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.
- SourceAvailable from: Ishtiaq Qadri[Show abstract] [Hide abstract]
ABSTRACT: Hepatitis viral infection is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Over one million people are estimated to be persistently infected with hepatitis C virus (HCV) worldwide. As capsid core protein is the key element in spreading HCV; hence, it is considered to be the superlative target of antiviral compounds. Novel drug inhibitors of HCV are in need to complement or replace the current treatments such as pegylated interferon's and ribavirin as they are partially booming and beset with various side effects. Our study was conducted to predict 3D structure of capsid core protein of HCV from northern part of India. Core, the capsid protein of HCV, handles the assembly and packaging of HCV RNA genome and is the least variable of all the ten HCV proteins among the six HCV genotypes. Therefore, we screened four phytochemicals inhibitors that are known to disrupt the interactions of core and other HCV proteins such as (a) epigallocatechin gallate (EGCG), (b) ladanein, (c) naringenin, and (d) silybin extracted from medicinal plants; targeted against active site of residues of HCV-genotype 3 (G3) (Q68867) and its subtypes 3b (Q68861) and 3g (Q68865) from north India. To study the inhibitory activity of the recruited flavonoids, we conducted a quantitative structure-activity relationship (QSAR). Furthermore, docking interaction suggests that EGCG showed a maximum number of hydrogen bond (H-bond) interactions with all the three modeled capsid proteins with high interaction energy followed by naringenin and silybin. Thus, our results strongly correlate the inhibitory activity of the selected bioflavonoid. Finally, the dynamic predicted capsid protein molecule of HCV virion provides a general avenue to target structure-based antiviral compounds that support the hypothesis that the screened inhibitors for viral capsid might constitute new class of potent agents but further confirmation is necessary using in vitro and in vivo studies.Bioinformatics and biology insights 01/2014; 8:159-68.
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ABSTRACT: Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NF¿B/COX-2 signal pathway. Additionally, the pre-S2 mutant protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.Journal of Biomedical Science 10/2014; 21(1):98. · 2.74 Impact Factor
- International Journal of Pharmacy and Pharmaceutical Sciences 01/2014; · 1.59 Impact Factor
Identification of hepatoprotective flavonolignans
Stephen J. Polyaka,b,c,1, Chihiro Morishimaa, Volker Lohmannd, Sampa Pala, David Y. W. Leee, Yanze Liue, Tyler N. Graff,
and Nicholas H. Oberliesf
Departments ofaLaboratory Medicine,bMicrobiology andcGlobal Health, University of Washington, Seattle, WA 98104;dDepartment of Molecular Virology,
University of Heidelberg, 69120 Heidelberg, Germany;eBio-Organic and Natural Products Laboratory, McLean Hospital, Belmont, MA 02478; andfDepartment
of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402
Edited* by Harvey J. Alter, The Warren G. Magnuson Clinical Center, Bethesda, MD, and approved February 22, 2010 (received for review December 11, 2009)
Silymarin,alsoknownasmilk thistle extract,inhibitshepatitisCvirus
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-α-induced NF-κB
transcription, virus-induced oxidative stress, and T-cell proliferation.
All compounds were well tolerated by Huh7 human hepatoma cells
silymarin in all assays tested except in T cell proliferation. Pure com-
pounds inhibited JFH-1 NS5B polymerase but only at concentrations
above 300 μM. Silymarin suppressed TNF-α activation of NF-κB
dependent transcription, which involved partial inhibition of IκB
location, without affecting binding of p50 and p65 to DNA. All com-
pounds blocked JFH-1 virus-induced oxidative stress, including
across multiple assays were taxifolin, isosilybin A, silybin A, silybin B,
silymarin- and silymarin-derived compounds may influence HCV dis-
dosed to identify specific clinical endpoints are urgently needed.
hepatitis C|liver disease|milk thistle|botanical medicine|
the number of patients with HCV-induced end-stage liver disease
is growing (1), and this condition is already the leading indication
for liver transplantation (2). The current standard of care for
chronic hepatitis C, pegylated IFN-α and ribavirin, results in sus-
tained elimination of virus in 55% of treated patients (3, 4).
are intolerant to, have contraindications to, or opt out of therapy.
