25 The water-soluble versions of silybin A and silybin B found in Legalon-SIL contain two succinate moieties that increase the molecular weight of the compound by over 244 atomic mass units, from 482 to 726. Thus, the water-soluble molecules are quite different chemically from the natural compounds, which are insoluble in water, and as a result the metabolism and biological effects of the compounds may differ. However,

in our study, silymarin did not inhibit HCV RNA and protein expression in multiple independent replicon cell lines that did not produce infectious progeny viruses, in agreement with a recent study showing that silymarin did not inhibit HCV AZD8055 NS5A protein or RNA expression in a subgenomic replicon cell line.36 The data suggest that blockade of polymerase activity is not a major antiviral mechanism, at least in the HCVcc system. Instead, we provided evidence to suggest that inhibition of virus entry and virus transmission contribute to the antiviral effects of silymarin. Indeed, silymarin blocked the entry of three different enveloped pseudoviruses and also potently inhibited the fusion of liposome membranes. Silymarin flavonolignans belong to the family of phytoestrogens and are composed of a phenylbenzopyrone structure.4 The structures of these molecules are relatively hydrophobic, so it is possible that silymarin may act by incorporating into lipid membranes of both viruses and target cells, or at least

may display partition into lipid bilayers, similar to other plant flavonoids.37 This would signaling pathway lead to the stabilization of membranes by silymarin, which would in turn become less prone to fusion.

This behavior is reminiscent of arbidol, a broad-spectrum antiviral inhibiting HCV entry, membrane fusion, and replication.24 This hypothesis is further corroborated MCE by our observations that silymarin blocks cell entry of pseudotyped particles of other enveloped viruses such as VSVpp and MLVpp. Future studies will focus on further dissecting these mechanisms. We also showed that silymarin inhibits MTP activity, apoB secretion, and production of infectious virus particles. In support of this argument and in agreement with the results obtained in the current report, the flavonoid taxifolin, which is present in silymarin, has been shown to block MTP activity and apoB secretion.38 Silymarin has also been shown to alter lipid profiles,39 so it is possible that the botanical may block virus transmission by targeting multiple components of lipid metabolism. Silymarin does many things to cells, including modulation of signal transduction,40 the redox state,41 T-cell function,6, 31 and nuclear factor kappa B.42 These studies suggest that direct effects of silymarin on cell functions are responsible for the prevention of liver disease in many animal models.33-35 We therefore hypothesize that silymarin’s blockade of virus entry and transmission occurs by targeting the host cell.