Identification of hepatoprotective flavonolignans from silymarin

Abstract

Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-alpha-induced NF-kappaB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 muM, except for isosilybin B, which was toxic to cells above 10 muM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 muM. Silymarin suppressed TNF-alpha activation of NF-kappaB dependent transcription, which involved partial inhibition of IkappaB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.

Fig. 1.

Antiviral effects of silymarin-derived flavonolignans. (A) Effects on HCV protein expression. JFH-1 virus at a multiplicity of infection of 0.05 was adsorbed to Huh7.5.1 cells for 5 h. After the inoculum was removed, fresh medium was added containing DMSO or 20.7, 41.4, 82.8, or 124.2 μM of pure flavonolignan (mw 482), or 32.9, 65.8, 131.6, or 197.4 μM of taxifolin (mw 304). Protein lysates were harvested 72 h later and HCV protein expression detected by Western blot analysis. Actin served as a loading control. (B) Effects on HCV RNA. JFH-1 virus at a multiplicity of infection of 0.05 was adsorbed to Huh7.5.1 cells for 5 h. After the inoculum was removed, fresh medium was added containing DMSO or pure flavonolignan or taxifolin at the concentrations listed above. Total RNA was harvested 72 h later and HCV RNA expression quantitified by real-time RT-PCR. The y axis presents the number of copies of HCV RNA per 10 ng of total RNA. Silydianin (SD), silychristin (SC), isosilychristin (ISC), silybin A (SA), and silybin B (SB), taxifolin (TAX), isosilybin A (ISA), isosilybin B (ISB), silibinin (SbN), and silymarin (SM).

Fig. 2.

Silymarin and silymarin-derived flavonolignans block HCV induced oxidative stress. (A) Huh7.5.1 cells were infected or mock-infected with JFH-1 at a multiplicity of infection of 0.01 and 72 h later, cells were labeled with the dye H2DCFDA for 30 min using the Image-IT Live Green Reactive Oxygen Species kit (Molecular Probes/Invitrogen). On oxidation, H2DCFDA becomes highly fluorescent. Images were captured on a Nikon Microscope using MetaMorph software. (B) Huh7.5.1 cells were treated as described above except in addition to silymarin (SM), cells were also treated with 41.4 μM of each flavonolignan, 65.8 μM of taxifolin, or 100 U/mL of IFN-α. ROS were quantitated by pixel intensity. Abbreviations of compounds are listed in the legend to Fig. 1.

Fig. 3.

Silymarin flavonolignans inhibit NF-κB transcription. (A and B) Silybin A and silybin B inhibit NF-κB transcription. Huh7 cells were transfected with a luciferase reporter gene under control of the pRDII domain from the IFN-B promoter and 24 h later, cells were treated with the indicated micromolar doses of each flavonolignan or DMSO control for 30 min before addition of 10 ng/mL TNF-α. Luciferase activity was measured 3.5 h later. Error bars represent SDs. (C) Comparison of NF-κB inhibitory profiles of silybin A, silybin B, taxifolin, and silychristin.