In patients with well-compensated liver cirrhosis, silybin was administered as silybin phytosome at a dose of 120 mg × 3 /d, or as silymarin containing 84 mg of silybin 4 fold/d. The results showed that liver cirrhosis does not modify the kinetics of silybin and that in liver patients, the bioavailability of silybin is higher when it is administered as a phytosome adduct[44
]. Similar data were obtained in rats with experimentally induced cirrhosis[45
]. Table summarizes the data obtained in humans.
Table 3 Comparison between silybin phytosome and silymarin pharmacokinetics in patients with liver cirrhosis
More recently, the pharmacokinetics of silybin has been evaluated in patients with chronic hepatitis [hepatitis C virus (HCV) or non-alcoholic fatty liver disease (NAFLD)] and compensated liver cirrhosis[46,47
]. A single, 600 mg p.o. dose of milk thistle extract was administered to healthy volunteers and to three patient cohorts, and blood samples were obtained over 24 h. Silybin A and B accounted for 43% of the exposure to the sum of total silymarin flavonolignans in healthy volunteers and only 31% to 38% in liver disease cohorts as a result of accumulation of silychristin (20%-36%). Area under the curve (AUC, 0-24 h) for the sum of total silymarin flavonolignans was 2.4, 3.3, and 4.7-fold higher for the HCV or NAFLD (P
≤ 0.03) and HCV cirrhosis cohorts (P
≤ 0.03), respectively, compared with healthy volunteers. Silymarin kinetics was correlated with plasma levels of caspases as an index of liver inflammation; caspase 3/7 activity correlated with the AUC (0-24 h) for the sum of all silymarin conjugates among all participants (R2
= 0.52) and was 5-fold higher in the HCV cirrhosis cohort (P
≤ 0.005 versus healthy participants). These findings suggest that the presence of liver damage, particularly as chronic inflammation, may affect the bioavailability of the different components of silymarin, possibly explaining the low beneficial effects of flavonoids in patients with liver damage.
Interactions and toxicity
There are substantial differences between silymarin and silybin in their interactions with metabolizing enzymes, and the reasons for these differences remain unknown. Silybin alone or as silybin β-galactoside, β-glucoside, β-lactoside, or β-maltoside at a final concentration of silybin 100 μmol/L has been tested in vitro
. Under these conditions, silybin showed slow inhibitory effects (IC50
> 200 μmol/L) on marker substrates of CYP2E1, CYP2D6, CYP2C19, and CYP2A6. Silybin and silybin β-glycosides did not induce expression of CYP1A2 and CYP3A4 and did not affect the inducible expression of either of these enzymes[48-53
In in vivo
work, Sridar et al[54
] investigated metabolic interactions involving silybin at doses ranging from 25 to 250 μmol/L and substrates metabolized by CYP3A4 or CYP2C9, showing that silybin may be a modulator/inactivator of P450s 3A4 and 2C9. What remains to be elucidated is whether this effect is brought about by competition at the site or inhibition for substrate binding and for metabolism at a particular binding pocket in the active site; another possibility is that it results from a conformational change in the active site. Despite these results, silybin has shown no effect on the metabolism of indinavir[55,56
], which is mediated by CYP3A4, and others[57-60
] have documented that silybin, at a concentration of 100 μmol/L, has no effect on either basal or inducible expression of CYP3A4 mRNA.
In any case, the silybin-drug interaction of these enzyme substrates is not clinically relevant, and the inhibitory effects of silybin occur only at concentrations that massively exceed the physiologically used doses[60-62
]. In vitro
, silybin inhibits the UGT glucuronyl transferases UGT1A6 and UGT1A9, but it is 14- to 20-fold more selective for UGT1A1[48,49,61-64
] (see Table as summary). The clinical relevance of this phenomenon is presently unknown because there are no published reports indicating the existence of a clinical interaction with bilirubin. UGT1A1 is the only enzyme responsible for bilirubin glucuronidation, and it contributes to the glucuronidation of several drugs. In animals, silybin affects the hepatobiliary elimination of various drugs[52,53,57,58,64
]; oral feeding of pure silybin at doses of 100 and 200 mg/kg/bw/d showed a moderate to highly significant increase in both glutathione-S-transferase and quinine reductase activities in the liver, lung, stomach, skin, and small bowel in a dose- and time-dependent manner[58
]. Recently, Flaig et al[27
] provided the best evidence that silybin can be administered to humans at doses producing anticancer-relevant concentrations, with minimal or no side effects. That study employed the largest doses ever used, ranging from 2.5 to 20 g of silybin-phosphatidylcholine (Indena’s Siliphos®
“silybin phytosome”), given daily in three divided doses for 4 wk to 13 men with a history of prostate carcinoma. At a dose escalation from 15 to 20 g/d, silybin was discontinued because of asymptomatic hyperbilirubinemia, most likely because of inhibition of the glucuronyl transferase UGT1A1. However, in all patients, this mild hyperbilirubinemia improved with treatment cessation.
