Hepatitis C Virus and Natural Compounds: a New Antiviral Approach?
Submitted by admin on Wed, 02/05/2014 - 18:00
C is a major global health burden with an estimated 160 million
infected individuals worldwide. This long-term disease evolves slowly,
often leading to chronicity and potentially to liver failure. There is
no anti-HCV vaccine, and, until recently, the only treatment available,
based on pegylated interferon and ribavirin, was partially effective,
and had considerable side effects. With recent advances in the
understanding of the HCV life cycle, the development of promising direct
acting antivirals (DAAs) has been achieved. Their use in combination
with the current treatment has led to encouraging results for HCV
genotype 1 patients. However, this therapy is quite expensive and will
probably not be accessible for all patients worldwide. For this reason,
constant efforts are being made to identify new antiviral molecules.
Recent reports about natural compounds highlight their antiviral
activity against HCV. Here, we aim to review the natural molecules that
interfere with the HCV life cycle and discuss their potential use in HCV
have been used for centuries for the treatment of human diseases.
Historically, numerous important modern drugs have been developed from
molecules originally isolated from natural sources [1,2]. Among them, the most popular is aspirin, based on a natural product salicin isolated from Salix alba. Morphine and codeine were extracted from opium poppy Papaver somniferum,
and quinine, traditionally used as an anti-malaria treatment, from
cinchona tree. In the past decades, taxol, a molecule extracted from the
bark of the Pacific yew tree, Taxus brevifolia, has become one of the most used anti-cancer agent worldwide .
search for new bioactive molecules in plants in key therapeutic areas
such as immunosuppression, infectious diseases, oncology and metabolic
diseases is still an active part of pharmaceutical research .
About 40 new drugs launched on the market between 2000 and 2010,
originate from terrestrial plants, terrestrial microorganisms, marine
organisms, and terrestrial vertebrates and invertebrates .
The World Health Organization (WHO) estimates that approximately 80% of
the world’s population rely mainly on traditional medicine,
predominantly originated from plants, for their primary health care.
medicines, including Chinese herbal formulations, can serve as the
source of potential new drugs. Active plant compounds used either for
prophylactic or therapeutic treatments are orally administrated to
patients as teas, powders, and other herbal formulations [6,7].
Phenolic compounds are often responsible for the bioactivities of the
plant crude extracts. During the last decades, people have tried to
identify more precisely the active molecules of these traditional
medicines. Another approach was to systematically screen natural
molecules present in plant extracts and test the activity of these
phytochemicals using the appropriate assays (depending on the pathology
The main advantage of using natural molecules
from plant extracts is a reduced cost of production, with no need of
chemical synthesis. This mode of production might lead to less expensive
treatments, available for populations of low-income countries.
Some natural medicines have been shown to possess antiviral activities against herpes simplex virus [8,9], influenza virus, human immunodeficiency virus [10,11,12], hepatitis B and C viruses [13,14]. The screening of natural products has led to the discovery of potent inhibitors of in vitro viral growth .
Antiviral activities of several hundred natural compounds have been
identified worldwide. In addition, dozens of herbs are known to have
hepatoprotective activities. During the last decades, scientists have
tried to analyze more precisely the active molecules present in this
traditional medicine that is frequently used for the treatment of
hepatitis in China .
last few years have seen a flurry of reports on the identification of
natural molecules of plant origin with anti-hepatitis C activities. The
aim of this review is to give an overview of these different compounds
with a special focus on the most promising molecules.
2. Hepatitis C Virus
C is a major healthcare problem worldwide caused by a viral infection
with a high tendency to become chronic. Chronic hepatitis C is linked to
the development of cirrhosis and hepatocellular carcinoma. The virus
responsible for this disease was discovered more than 20 years ago .
Its transmission is thought to be essentially parenteral, and has been
linked to blood transfusions before its discovery. Since hepatitis C
virus (HCV) discovery, blood screening diagnostics have greatly reduced
the blood-borne transmission of the virus. However, the transmission
still occurs through other modes of contamination and the slow
development of the disease results in many persons not knowing their
infected status. It is estimated that about 160 million persons (2.35%
of the world population) are infected with HCV .
there is no vaccine against HCV and the high diversity of viral
isolates will probably make it very difficult to develop a vaccine. On
the other hand, we know that, in contrast to hepatitis B and human
immunodeficiency viruses, HCV can be eradicated from chronically
infected patients with antiviral treatments. However, the standard
therapy, which is based on a combination of pegylated interferon alpha
(IFN-?) and ribavirin , results in highly variable outcomes ,
is very expensive and has severe side effects that are difficult to
endure for the patients. Nevertheless, it is currently thought that
efficient anti-HCV therapies will be achieved with direct acting
antivirals (DAAs) .