Furthermore, because emerging specifically targeted antiviral
this therapy. Thus, there are many patients who have no other
Food and Drug Administration-approved options to eliminate
HCV and prevent progression of liver disease. As a result, many
individuals have opted for complementary and alternative medi-
cine-basedapproaches,including botanicals, totreattheirchronic
hepatitis C. Indeed, as many as 13 to 23% of American patients
with chronic liver disease use botanical medicines, with silymarin
being the most popular (6, 7).
Silymarin, an extract from the seeds of the milk thistle plant,
Silybum marianum, has been used for centuries as a hepato-
protectant. Although silymarin has been described to possess anti-
oxidant, immunomodulatory, antiproliferative, antifibrotic, and
defined. Although its clinical efficacy is currently uncertain (8, 10),
hronic hepatitis C virus (HCV) is a major global medical
interest in this botanical medicine has been piqued by studies
showingsilymarin blocksHCVcellculture(HCVcc)infection (9).
of silybin A and silybin B, causes dose-dependent reduction of
viral load in patients with chronic hepatitis C (11).
We previously showed that silymarin blocked HCVcc infection
of human hepatoma cultures, inhibited TNF-α and TCR induced
NFκB-dependent transcription, and suppressed TCR-mediated
proliferation and inflammatory cytokine production from T cells
(9, 12). Thus, silymarin displays antiviral, anti-inflammatory, and
immunomodulatory functions in human liver and immune cells.
We (13) and others (14) have recently shown that silymarin can
also block in vitro HCV NS5B polymerase activity at high con-
centration. Therefore, in the current study, we screened the seven
major flavonolignans and one flavonoid that comprise silymarin
for antiviral, antipolymerase, anti-NF-κB, and immunomodula-
tory actions. In addition, we assessed the antioxidant potential of
the pure compounds.
Silymarin is a complex of eight major compounds, including seven
flavonolignans: silybin A, silybin B, isosilybin A, isosilybin B, sily-
christin, isosilychristin, silydianin, and one flavonoid, taxifolin
(15). Compounds were purified as previously described (16) (Fig.
assays for HCVcc infection, NS5B polymerase activity, HCVcc-
TCR-mediated induction of T-cell proliferation.
We first performed cytotoxicity dose-response experiments by
measuring ATP levels, a sensitive marker of cell viability. All
compounds were well tolerated by Huh7.5.1 human hepatoma
cells up to 80 μM, except for isosilybin B, which was toxic to cells
above 10 μM (Fig. S2). Silydianin, silychristin, isosilychristin, and
taxifolin were not toxic at 100 μM.
Fig. 1A depicts the antiviral effects of the pure compounds
against in vitro HCV infection, based on HCVprotein expression.
shown in Table 1. Taxifolin, isosilybin A, and silybin A were the
were more potent than silymarin extract. Silybin B and silibinin, a
with potencies similar to silymarin. Isosilychristin, silydianin, and
isosilybin B did not inhibit HCV protein expression.
Y.L., T.N.G., and N.H.O. performed research; V.L., T.N.G., and N.H.O. contributed new
reagents/analytic tools; S.J.P., C.M., V.L., S.P., D.Y.W.L., Y.L., T.N.G., and N.H.O. analyzed
data; and S.J.P., C.M., V.L., S.P., D.Y.L., and N.H.O. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| March 30, 2010
| vol. 107
| no. 13
Fig. 1B and Table 1 summarize inhibition of HCV RNA repli-
cation among the different compounds. Taxifolin was the most
potentatblocking HCV RNA replication, followed by isosilybin A,
silybin B, silybin A, silibinin, and silychristin, which were all more
potent than silymarin. Silydianin and isosilychristin were inactive.
