In the current study, we characterized the biological activities of oral (natural, ie silibinin) and intravenous (semi-synthetic, ie SIL) preparations from milk thistle on the HCV life cycle and inflammatory cellular functions that collectively contribute to development and progression of liver disease in hepatitis C. We demonstrate for the first time that SIL and silibinin elicit very different antiviral and anti-inflammatory actions in human liver and T cell cultures. Both mixtures inhibited virus fusion potently and NS5B RdRp activity weakly. SIL inhibited NS5B binding to RNA, whereas silibinin did not. Silibinin did not inhibit HCV RNA and protein expression from subgenomic genotype 1b or 2a replicons, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection at much higher doses than required for RdRp inhibition. Both silibinin and SIL inhibited progeny virus production from JFH-1 infected cells. Silibinin but not SIL inhibited innate inflammatory and antiviral signaling from NF-κB and IFN-B promoters. Both SIL and silibinin suppressed T cell proliferation to similar degrees. Overall, silibinin activity was similar to silymarin in all assays tested 
, while SIL had a unique profile, requiring higher concentrations for antiviral activity in the JFH-1 system and was devoid of anti-inflammatory activity in hepatocyte cultures.
The effects of SIL and silibinin were tested against a widely used HCV genotype 1b isolate, BK, plus four patient-derived isolates with widely varying specific activities for RNA synthesis. Overall, both SIL and silibinin could inhibit the RdRps, with SIL being somewhat better than silibinin. However, inhibition plateaued at moderate drug concentrations, leading to maximal inhibition levels in most cases of only about 2–3 fold. The reasons for this unusual inhibition pattern are unknown. What is clear, however, is that SIL and silibinin do not shut down in vitro polymerase activity like nucleoside analog drugs do. Even at high concentrations of SIL or silibinin, the net RdRp activity is only marginally suppressed. We therefore posit that inhibition of NS5B polymerase elongation provides only a minor contribution to the log-fold suppression of viremia seen in patients receiving intravenous SIL. This conclusion is supported by the observation that in vitro HCV RdRp activity does not appear to be limiting for viral production in vivo, because no correlation was found between in vitro RNA polymerase activity and serum HCV titer in the patients from which the polymerases were derived 
Silibinin as well as Legalon-SIL are mixtures of two diastereoisomers, silybins A and B. These molecules are arranged around a flavonoid skeleton, and their overall structure is relatively hydrophobic. It is therefore possible that these molecules may act, at least in part, by incorporating or partitioning in the hydrophobic core of lipid membranes of both viruses and target (cell) membranes, as shown for similar compounds 
. This might lead to the stabilization of membranes by both molecules, which would in turn become less prone to fusion. This behavior would be reminiscent of that of arbidol, a broad-spectrum antiviral inhibiting HCV entry, membrane fusion and replication, as recently described by our group (
; Teissier & Pécheur, unpublished observations).
SIL was as efficient as silymarin at inhibiting HCV-mediated fusion, with IC50 of ca. 6 µM and 5 µM respectively (see also 
). Interestingly SIL displayed a much more potent inhibitory effect on HCV fusion than silibinin (IC50 6 µM vs ca. 25 µM, respectively). This indicates that silybins A and B by themselves are at least as efficient at blocking fusion as is silymarin, the mixture of multiple flavonolignans (including silibinin). However the chemical formulation of these molecules appears to alter pharmacological and antiviral activity, since SIL, the disuccinate form of silibinin, appears to be a more potent antiviral in the clinic than silibinin. Further studies are needed to determine if the chemical composition of SIL enhances its interaction with membranes.
HCV infection induces inflammation via hepatocellular sensing of virus by PRRs 
, induction of oxidative stress 
, and induction of inflammatory cytokines and chemokines 
. Unfortunately, this response, which is usually beneficial to the host, is deregulated in chronic hepatitis C because the virus is not cleared. Inflammatory events such as T cell infiltration of the liver, and release of inflammatory cytokines and chemokines damage the liver via further induction of oxidative stress. Perpetuation of this inflammatory cascade and immune cell mediated liver damage is thought to induce subsequent fibrosis. Thus, chronic hepatitis C may be thought of as a disease caused by an inflammatory response gone awry 
. In this harsh environment, hepatocytes die and regenerate more frequently. Since chronic inflammation is mechanistically involved in the establishment of cancer 
, and in particular hepatocellular carcinoma 
, the deregulated cellular responses are an integral part of the complex processes that lead to HCV-induced liver disease. We have shown that silymarin displays antioxidant, anti-inflammatory, antiviral, and immunomodulatory properties 
. Therefore, we propose that silymarin elicits hepatoprotection by multiple actions that collectively reduce inflammation by several mechanisms including inhibition of NF-κB signaling, T cell proliferation and inflammatory cytokine production, and virus infection. Regarding anti-HCV effects, although silymarin and purified flavonolignans can inhibit NS5B polymerase activity, we propose that blockade of viral targets is not the dominant antiviral mechanism. Instead, silymarin blockade of cellular targets may confer antiviral effects by blocking virus entry, HCV RNA and protein expression, and virus transmission 
In summary, our data clearly demonstrate that silibinin and SIL function in different ways to induce hepatoprotection. SIL has shown antiviral efficacy during liver transplantation for end-stage hepatitis C 
and for prior non-responders to pegylated IFN plus ribavirin therapy 
. However, silymarin, silibinin, and SIL have been shown to modulate the expression and activity of various drug-metabolizing enzymes 
. Given that much higher plasma and presumably liver levels of silymarin flavonolignans can now be achieved via oral 
and intravenous 
dosing, careful considerations should be given to the routes of administration, chemical composition, and possible interactions of pharmaceuticals with silymarin-derived flavonolignans 
. Our studies suggest that future clinical and basic research studies of specific silymarin components, including those that are chemically modified, will be the key to understanding their clinical effects and developing novel and effective natural product-derived medicines for liver disease.