The recent addition of protease inhibitors to the standard anti-HCV
therapy has already improved sustained virological response rates in
patients infected with genotype 1 HCV. New drugs targeting other viral
proteins are in clinical trials and will probably also help improving
response to HCV therapy [22,23].
A combination of DAAs will reduce the risk of selecting viral escape
mutants. DAAs combinations in the absence of interferon will probably
enable to greatly reduce side effects of the therapy, which are mainly
associated with the use of interferon and contribute to the failure of
the treatment. Ideally, such a combination should include DAAs targeting
different steps of the HCV life cycle and should be efficient against
all HCV genotypes. Moreover, to have a chance of eradicating HCV, the
therapy should be cheap so as to be able to cure infected patients from
low-income countries and stop the transmission of the virus.
Hepatitis C virus is a small, enveloped virus belonging to the Hepacivirus genus of the Flaviviridae family .
Its single-stranded genomic RNA contains a single open reading frame
surrounded by two untranslated regions (UTR) that are necessary for the
translation and the replication of the viral genome [25,26,27].
The translation of the open reading frame is under the control of an
internal ribosome entry site (IRES), located in the 5’UTR. It gives rise
to a polyprotein precursor, which is cleaved by host- and viral-encoded
proteases into ten polypeptides. The N-terminal part of the polyprotein
contains structural proteins: the core protein C, a component of the
viral capsid, and the two envelope glycoproteins E1 and E2. The
C-terminal part of the polyprotein contains non-structural proteins
required for RNA replication: NS3, which has protease and helicase
activities; NS4A, a co-factor of NS3 protease; NS4B, a polytopic
membrane protein; NS5A, a phosphoprotein; and NS5B, the viral
RNA-dependent RNA polymerase. Between the structural proteins and the
non-structural proteins involved in RNA replication, the polyprotein
also contains two additional polypeptides required for viral assembly,
which are dispensable for RNA replication: the viroporin p7 and the NS2
protein, which has an autoprotease activity during the maturation of the
The structure of the viral
particle is still unknown. In patients, circulating HCV particles are
associated with apolipoproteins (Apo) B and E, and have highly variable
buoyant densities, the lighter ones being the most infectious .
It is currently thought that infectious HCV particles are initially
secreted as very low-density lipoprotein (VLDL)-like particles by
infected hepatocytes and then potentially undergo lipolysis in the
bloodstream, which progressively converts them into intermediate density
lipoprotein (IDL)- and LDL-like particles. However, it is not yet clear
what in this process reduces the specific infectivity of HCV viral
Our knowledge of the HCV life cycle has
greatly improved in recent years, following the finding of a viral
strain (JFH-1) able to replicate in cell culture [29,30,31].
The JFH-1-based cell culture model has been named HCVcc. For reasons
that are still unknown, other viral isolates do not efficiently
replicate in cell culture. Before the HCVcc model was established,
specific steps of the HCV life cycle had been studied with other
experimental systems recapitulating RNA replication, with the subgenomic
replicon model [32,33], or viral entry, with the HCV pseudoparticles (HCVpp) model [34,35,36].
The replicon model is based on a modified HCV genome, in which the
coding region of the structural proteins is replaced by a selection
marker. In vitro synthesized subgenomic replicon RNA is
introduced in cells by electroporation and the cells replicating it
express the selection marker and can thus be selected. There is no
release of viral particles, and this model only allows studying cellular
and molecular mechanisms involved in viral RNA replication. Hepatitis C
virus pseudoparticles are retroviral particles pseudotyped with HCV
envelope glycoproteins E1E2. In this system, only E1E2-dependent, early
entry steps (virus binding, uptake and fusion) are HCV specific, whereas
later steps depend on retroviral function.
cycle of HCV can be divided into three major steps: entry of the virus
into its target cells by receptor-mediated endocytosis, cytoplasmic and
membrane-associated replication of the RNA genome, and assembly and
release of the progeny virions (). Hepatitis C virus entry is a very complex process, which involves a series of host entry factors .