The limitations of available clinical trials with regard to establishing safety are the same as those for establishing efficacy. Clinical trials testing safety are poor predictors of the fate of extracts in real-world settings, where patients ingest multiple drugs and herbs, take different formulations of the same product, and add alcohol and other compounds, often for extremely long periods. In the randomized trials that reported adverse effects, their frequency was approximately equal in the silybin and control groups. The majority of the observed adverse events were unrelated to the product or difficult to separate from the concomitant disease, and in available reports, causality is rarely addressed. There are no safety data in children or older adults, as there are no reported studies in children and very few studies that included patients older than 65 years. Adverse effects associated with oral ingestion of silybin include mainly gastrointestinal problems, but these are rare. Headache/dizziness and pruritus were reported in one clinical trial. Asymptomatic liver toxicity has been observed in recent clinical trials performed in cancer patients, in whom hyperbilirubinemia and increases in alanine aminotransferase (ALT) levels were observed; however, these effects were present only when very high dosages of silybin phytosome (between 10 and 20 g/d) were used. At high doses, a laxative effect of silybin phytosome is possible because of increased bile secretion and bile flow. Mild allergic reactions have also been noted, but they were not serious. Therefore, the available data indicate that milk thistle has few side effects when doses lower than 5 g/d are used, with possible adverse effects at doses greater than 10 g/d[6,7,62
The documented effects of silybin in basic research
In liver cells, as well as in other types of cells, the common effects of silybin may be summarized as follows: (1) Antioxidant; (2) Direct and/or indirect (through the antioxidant capability) modulator of inflammation and fibrogenesis; and (3) indirect and/or direct modulator of some intrahepatic metabolic pathways.
The antioxidant effects of silybin have been demonstrated in all cells studied. Silybin acts as an antioxidant because it inhibits radical formation, binds some radical species (scavenger), interferes with lipid peroxidation of membranes (and therefore modulates membrane permeability), and increases the intracellular content of scavengers[65
]. In fact, in the presence of oxidative and nitrosative stress, silybin inhibits the formation of superoxide anion radicals and of nitric oxide (NO), increases ATP content through the phosphorylation of ADP, decreases the content of malondialdeyde (MDA) and totally abolishes the decrease of glutathione, of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase[66-73
]. These results, which are dose-dependent, have been documented in isolated rat Kupffer cells, hepatocytes, HEPG2 cells, isolated mitochondria from rat hepatocytes, and in models of ischemia-reperfusion of rat liver. In experimentally induced liver damage, one hour after intragastric administration to rats of conjugated silybin (0.6 g/kg bw), the content of silybin in the microsomes measured using a specific HPLC assay was approximately 2.5 μg/mg of protein, corresponding to a final concentration of 10 μmol/L of silybin. Under these conditions, lipid peroxidation, mitochondrial permeability and respiration, and membrane potential as well as cell death were reversed by silybin, particularly if it was conjugated as silybin + phosphatidylcholine. This last compound, at a dose of 20 μmol/L, directly scavenges hydroxyl, hydroxyethyl, lipodienyl, methyl, and trichloromethyl radicals[74,75
Finally, silybin acts as an antioxidant because it also serves as an iron chelator[76-78
]. More recently, it has been suggested that dehydrosilybin (DHS), an oxidized form of silybin, has greater antioxidant activity than silybin (about three times better), probably because of the presence of unsaturated bonds that contribute to hydrogen-donating capacity. The better scavenger activity of DHS may also be the result of its greater ability to react with cell membranes because it has higher lipophilicity than silybin[32,65
Table summarizes the main antioxidant effects of silybin.
