product used for hepatoprotection and chemoprevention. Two commercially available formulations are the crude extract, silymarin, and the semipurified product, silibinin. Silymarin consists of at least seven flavonolignans, of which the most prevalent are the diastereoisomers silybin A and silybin B; silibinin consists only of silybin A and silybin B. Based on a recent clinical study showing an interaction between a silymarin product and the CYP2C9 substrate losartan, the CYP2C9 inhibition properties of silybin A and silybin B and corresponding regioisomers, isosilybin A and isosilybin B, were evaluated using human liver microsomes (HLMs), recombinant CYP2C9 (rCYP2C9) enzymes, and the clinically relevant probe, (S)-warfarin. Silybin B was the most potent inhibitor in HLMs, followed by silybin A, isosilybin B, and isosilybin A (IC(50) of 8.2, 18, 74, and >100 microM, respectively). Next, silybin A and silybin B were selected for further characterization. As with HLMs, silybin B was more potent than silybin A toward rCYP2C9 1 (6.7 versus 12 microM), rCYP2C9 2 (9.3 versus 19 microM), and rCYP2C9 3 (2.4 versus 9.3 microM). Using a matrix of five substrate (1-15 microM) and six inhibitor (1-80 microM) concentrations and HLMs, both diastereoisomers inhibited (S)-warfarin 7-hydroxylation in a manner described best by a mixed-type inhibition model (K(i) values of 4.8 and 10 microM for silybin B and silybin A, respectively). These observations, combined with the high systemic silibinin concentrations (>5-75 microM) achieved in a phase I study involving prostate cancer patients, prompt clinical evaluation of a potential warfarin-milk thistle interaction.

Large-scale isolation of flavonolignans from Silybum marianum extract affords new minor constituents and preliminary structure-activity relationships.

The gram-scale isolation of the major flavonolignan diastereoisomers from milk thistle ( Silybum marianum) extract provided an entree into the isolation of two related analogues that are present in extremely minute quantities. The isolation and structure elucidation of these two new compounds, which we have termed isosilybin C and isosilybin D due to their structural similarities to isosilybin A and isosilybin B, respectively, afforded a preliminary analysis of structure-activity relationships toward prostate cancer growth, survival, and apoptotic endpoints.

[Silymarin in the treatment of chronic liver diseases: past and future]. / Silymarin az idült májbetegségek kezelésében: múlt és jövo.

In the treatment of chronic liver diseases adequate therapy can be chosen only in the knowledge of pathogenetic processes. In the liver diseases caused by oxidative stress (alcoholic and non-alcoholic fatty liver and steatohepatitis, drug and compound induced liver toxicity) the antioxidant drugs, like silymarin, in chronic hepatitis caused by hepatitis B and hepatitis C virus, combined peginterferon and nucleosid treatments are the primary therapy modalities to be selected. The main effects of silymarin are the membrane stabilising and antioxidant effects, it is able to help the liver cell regeneration, it can decrease the inflammatory reaction and inhibit the fibrogenesis in the liver. These results have been established by experimental and clinical trials. According to open studies, the long administration of silymarin significantly increased the survival time of patients with alcohol-induced liver cirrhosis. Recently it was demonstrated that high-dosage silibinin infusion treatment could significantly decrease the number of hepatitis C viruses after four-week application. On the basis of the results with the methods of molecular biology, silymarin is able to decrease significantly tumor cell proliferation, angiogenesis as well as insulin resistance. These results support the administration of silymarin preparations in the therapy of chronic liver diseases, especially in alcoholic and non-alcoholic steatohepatitis in current clinical practice, and as it can be awaited, also in the future. In some neoplastic diseases they could also be administered as adjuvant therapy.

Synchronized and sustained release of multiple components in silymarin from erodible glyceryl monostearate matrix system.

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.

A phase I and pharmacokinetic study of silybin-phytosome in prostate cancer patients.

Silibinin is a polyphenolic flavonoid isolated from milk thistle with anti-neoplastic activity in several in vitro and in vivo models of cancer, including prostate cancer. Silybin-phytosome is a commercially available formulation containing silibinin. This trial was designed to assess the toxicity of high-dose silybin-phytosome and recommend a phase II dose. Silybin-phytosome was administered orally to prostate cancer patients, giving 2.5-20 g daily, in three divided doses. Each course was 4 weeks in duration. Thirteen patients received a total of 91 courses of silybin-phytosome. Baseline patient characteristics included: median age of 70 years, median baseline prostate specific antigen (PSA) of 4.3 ng/ml, and a median ECOG performance status of 0. The most prominent adverse event was hyperbilirubinemia, with grade 1-2 bilirubin elevations in 9 of the 13 patients. The only grade 3 toxicity observed was elevation of alanine aminotransferase (ALT) in one patient; no grade 4 toxicity was noted. No objective PSA responses were observed. We conclude that 13 g of oral silybin-phytosome daily, in 3 divided doses, appears to be well tolerated in patients with advanced prostate cancer and is the recommended phase II dose. Asymptomatic liver toxicity is the most commonly seen adverse event.

Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography-electrospray ionization mass spectrometry.

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.

Separation and characterization of silybin, isosilybin, silydianin and silychristin in milk thistle extract by liquid chromatography-electrospray tandem mass spectrometry.

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.