On the viral particle, envelope glycoproteins E1E2 play a major role
during entry. The viral particle probably initially binds to
glycosaminoglycans (GAG) on the surface of the target cell. It has been
proposed that interactions between the LDL receptor (LDL-R) and
apolipoproteins of the viral particle might also participate in the
initial binding to the cell surface. Following these rather non-specific
initial binding events, several host entry factors are specifically
involved in the entry process . The tetraspanin CD81 , the scavenger receptor class B type I (SR-BI) , and the tight junction proteins claudin-1 (CLDN1)  and occludin (OCLN) [42,43] are mandatory for HCV entry. Epidermal growth factor receptor, ephrin receptor A2 , and the cholesterol transporter Niemann-Pick C1-like 1 also participate to the entry process . The particle is internalized by clathrin-mediated endocytosis 
and the viral genome is released into the cytosol of the cell following
the fusion of the viral envelope and the endosomal membrane.
in the cytosol, the viral genome is translated. Non-structural viral
proteins NS3/4A, NS4B, NS5A, and NS5B assemble into replication
that generate new viral genomic RNA molecules through the prior
synthesis of negative RNA strands, complementary to the genomic RNA.
Much like for many positive stranded RNA viruses, HCV replication occurs
in host cell cytoplasm in association with rearranged membranes, named
‘membranous webs’ .
A large number of host factors probably participate to the formation
and the functioning of HCV replication complexes, which are recruited
through interactions with viral proteins. A major host cell factor
regulating HCV replication recently identified is the class III
phosphatidylinositol 4-kinase alpha [49,50].
The protease NS3/4A and the RNA polymerase NS5B are the two major
druggable viral factors involved in HCV replication, which have been
used in antiviral screens.
C virus (HCV) life cycle and targets of the most potent natural
inhibitors. First, HCV binds to non-specific factors glycosaminoglycans
(GAG) and LDL receptor (LDL-R) present at the cell surface (attachment
step). Then, the viral particle is directed to specific entry factors
(entry step), the scavenger receptor class B type I (SR-BI), the
tetraspanin CD81 and the tight junction proteins claudin-1 (CLDN1) and
occludin (OCLN). The virus is internalized by endocytosis and the viral
genome is released into the cytosol of the cell after fusion with
endosomes (fusion step). Next, the translation and the polyprotein
processing take place and RNA is replicated (replication step). In the
late stages of the cycle, the virion is assembled (assembly step) in the
vicinity of cytoplasmic lipid droplets (LD) and is released from the
cell. Finally, the released virions can infect adjacent cells by
cell-free transmission or cell-to-cell transmission. The affected steps
of the viral cycle are in black. The natural compounds are in red. EGCG:
epigallocatechin-3-gallate; ER: endoplamic reticulum.
assembly step of the HCV life cycle occurs in the vicinity of
cytoplasmic lipid droplets (LD). The core protein, which is localized on
the surface of LD , recruits replication complexes through interaction with NS5A .
It was recently shown that p7 and NS2 are involved in the assembly step
by interacting with E1E2 envelope glycoproteins and non-structural
proteins, mainly NS3, and that these interactions are crucial for the
formation of assembly sites [53,54,55,56,57].
Host factors critical for HCV assembly include diacylglycerol
acyltransferase-1, a triglyceride-synthesizing enzyme required for core
trafficking to LD , and VLDL secretion machinery [59,60].
years, the production of HCV in cell culture has been impossible and
the search for DAAs was essentially limited to host and viral targets
involved in the replication step of the virus. Most of the early screens
were performed based on the in vitro protease activity of
NS3/4A. With the recent introduction of various assays based on the
HCVcc system, the search for DAAs has been highly stimulated and can now
be performed in the context of a complete HCV life cycle. During the
last few years, this led to a substantial increase of reports on natural
compounds displaying an anti-HCV activity. The identified molecules
belonging to different chemical families are summarized in , and the different affected steps of the HCV life cycle depicted in .
In this review, we have chosen to classify these molecules according to
the advances in their proof of concept and to their chemical family.