In general terms, silymarin and silybin interfere with the NF-κB-controlled transduction cascade. NF-κB is an inducible and ubiquitously expressed DNA-binding protein, acting as a transcription factor for genes involved in inflammation, cell survival, differentiation, and growth[79,80
]. In unstimulated cells, NF-κB is sequestered in the cytoplasm by interaction with inhibitory protein 1 κ B α (IκBα). Upon activation from oxidative stress, NF-κB dissociates from IκBα, and IκBα is degraded. NF-κB translocates to the nucleus and, through kinase phosphorylation, drives the activation of genes supporting inflammation. Consistent with its antioxidant activity, silybin has been demonstrated to inhibit NF-κB activation and translocation through suppression of IκBα phosphorylation and degradation[81-83
]. In an acute model of liver damage in which mice were treated with concanavalin A, silybin reduced plasma levels of transaminases and the plasma and liver content of pro-inflammatory cytokines, inhibited hepatic NF-κB activation, and increased plasma and tissue levels of IL-10[84
]. In rats with dimethyl-nitrosamine-induced chronic liver damage, silybin conjugated with phosphatidylcholine and vitamin E (Realsil®
, Ibi-Lorenzini, Italy) administered by gastric gavage could prevent loss of body and liver weight, as well as reducing the degree of liver injury, as determined by ALT values and necroinflammatory scores. This outcome was associated with reduced hepatic stellate cell activation and proliferation both after 1 and 5 wk of treatment[85
]. The anti-inflammatory action of silybin is also related to its interference with multiple cytokine-induced signaling pathways to down regulate inducible nitric-oxide synthase (iNOS) expression[86-88
]and to the inhibition of cyclooxygenase (COX)-2 expression and activity and leukotriene formation in human platelets, white blood cells, and endothelial cells. Finally, silybin inhibits activation of the protein kinases and of a c-jun N-terminal kinase[89-92
In addition to its antioxidant and anti-inflammatory actions, silybin also shows an antiviral effect. In fact, at a concentration of 20 μmol/L, it inhibits the protein expression and the replication of HCV virus in infected polymorphonucleated cells derived from patients with chronic HCV infection[93-95
In an in vitro
model of human hepatic fibrogenesis, silybin demonstrated both direct and indirect antifibrotic properties. In fact, in stellate cells from human liver, silybin reduced platelet-derived growth factor (PDGF)-induced DNA synthesis and cell proliferation at a dose of 25 μmol/L. Silybin also reduced PDGF-induced cell migration in a dose-dependent fashion. Finally, pre-treatment with 25-50 μmol/L of silybin significantly reduced the TGF-β-induced de novo
synthesis of procollagen type I in cell supernatants[96
To investigate the role of silybin in modulating the pro-inflammatory properties of hematopoietic stem cells, cells were stimulated with IL-1β (20 ng/ml), a potent pro-inflammatory cytokine; silybin inhibited, in a dose-dependent manner, IL-1-induced synthesis of human MCP-1 (monocyte chemoattractant protein 1) and human IL-8 as detected in cell supernatants. This effect was related to the effect of silybin on the inhibition of IκBα phosphorylation and to its capability to inhibit ERK, MEK, and Raf phosphorylation at any concentration used[96
]. Antifibrotic effects were also documented in experimental animals and in humans[85,97-99
Table summarizes the main anti-inflammatory and antifibrotic effects of silybin.
Table 6 Anti-inflammatory/antifibrotic effects of silybin[79-99]
Silybin interferes with some mechanisms of action of insulin. In fact, it modulates the uptake of glucose in adipocytes by blocking the insulin-dependent glucose transporter 4. In rat hepatocytes, silybin, in concentrations ranging from 25 to 100 μmol/L, lowers glucose formation from different gluconeogenic substrates through an inhibitory effect on pyruvate kinase activity[100
]. As previously reported in cultured hepatocytes, low doses of silybin reduce reactive oxygen species formation from mitochondria; this reduction leads to a decrease in oxidation of carbons arising from glycolysis[101
]. Moreover, silybin inhibits, in a dose-dependent manner, gluconeogenesis and glycogenolysis, both in basal conditions and after a glucagon-dependent stimulation, by blocking glucose-6-phosphate hydrolysis. This effect was demonstrated using different substrates, such as dihydroxyacetone, lactate/pyruvate, glycerol, and fructose[102
] (Table ).