Silymarin inhibits HCV NS5B polymerase activity, albeit at
concentrations 5- to 10-fold higher than required for HCVcc
inhibition (13). We therefore tested the pure compounds for their
ability to inhibit JFH-1 NS5B polymerase activity. NS5B poly-
merase inhibition was only observed at very high concentrations,
with silymarin and silybin B displaying the most potent activities,
with IC50 values of 300 to 600 μM (Table 1).
In summary, the data indicate that certain silymarin-derived
pure flavonolignans can inhibit HCV infection as measured by
blockade of HCV protein and RNA expression, but that inhib-
ition of NS5B RdRp activity can only partly explain the antiviral
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).
Antiviral effects of silymarin-derived flavonolignans. (A) Effects on HCV protein expression. JFH-1 virus at a multiplicity of infection of 0.05 was
Table 1.Hepatoprotective effects of silymarin-derived flavonolignans
Compound HCV Protein*HCV RNA†
RdRp*NFkB* ROS (%)T-cell proliferation (%)
40 40 6.8
IC50 data are expressed as concentrations in micromolars, both estimated* and actual†. Antioxidant data reflect the percent inhibition of JFH-1-induced
oxidative stress for flavonolignan treated infected cells versus DMSO solvent controls. For immunomodulatory function, data are expressed as percent-
inhibition of CD3 induced proliferation at a compound concentration of 40 μM.
‡Note that isosilybin B was toxic above 10 μM, so the concentrations for the reported activities are largely because of toxicity rather than a specific
| www.pnas.org/cgi/doi/10.1073/pnas.0914009107Polyak et al.
activity. Indeed, we have shown that silymarin blocks virus entry
and fusion, and virus production (13).
To determine if silymarin contains antioxidant functions in the
context of HCV infection, we treated infected cells with silymarin,
then labeled cells with the oxidant reactive dye, H2DCFDA, and
cells caused large increases in fluorescently labeled cells compared
with mock-infected cells, indicative of oxidative stress (Fig. 2A).
Treatment of cells with three different preparations of silymarin
(MK-001, USP, and Legalon) caused significant reductions of cel-
lular fluorescence (Fig. S3). Treatment with all eight pure com-
pounds inhibited JFH-1-induced oxidative stress (Fig. 2B). IFN, a
known antiviral for HCV, also inhibited JFH-1-induced oxidative
stress. Silibinin (a 1:1 mixture of silybin A and silybin B), taxifolin,
and isosilybin A had antioxidant activity equal to or greater than
silymarin, causing greater than 90% reduction in JFH-1-induced
oxidative stress. Compounds that lacked antiviral action, such as
silydianin and isosilychristin, still possessed antioxidant function.
NF-κB is the key transcription factor that is central to the
induction of an inflammatory response (17). NF-κB transcription
factors are homo- and heterodimeric complexes of proteins with
molecular weights of 50 and 65 kDa (18). Inactive NF-κB is found
in the cytoplasm via its association with inhibitory proteins of the
IκB family. Stimulation of NF-κB activity involves phosphor-
ylation of IκB-α on serine amino acid 32, phosphorylation of NF-
κB on serine 536, followed by degradation of IκB-α, permitting
NF-κB translocation to the nucleus and expression of inflamma-
tory response genes (19, 20).
Silymarin blocks NF-κB-dependent transcription from a can-
onical NF-κB promoter (9). Silymarin also blocked TNF-α acti-
vation of NF-κB dependent transcription from the positive
regulatory domain (pRDII) domain of the IFN-B promoter, a
key gene in antiviral defense (21) (Fig. S4A). Silymarin reduced
both p65 and IκB-α serine phosphorylation by about 20% and
caused an approximate 20% decrease in TNF-α induced p50 and
p65 nuclear translocation (Fig. S4 C and D). However, silymarin
did not affect the ability of p50 or p65 to bind to the NF-κB
promoter (Fig. S4E). The data suggest that silymarin partially
inhibits phosphorylation of IκB-α and p65/RelA and nuclear
translocation of p50 and p65 subunits of NF-κB, but does not
impair the ability of NF-κB to bind to DNA.