Natural molecules with anti-HCV activities tested in vivo or in cellular models.
or bioflavonoids are a class of plant secondary metabolites. They are
naturally present in numerous plants. More than 4,500 flavonoids have
been characterized so far. They have been classified according to their
chemical structure and are usually subdivided into different subgroups.
Some of them are described as potential anti-HCV molecules.
Silymarin is extracted from the seeds of milk thistle Silybum marianum.
This plant native of Southern Europe and Asia is now found throughout
the world. The seed extract of milk thistle is an ancient herbal remedy
used as hepatoprotectant and to treat liver disease. It contains at
least seven flavonolignans (silybin A, silybin B, isosilybin A,
isosilybin B, silychristin, isosilychristin, silydianin) and one
flavonoid (taxifolin). Flavonolignans are natural polyphenols composed
of flavonoid and lignan moieties. The major component of silymarin,
silibinin (a mixture of the two diastereoisomers silybin A and silybin
B) has also been reported to have anti-HCV activity.
has multiple effects on HCV. Silymarin appears to inhibit HCV infection
at least at two different levels: it inhibits HCV replication in cell
culture  and it also displays anti-inflammatory and immunomodulatory actions that may contribute to its hepatoprotective effects . By screening the seven major flavonolignans, Polyak et al. showed that specific compounds present in silymarin are responsible for the different anti-HCV activities [61,64].
The inhibition of HCV replication was attributed to the inhibitory
action of silibinin on the NS5B RNA-dependent RNA polymerase [63,65]. Half inhibitory concentrations (IC50)
in the order of 75–100 µM and 40–85 µM were reported in these studies
for a succinate-conjugated form of silibinin, which is more soluble in
aqueous solutions than natural silibinin. An inhibition of entry was
also reported with HCVpp and liposome fusion assays [62,63]. Another potential anti-HCV activity of silymarin has been described by Ashfaq et al. .
Using a heterologous expression system, they reported an
NS5B-independent inhibition of HCV genotype 3a core expression by
Low bioavailability of silymarin components has been reported .
This is probably the reason why clinical studies dealing with oral
administration of silymarin have been unsuccessful in curing patients
from HCV [85,86,87]. Because silibinin is rapidly metabolized after oral administration ,
clinical studies were also attempted with the water-soluble,
succinate-conjugated silibinin formulated for intravenous injection. In
this case, silibinin monotherapy showed a substantial antiviral effect
in patients with chronic hepatitis C not responding to standard
pegylated interferon/ribavirin therapy [67,68].
Two cases of successful prevention of liver graft infection with
silibinin monotherapy in patients with chronic hepatitis C have also
been reported [69,70],
and a case of sustained virological response after treatment with
intravenous silibinin was reported for a HCV/HIV co-infected patient not
responding to the standard HCV therapy .
Therefore, although oral administration of silymarin is not effective
for the treatment of HCV patients, intravenous silibinin formulation may
represent a potential therapeutic option.
3.2. (?)-Epigallocatechin-3-gallate (EGCG)
is the most abundant flavonoid from the subclass of catechin present in
green tea extract. It has been shown that a single cup of tea contains
up to 150 mg of this molecule and its administration is safe in healthy
individuals . Very recently, three different groups have independently identified EGCG as a new inhibitor of HCV entry [72,73,74].
These studies showed that EGCG present during infection of Huh-7 cells
with HCVcc resulted in dose-dependent inhibition of infection. Different
IC50 (between 2.5 µg/mL and 9.7 µg/mL, corresponding to 5 µM
and 21 µM) were obtained in the three studies, probably at least in
part reflecting differences in experimental setups. The half cytotoxic
dose was comprised between 150 and 175 µM in Huh-7 cells, depending on
the exposure time. Two groups found no additional effect of EGCG on HCV
RNA replication and on release of HCV infectious particles [72,73], despite reported inhibitory activities of EGCG on NS3 and NS5B in in vitro assays [90,91], while the third group reported an additional activity of EGCG on the RNA replication step .
Hepatitis C virus pseudoparticles were used to further confirm the
impact of EGCG on HCV entry. EGCG inhibited HCVpp entry in a
genotype-independent manner in hepatoma-derived cells [72,73,74], as well as in primary human hepatocytes .
mechanism of action of EGCG on HCV entry is still being investigated.
EGCG inhibits HCV entry only when it is present during the inoculation
period, or when viral particles have been pre-incubated with it [72,73,74].