Table 7 Effects of silybin on hepatic metabolism of glucose at doses of 25-100 μmol/L in hepatocytes[100-102]
In an animal model of type 1 diabetes mellitus[103
], in a 6-mo, double-blind, randomized trial in patients with poorly controlled non-insulin-dependent diabetes mellitus and alcoholic liver disease[104
], and in a randomized, double-blind, placebo-controlled trial in patients with type II diabetes[105
], silybin significantly affected plasma levels of glucose and triglycerides, with a trend toward lower hemoglobin A1c levels.
Other effects on cellular signaling
In cancer cells, silybin alters cell cycle regulators and induces apoptosis, both through antioxidant and anti-inflammatory properties and through the inhibition of growth factor receptor-mediated mitogenic and cell survival signaling, particularly related to the activation of tyrosine kinases[106-111
]. PDGF receptor (PDGFR), epidermal growth factor receptor (EGFR), Bcr-Abl, and KIT are examples of tyrosine kinases overexpressed in most human cancers. Silymarin or silybin are particularly effective in inhibiting EGFR signaling with suppression of cyclin-dependent kinase expression (i.e. CDK4) and up regulation of CDK inhibitors (CDKIs). These effects lead to G1 and G2-M arrest in cancer cells[112-119
], as recently confirmed in a model of human colon cancer[120,121
]. In fact, at a dose ranging from 40-75 μmol/L, silybin significantly inhibited cell proliferation through cell cycle arrest via
inhibition of cyclin promoter activity.
Because it inhibits constitutive NF-κB activation, silybin can induce apoptosis, consistent with a significant decrease in its nuclear level of p65 subunit. In addition, it activates caspase 3 and caspase 9 and decreases survivin levels[122
Angiogenesis refers to the growth of capillary vessels from existing blood vessels and is considered obligatory for the growth and progression of solid tumors. Angiogenesis critically depends on several conditions; in fact, endothelial cells must: (1) proliferate to provide the necessary number of cells for the growing vessels; (2) secrete matrix metalloproteinases (MMPs), which are required to break down surrounding tissue matrix; and (3) be capable of movement and migration. In addition, the angiogenic stimuli like hypoxia and the production of angiogenic cytokines, such as vascular endothelial growth factor (VEGF), must be sustained. Silybin treatment decreases secreted VEGF levels and shows a strong, concentration-dependent inhibition of capillary tube formation on matrigel, retraction, and disintegration of preformed capillary network, inhibition of matrigel invasion and migration, and a decrease in MMP-2 secretion[92,106-122
Table summarizes the main cellular signaling affected by silybin.
The use of silybin in the clinical setting: efficacy and suggestions
The use of silymarin in the clinical setting has a long history; in its native Mediterranean region, it has been employed for liver damage since the Greco-Roman era. As mentioned in the introduction, “herbal therapy” use is consistently increasing worldwide, and some of the most common herbal supplement preparations are derived from the milk thistle plant (Silybum marianum
). Despite a long history of its use and the large number of people who consume this substance, no conclusive data on its clinical efficacy can be identified. In fact, only a few well designed clinical trials have been performed. Most studies have been conducted using silymarin and with inclusion of patients with alcoholic or viral cirrhosis. Stickel et al[8
] identified the 10 main controlled trials conducted in liver patients, with numbers of patients ranging from 20 to 200 and doses of silymarin ranging from 210 to 450 mg/d for 7 to 730 d. These clinical studies generally have suffered from the same shortcomings found in many other trials on herbal medicines, such as a small sample size, lack of appropriate randomization and of allocation concealment or blinding, very different periods of treatment, lack of information about type and dose of extract used as well as product characterization, ill-defined patient population, and a lack of etiology, severity of disease, and discussion of potential confounders.
We could argue that the history of studies on silybin reflects, in part, the epidemiological history of liver diseases. When liver cirrhosis was the only clearly identified manifestation of liver damage and alcohol was the only known pathogenetic factor, silymarin and other plant extracts represented the most frequently used drugs. Knowledge about the pathogenetic relationships between hepatitis viruses and liver damage induced researchers and industry to focus attention on antiviral drugs. In response to the discovery of metabolic liver diseases, as well as increased knowledge about the cellular and subcellular mechanisms of both induction and progression of liver damage, researchers in academia and industry have begun to reappraise natural products and to evaluate their therapeutic efficacy using appropriate methods.