Fig. 3 depicts examples of the effects on the pure compounds’
abilities to block TNF-α induced NF-κB-dependent transcription.
because the flavonolignans were only incubated with cells for 4 h.
Silybin A and silybin B caused dose-dependent blockade of TNF-α
inducedNF-κB dependenttranscription,with IC50s of40 μM (Fig.
3 A and B and Table 1). Compared with silybin A and B, taxifolin
induced the most-pronounced inhibition of NF-κB transcription at
the lowest doses, but its effect appeared to plateau (Fig. 3C).
Table 1 summarizes the effects of silymarin at 40 μM on T-cell
proliferation inducedbyCD3 ligation. Silymarin, silibinin, silybin
A, and isosilybin A displayed the most potent suppression of
T-cell proliferation. Isosilychristin, silydianin, and taxifolin were
essentially inactive. Isosilybin B also blocked T-cell proliferation,
but this was because of toxicity. In contrast to the other measures
of hepatoprotection, where some pure compounds were more
active than silymarin, silymarin was the most potent in blocking
T-cell proliferation. Silymarin and pure compounds also inhibi-
ted inflammatory cytokine expression from T cells (Fig. S5).
To our knowledge, this report is unique in characterizing silymarin
and silymarin-derived pure compounds in four different measures
activity in the five hepatoprotective assays. Isosilybin A, taxifolin,
andsilibininwere themosteffective hepatoprotectorsbecausethey
displayed potent activity in four of five assays, followed by silybin A
particularly noteworthy because hepatoprotection was observable
at lower doses than the other compounds, and tended to plateau
showed more linear dose-response relationships. Intriguingly, all
compounds inhibited virus-induced oxidative stress regardless of
whether they had antiviral activity.
With the exception of T cell proliferation, several purified
compounds were more potent than the parent silymarin extract.
Although some complementary and alternative medicine prac-
tioners may emphasize that it is the combination of bioactive
molecules in a botanical extract that defines its unique biological
properties, our data suggest that combinations of flavonolignans
may provide optimal hepatoprotection. Because the structures of
the flavonolignan stereoisomers are similar, there may be com-
petitive interactions between flavonolignans for cellular targets.
This possibility implies that the hepatoprotective potential of
the silymarin extract itself. Moreover, different combinations of
flavonolignans might be selected, depending on the hepatopro-
tective functions one is targeting. Given that isosilybin B is very
toxic in cell culture, as shown here, select combinations may be
especially relevant if one is trying to minimize cytotoxicity for
treatment of liver diseases of both viral and nonviral origin. Fur-
ther studies that examine combinations and define the cellular
targets of the pure compounds are needed to resolve these issues.
In the current study, although silymarin and silybin B appeared to
be the most potent polymerase inhibitors, the IC50 concentrations
were much higher than the concentrations that caused cytotoxicity.
clinical isolates of genotype 1b HCV RdRps, and silymarin does not
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.
Silymarin and silymarin-derived flavonolignans block HCV induced
Polyak et al.PNAS
| March 30, 2010
| vol. 107
| no. 13
inhibit HCV replication in noninfectious replicon cell lines (13).
Therefore, we propose that polymerase inhibition by the natural
compounds, although demonstrable in vitro, may not contribute to
in patients with chronic hepatitis C when silymarin is taken orally.
ofsilybinA andsilybinB.This chemicalmodificationmakes silibinin
and mechanism of action than the natural compounds.
NF-κB serine phosphorylation is required for maximal transcrip-
tional activation by NF-κB (22). We showed that silymarin partially
inhibits NF-κB-dependent transcription via partial blockade of
on IκB-α. Silymarin also partially inhibited nuclear translocation of
block p50/p65 nuclear translocation and IKK-α activity (23).