In contrast, the pre-incubation of target cells has no impact on HCV
infection. Moreover, EGCG does not change the expression levels of
cellular entry factors (CD81, CLDN1, OCLN, SR-BI) [72,74].
Therefore it is very likely that EGCG acts directly on the viral
particle. EGCG inhibits the binding of the virus to the cell surface [72,73]
and has no effect when added post-binding. It does not appear to alter
physical properties of HCV virions, such as their density profile or
lipoprotein association .
Although antiviral activities of EGCG have also been reported against
other viruses, such as herpes simplex virus and influenza virus [92,93,94], the antiviral effect of EGCG on HCV cannot be generalized to the other members of the Flaviviridae
family, because this molecule is inactive against bovine viral diarrhea
virus (BVDV, a pestivirus) or yellow fever virus (YFV, a flavivirus) .
Based on its action on both HCVcc and HCVpp, it can be speculated that
EGCG interacts with E1E2 glycoproteins and that this interaction
inhibits virion binding to target cell surface. However a direct
experimental evidence for this interaction is still missing.
Interestingly, EGCG could inhibit cell-to-cell transmission [72,73,74]
in addition to its action on cell-free particle binding. Cell-to-cell
transmission is probably a major route of spreading of HCV in the liver
of infected patients. It was also reported that EGCG could be used in
combination with boceprevir or cyclosporin A (two known inhibitors of
HCV replication), with an increased efficiency .
Finally, it was shown that the anti-HCV effect of EGCG can lead to
undetectable levels of virions in the supernatant of Huh-7
infected-cells after a few passages [73,74]. Recently, Fukazawa et al. 
by developing a new anti-HCV molecule-screening assay, have confirmed
the anti-HCV activity of EGCG. Moreover, consumption of up to 800 mg of
EGCG is safe and increases the concentration of EGCG detected in the
plasma , indicating its potential use in clinical trials.
these data indicate that EGCG is a new anti-HCV molecule with
interesting properties. It directly inactivates HCV particle, is not
genotype-specific (unlike currently used protease inhibitors), and also
prevents cell-to-cell transmission. These properties make it an
especially interesting molecule to prevent HCV recurrence and spread in
chronically infected liver transplant patients. Future clinical trials
should investigate whether it could actually prevent the re-infection of
patients undergoing orthotopic liver transplantation, and whether it
could be used in combination with other DAAs to treat infected patients.
Recently, Haid et al. have isolated a molecule with anti-HCV activity in a screen of a library of natural phenolic compounds from plant extracts .
From the most active plant extract, they characterized and
re-synthesized the component exhibiting the highest antiviral activity.
Ladanein (and its synthetic equivalent BJ486K) was identified as the
active anti-HCV component. Ladanein, extracted from Marrubium peregrinum L. (Lamiaceae), is a flavone, a molecule belonging to a subgroup of the flavonoid family. Ladanein inhibited HCV entry with an IC50
of 2.5 µM in a genotype-independent manner. In contrast to EGCG,
ladanein did not appear to inhibit the binding of the viral particle
(although results of a direct binding assay were not reported), but
rather inhibited a later, yet uncharacterized step of viral entry.
Interestingly, when used in combination with cyclosporin A, a known
inhibitor of HCV replication, ladanein acted synergistically on HCV
infection. Ladanein also exhibited an antiviral activity in primary
human hepatocytes, but with an increased IC50 (10 µM). Very
importantly, this molecule was shown to be orally bioavailable in mice
with a peak of plasma level of 329 nM after a single oral dose of 0.25
mg/kg. These data are encouraging for a potential use of ladanein as an
anti-HCV molecule in patients.
is a dietary supplement demonstrated to possess anti-oxidant,
anti-inflammatory, and anti-carcinogenic properties both in vitro and in vivo.
This molecule belongs to the flavonoid family. It is the predominant
flavanone present in the grapefruit and is responsible for its bitter
taste. Naringenin has been previously shown to reduce cholesterol levels
both in vitro  and in vivo .
Furthermore, naringenin inhibits ApoB secretion by reducing the
activity and the expression of the microsomal triglyceride transfer
protein (MTP) and the acyl-coenzyme A cholesterol acyltransferase 2
(ACAT) [96,98]. Due to the close link between HCV assembly/secretion and lipoprotein metabolism, Nahmias et al. have studied the impact of naringenin on the secretion of HCV particles .