The clinical experience with silybin is mainly related to its properties as a detoxifying agent and as a hepatoprotective compound in different acute and chronic liver diseases.
Silybin as a detoxifying and hepatoprotective substance
Several chemotherapeutic agents are metabolized by the liver and can exert hepatotoxicity, with the net result of drug reductions or withdrawal. Chemotherapeutic agents that are likely to produce hepatotoxicity include dactinomycin, daunorubicin, docetaxel, gemcitabine, imatinib, 6-mercaptopurine, methotrexate, and oxaliplatin. Cancer patients taking these therapies often self-medicate with milk thistle because of its reputation as a liver protectant. Clinicians also prescribe it to cancer patients for the same purpose. The rationale of milk thistle use is to provide support to the liver while it performs multiple functions, including responding to the increased metabolic demands caused by tumor growth, assisting in metabolizing products generated when a tumor is killed or reduced by chemotherapy and radiation, and aiding in the processing of drugs prescribed to cancer patients.
Silybin is also considered a potent inhibitor of human intestinal β-glucuronidase, blocking the release and reabsorption of free xenobiotics and their metabolites from their glucuronide conjugates. Because the liver is the primary organ that cleanses and detoxifies the blood, many detoxification programs include a component of liver support or what is often called liver cleansing. For these reasons, silybin is commonly included in detoxification regimens[6,7,62,108-111
]. However, only one randomized, double-blind study has reported the effects of milk thistle in patients receiving cancer therapy; 50 children with acute lymphoblastic leukemia and grade 2 or higher hepatic toxicity were randomized to receive a milk thistle supplement (Siliphos, Thorne Research, Dover, Idaho) (5.1 mg/kg/day) or placebo for 28 days. The authors reported significant reductions in AST levels (P
< 0.05) and a trend toward a significant reduction in ALT levels (P
< 0.07). A significantly larger number of children in the milk thistle group developed a > 50% reduction in total bilirubin at day 28 compared to placebo (P
Silybin in acute liver damage
The administration of silybin within approximately 48 h after poisoning produced by the mushroom Amanita phalloides
(death cap) seems to be an effective measure to prevent severe liver damage[124
]. A retrospective analysis of 205 cases of clinical poisoning from 1971 to 1980 documented the efficacy of milk thistle silybin extract in increasing survival rates in adults and children exposed to this potentially lethal mushroom[125
]. In January 2007, six family members in California suffering from aflatoxin poisoning caused by A. phalloides
mushrooms were treated with intravenous milk thistle, provided by Madaus Pharma (Brussels, Belgium; division of Madaus AG, Cologne, Germany). The US Food and Drug Administration granted permission for the use of milk thistle after considering that all patients were at high risk of dying of liver failure. Ultimately, one of the six patients died, while the others had a full recovery after treatment. A case report mentions that silybin may offer protection from liver toxicity caused by the pharmaceutical drug phenytoin[126
Silybin in chronic liver disease
The various flavonoid compounds that are included in the general term “milk thistle” have been recognized as a “safe and well-tolerated herb” with a limited adverse event profile[1-10
]. As previously reported, the majority of studies have been conducted using silymarin and including patients with alcoholic or viral cirrhosis. The Cochrane Collaboration group[4,127
] evaluated the studies related to the use of silymarin/silybin in patients with different types of acute and chronic liver diseases. Various items were analyzed to define the quality of trials, such as the number of participants, the criteria of randomization, the blinding, the modalities of follow-up, and the statistical methods. From 1,831 references, 67 publications addressed patients with alcoholic and/or hepatitis B or C liver diseases treated with milk thistle; of these, only 26 were selected according to the agreed criteria, and these were associated with 13 trials. The active treatment consisted of silymarin per os in 10 randomized clinical trials, silybin phytosome per os in two randomized clinical trials, and silybin + phosphatidylcholine in one randomized clinical trial. The Cochrane Collaboration studies concluded that milk thistle does not seem to significantly influence the course of the disease in treated patients. However, all causes of mortality were reduced by 50% in patients with alcoholic liver disease without HCV antibodies who took milk thistle extracts compared to placebo (P
< 0.05); in trials that studied liver-related mortality, there was a significant effect of silybin (relative risk, 0.50; 95% CI 0.29 to 0.88; P
Seeff et al[5,129
] examined the spontaneous use of herbal products in the US in patients affected by HCV chronic infection, including non-responders to a previous treatment with interferon and ribavirin. In 1145 patients, about 50% used herbal products; of these, silybin was used by 70%. Even if no changes in liver tests and/or in HCV-RNA serum levels were observed, the univariate analysis documented a lower incidence of symptoms and a better quality of life in patients who consumed silybin relative to those who did not. On multivariate analysis, adjusted for age, gender, educational status, alcohol use (n. drinks/d), physical activity, body mass index, and smoking, silybin positively affected more than one aspect of quality of life. An examination of the total number of patients treated with silymarin/silybin in both well-conducted trials and in pilot studies with a high quality identified about 2000 patients with liver cirrhosis or with chronic hepatitis of different etiology, with a mean duration of treatment of six months and a dose of silybin ranging from 160 to 360 mg/d (except in two cases, see below).