HCV infection induces oxidative stress and inflammation (24–
27). Unchecked oxidative stress can modify proteins and lipids,
damage DNA, alter mitochondrial membrane potential, and be a
in oxidative stress. These effects were abrogated by silymarin and
induced oxidative stress by at least two mechanisms. First, silymar-
in’s demonstrated antiviral actions could have reduced HCV rep-
lication and the ensuing oxidative stress. Second, flavonolignan
components of silymarin may have direct antioxidant functions
without antiviral functions. Evidence in support of both modes of
action was provided in the present study. These data extend sily-
marin’s previously documented antioxidant functions (28) to the
independently of suppression of virus, it is possible that the hep-
atoprotective actions of the botanical may be extended to liver
diseases of nonviral origin.
Although silibinin, which is a mixture of silybin A and silybin B,
has been largely touted to contain many of the hepatoprotective
functions (28, 29), we are unique in showing that there are other
antiviral activities that were superior to silibinin. Yet in T cell pro-
liferation silibinin, but not taxifolin, displayed significant activity.
These data underscore the importance of careful evaluation of all
flavonolignans and that the activity of each flavonolignan may vary
depending on the biological assay.
Our data suggest that silymarin and silymarin-derived com-
pounds could influence HCV disease course in some persons.
Although standardized silymarin has an exceptional safety record
(8), the effects observed in vitro occur at relatively high concen-
trations, so additional studies are necessary to assess if adverse
effects occur in humans at these levels. Therefore, we caution
particularly those purchased as dietary supplements, because there
is inconsistency in the standardization of these products, the entire
contents may not be known, they may contain contaminants, and
compoundssuggests greatpromiseforthe treatmentofhepatitisC,
for which previously unexplored therapeutics are urgently needed.
of biological activities. The four activities we measured: antiviral,
antioxidant, anti-inflammatory, and immunomodulatory, are all
likely to be directly related to its well-described hepatoprotective
(32). Although the latter may be primarily because of the metab-
olism and bioavailability of silymarin and a dearth of properly
designed clinical studies, it is clear that further studies are neces-
such pleiotropic effects.
Subjects. Subjects with and without chronic HCV infection were recruited at
a University of Washington Institutional Review Board–approved protocol.
PBMC Isolation and Proliferation Assay. Peripheral blood mononuclear cells
(PBMCs) were isolated within 24 h of venipuncture and immediately assayed.
PBMC were stimulated for 24 h at 37 °C 5% CO2using plate-bound anti-CD3
(UCHT1, 10 μg/mL, BD Biosciences) in RPMI medium 1640 supplemented with
10% human serum (Gemini Bio-Products). Cellular proliferation detected by
3H-thymidine incorporation into replicating DNA was measured by adding
1 μCi to each replicate well of 105PBMC for an additional 24 h before quan-
titative analysis using a Topcount Liquid Scintillation Counter (Perkin-Elmer).
cpm incorporated per condition tested. Percent inhibition was calculated using
the following formula: 100 × (vehicle-treated mean cpm − compound-treated
mean cpm)/vehicle-treated mean cpm.
Cells, Virus, and Plasmids. Huh7.5.1 cells were grown in Huh7 medium as
described (9). JFH-1 viral stock preparation, cell infection, and titration was
performed as described (33–35). For antiviral assays, cells were in infected at a
multiplicity of infection of 0.05 and protein and RNA harvested 72 h post-
infection. To measure NF-κB–dependent transcription, we used a luciferase
reporter gene under control of the positive regulatory domain (pRDII), which
contains the NF-κB site from the IFN-B promoter.
Silymarin Preparations, and Pure Compounds. Three preparations of silymarin
were used in the current study. The first was MK-001, prepared as described
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.
Silymarin flavonolignans inhibit NF-κB transcription. (A and B) Silybin A and silybin B inhibit NF-κB transcription. Huh7 cells were transfected with a
| www.pnas.org/cgi/doi/10.1073/pnas.0914009107 Polyak et al.