A concomitant dose-dependent decrease of core protein, HCV-positive
strand RNA, infectious particles, and ApoB was observed in the
supernatant of infected Huh-7 cells after naringenin treatment .
The inhibitory activity of naringenin was also observed in primary
hepatocytes in culture. Naringenin blocked the assembly of intracellular
infectious viral particles without affecting intracellular levels of
the viral RNA or protein. The maximal inhibition (74% of inhibition) of
secretion of both ApoB and HCV RNA is observed at 200 µM naringenin with
an IC50 of 109 µM .
mechanism of action of naringenin was proposed to be through the
inhibition of ApoB secretion. In a first study, this inhibition was
correlated to a reduction of the activity of the MTP and an inhibition
of the transcription of 3-hydroxy-3-methyl-glutaryl-coenzyme reductase
(HMGR) and ACAT, three enzymes involved in the production of VLDL .
The authors later observed that naringenin also induces peroxisome
proliferator-activated receptor alpha (PPAR?) and that naringenin
inhibition of HCV secretion can be reversed by a PPAR inhibitor .
Naringenin caused an increased expression of PPAR? and its target gene
acyl-CoA oxydase and a concomitant decrease in sterol regulatory
element-binding protein (SREBP) and its target HMGR. PPAR? induction is
known to inhibit cholesterol synthesis through SREBP and its target
gene, HMGR. These data suggest that naringenin effect is at least
partially mediated by PPAR? activation.
is a flavonol, a plant-derived flavonoid, present in fruits,
vegetables, leaves and grains. This molecule has been described as an
anti-HCV molecule by two different teams. In 2009, Gonzalez et al. were looking for novel cellular proteins that interact with the viral protein NS5A (from H77 strain (genotype 1a)) of HCV .
By co-immunoprecipitation and co-localisation assays, they detected an
interaction between NS5A and the heat shock proteins (HSP) HSP40 and
HSP70. In order to confirm the implication of these proteins in the HCV
life cycle, they tested the impact of quercetin, a known inhibitor of
HSP synthesis. Using a cell culture-based bicistronic reporter system,
quercetin was found to decrease IRES activity either in absence or in
presence of NS5A. Quercetin also had a strong inhibitory effect at 50 µM
on HCV production in cell culture. However, its mechanism of action is
not clear, because siRNA-mediated depletion of HSP proteins had no
effect on HCV particle production, and because quercetin had only a
modest effect on replication in the HCVcc system, and did not inhibit
HCV replication in a subgenomic replicon system. Therefore, the anti-HCV
action of quercetin could be related to an impairment of viral
morphogenesis or secretion, rather than to a direct action on the
replication step of the HCV life cycle.
By screening for NS3 inhibitors in traditional Indian medicinal plants, Bachmetov et al. recently identified quercetin as the active substance responsible for the inhibition of NS3 protease activity by Embelia ribes plant extracts .
This inhibition of NS3 was confirmed in cell culture with NS3
protease-dependent fluorescent reporter systems. Again, the authors
found a strong inhibition of HCV particle production at the dose of 10
µg/mL (?33 µM), and a weaker impact on replication.
in both studies, entry and secretion were not investigated with
specific assays in order to know if the effect observed on HCV
replication was only due to the single action of this molecule at the
replication step or if it results from an additional effect on other
steps of the HCV life cycle.
3.6. Luteolin and Apigenin
Luteolin and apigenin, two other natural flavone molecules, were identified as anti-HCV agents via a pharmacophore search .
A pharmacophore corresponds to a theoretical description of molecular
features, which can be used for probing specific interactions between a
ligand and a biological molecule. In this study, the designed
pharmacophore was established from eight NS5B inhibitors selected from
the literature according to different criteria. The resulting
pharmacophore was tested against 15,568 compounds from an in-house
database. Only 31 compounds were potentially relevant and were evaluated
for their anti-HCV activities in vitro. Finally, 20 compounds showed a significant activity against HCV (half maximal effective concentration, EC50
< 50 µM). Among them, the most potent molecules were luteolin and
apigenin. Luteolin and apigenin displayed an anti-HCV activity with EC50
values of 4.3 µM and 7.9 µM respectively in a cell-based antiviral
assay. Finally, the authors showed that luteolin exhibited a good
inhibition of NS5B polymerase enzymatic function with an IC50 of 1.12 µM according to the method used .
lignans are a group of chemical compounds found in plants. Lignans are
one of the major classes of phytoestrogen, which are estrogen-like
chemicals and act as antioxidants.