The main results regarding the efficacy of silybin in liver disease patients are summarized as follows.
In the past, most studies were focused on liver cirrhosis, particularly alcoholic cirrhosis, and the efficacy of silybin was evaluated in terms of improvement in liver test abnormalities and/or in mortality rate (see above for results)[126-128
]. Silybin β-cyclodextrin has been studied as an antidiabetic drug in patients with alcoholic liver disease and concomitant non-insulin-dependent diabetes mellitus; in these patients, the drug (at a dose of 135 mg/d) did not influence liver function test results or insulin secretion but significantly reduced fasting glucose (P
< 0.03) and serum triglyceride levels (P
< 0.01) compared to placebo. The effects seem to be related to a reduction in insulin resistance[104
More recently, studies have focused on chronic hepatitis, particularly that induced by HCV. Silybin phytosome, at a dose ranging from 240 mg/d to 942 mg t.i.d. (the highest dose used in patients with liver damage) was well tolerated without adverse effects and significantly improved liver damage as expressed by transaminase levels, oxidative stress (serum malondialdehyde levels) and both ferritin serum levels and iron global body storage[130-138
]. Today, the attention of researchers is focused on a possible antiviral effect of silybin against HCV infection.
Polyak et al[95
] tested the anti-inflammatory and antiviral action of different extracts from silymarin on polymorphonuclear cells from patients with chronic HCV infection, documenting an anti-inflammatory and antiviral effect of milk thistle extract, mainly for silybin, which presented the strongest anti NF-κB and anti-HCV replication effect. Ferenci et al[139
] and Biermet et al[140
] have demonstrated that silybin is a potent antiviral agent in patients with chronic hepatitis C not responding to pegylated interferon/ribavirin therapy. These studies also showed that very high doses of silybin i.v. (from 5 to 20 mg/kg/d for 14 d) were free of toxic effects.
Finally, non-alcoholic fatty liver disease (NAFLD) may occur as an expression of a metabolic syndrome or in association with HCV chronic infection. The simultaneous presence of NAFLD in this latter group of patients may negatively affect the progression of fibrosis and the response to antiviral treatment. Patients affected by primitive NAFLD and/or chronic HCV-related hepatitis in combination with NAFLD, all non-responders to previous antiviral treatment, were treated with four pieces/d of the complex silybin-vitamin E-phospholipids corresponding to about 200 mg of pure silybin/d for three or six months. The treatment induced a significant reduction of plasma markers of chronic inflammation (C-reactive protein and cytokines), metabolic parameters (triglycerides, cholesterol, insulin resistance), liver tests (transaminases and gamma-glutamyl transpeptidase), degree of ultrasonographic liver steatosis and, finally, of main indices of liver fibrosis TGF-β, hyaluronic acid and metalloproteinase 2)[99,141,142
In a rodent model of non-alcoholic steatohepatitis, the same silybin phospholipid complex prevented mitochondrial dysfunction[143
], and preliminary results in a large, multicenter Italian trial vs. placebo indicate that complex silybin-vitamin E-phospholipids significantly improved liver damage in patients with NAFLD and markers of liver fibrosis in patients with HCV chronic hepatitis[144
In Table the main studies performed with silybin are reported and discussed.
Main studies performed by using purified silybin as drug