(9, 36). The second preparation was commercially prepared (Legalon;
Madaus). The third preparation was purchased from US Pharmacopeia.
Silymarin was solubilized in DMSO at 50 mg/mL for Huh7 cell studies;
methanol was the solvent for PBMC studies. Pure flavonolignans were
solubilized at 50 mg/mL in DMSO for all studies except PBMC studies, where
methanol was used as the solvent.
Silymarin treatment of Huh7.5.1 cultures consisted of adding silymarin
to plate-bound anti-CD3 (T-cell receptor antibody).
Cytotoxicity Determinations. The toxicity of silymarin extracts and pure fla-
vonolignans on Huh7.5.1 cells was determined by measuring ATP levels using
the ATPlite system (Perkin-Elmer), as described (9).
Reporter Gene Assays. Reportergeneassayswereperformedasdescribed(37).
Endotoxin free plasmid DNA was purified (Endofree kit, Qiagen), and was
introduced into cells with Lipofectamine 2000 according to manufacturer’s
recommendations (Invitrogen). Next, 100 ng of the pRDII-luciferase gene was
transfected into cells in quadruplicate. Eighteen hours later, cells were pre-
mL; Sigma Aldrich) was added. Four hours later, luciferase activity was meas-
ured on cell lysates using the Britelite Assay System (Perkin-Elmer).
Western Blot Analysis. NS5A and core proteins in JFH-1 infected cells were
was detected with polyclonal antiserum (Santa Cruz Biotechnology). NF-κB
antibodies consisted of a monoclonal antibody to RelA/p65 Serine 536, IκBα
Serine 32 (Cell Signaling), and antibodies to p50 and p65 subunits (Upstate/
Millipore). For Western hybridizations and washings, both the standard seal a
meal bag approach and the Snap-ID system (Millipore) were used.
Oxidative Stress Measurements. Huh7.5.1 cells were incubated with 20 μg/mL
of silymarin or pure flavonlignans immediately after virus adsorption. Sev-
enty-two hours later, cells were labeled with the dye 2’,7’-dichlorodihydro-
fluorecein diacetate (H2DCFDA) for 30 min using the Image-IT Live Green
Reactive Oxygen Species kit (Molecular Probes/Invitrogen). H2DCFDA is a
cell-permeable indicator for reactive oxygen species that is nonfluorescent
until the acetate groups are removed by intracellular esterases and oxida-
tion occurs within the cell. On oxidation, H2DCFDA becomes highly fluo-
rescent Pharmacopeia. Images were captured on a Nikon Microscope using
MetaMorph software (Molecular Devices) with a 20× objective.
HCV NS5B Polymerase Assays. NS5BΔC21C-terminallyfusedtoahexa-histidine
tag was expressed and purified for HCV JFH1 and for the genotype 1b isolates
as described (38). JFH-1 RNA dependent RNA polymerase (RdRp) assays con-
tained buffer, 5 μCi of [α-32P]GTP, 50 μM GTP, 1 mM each ATP, CTP, UTP, 2 μg
polyC (GE Healthcare), 1 μg purified polymerase, and given amounts of puri-
fied compounds in DMSO in a total volume of 25 μL. All reaction components
except nucleotides and template were preincubated for 15 min at room
temperature; the reaction was started by adding the nucleotide mixture and
polyC and was incubated for 1.5 h at room temperature. Reaction products
were precipitated, passed through microfilters (GE Healthcare), washed five
air-dried. After addition of 6 mL of Ultima Gold (Perkin-Elmer), samples were
subjected to liquid scintillation counting. All measurements were performed
in triplicate and the IC50 values were calculated with GraphPad Prism.
ACKNOWLEDGMENTS. We thank Jessica Wagoner and Minjun Chung for
technical assistance. S.J.P. is partially supported by National Institutes of
Health Grant AT002895 from the National Center for Complementary and
Alternative Medicine. V.L. is supported by the Deutsche Forschungsgemein-
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Polyak et al.PNAS
| March 30, 2010
| vol. 107
| no. 13