Honokiol is a lignan present in the cones, the bark and the leaves of Magnolia officinalis. This plant has been used in the traditional Japanese medicine Saiboku-to. In 2011, Lan et al. showed that honokiol inhibits HCV infection .
The effect of honokiol on HCV infection, entry, translation and
replication was assessed in Huh-7 cells using HCVcc, HCVpp and
subgenomic replicons. Honokiol strongly inhibited HCVcc infection (EC50 = 1.2 µg/mL, corresponding to 4.5 µM, and EC90
= 6.5 µg/mL) at non-toxic concentrations (median lethal dose = 35
µg/mL). Combined with IFN-?, its inhibitory effect on HCVcc was more
profound than that of ribavirin combine with interferon.
Honokiol-mediated inhibition of HCV infection was shown to result from
multiple effects on the HCV life cycle. Honokiol inhibited the entry of
HCVpp from genotypes 1a, 1b and 2a. Honokiol dose-dependently inhibited
the expression levels of NS3, NS5A and NS5B. It also inhibited the
replication of genotypes 1a and 2a subgenomic replicons in a
dose-dependent manner. The authors conclude that the inhibition of both
entry and replication by honokiol provides the impetus to fully explore
the clinical utility of honokiol as an adjunct to current standards of
treatment for HCV infection.
4.2. 3-Hydroxy Caruilignan C
In 2012, Wu et al. reported the anti-HCV effect of 3-hydroxy caruilignan C (3-HCL-C) isolated from Swietenia macrophylla stems . Swietenia macrophylla belongs to the Meliaceae
family and its fruits are used as a folk medicine in Malaysia. 3-HCL-C
reduced both protein (NS3) and RNA levels of HCV with an EC50
value of 10.5 µg/mL (corresponding to 37.5 µM) in the subgenomic
replicon system. Moreover, combinations of 3-HCL-C and IFN-?,
2'-C-methylcytidine (NM-107, an NS5B polymerase inhibitor) or telaprevir
(VX-950, an NS3/4A protease inhibitor) increased the suppression of HCV
RNA replication. 3-HCL-C interfered with HCV replication by inducing
IFN-stimulated response element transcription and IFN-dependent
anti-viral gene expression. Therefore, 3-HCL-C has the potential to be
developed into a potent adjuvant for anti-HCV therapy.
5. Other Polyphenols
Other polyphenols have been reported to have potential anti-HCV activity using in vitro assays. Suzuki et al.
identified 3',4',5,6,7,8-hexamethoxyflavone, also known as nobiletin,
as the active compound responsible for the anti-HCV activity of Citrus
unshiu peel (Aurantii nobilis pericarpium) extract, an ingredient of traditional Japanese Kampo medicine . Nobiletin displayed an anti-HCV activity at 10 µg/mL in MOLT-4 cells infection assay. Hegde et al. isolated and characterized two novel oligophenolic compounds, named SCH 644343, and SCH 644342 from the Peruvian plant Stylogne cauliflora . These two compounds were identified as inhibitors of HCV NS3 protease activity in vitro with IC50 of 0.3 µM and 0.8 µM respectively. SCH 644343 was also active in an NS3 binding assay (IC50 = 2.8 µM). Zuo et al. identified the 1,2,3,4,6-penta-O-galloyl-?-D-glucoside as a potent inhibitor of NS3 protease activity from the plant Saxifraga melanocentra Franch . The IC50 was 0.68 µM and the molecule was not toxic up to 6 mg/mL (corresponding to 6.4 mM) on COS cells. Duan et al. identified three polyphenol components from the ethyl acetate fraction of the traditional Chinese medicine Galla Chinese . These polyphenols molecules, 1,2,6-tri-O-galloyl-?-D-glucose, 1,2,3,6-tetra-O-galloyl-?-D-glucose and 1,2,3,4,6-penta-O-galloyl-?-D-glucose, were shown to inhibit NS3 protease in vitro with IC50 values of 1.89, 0.75 and 1.60 µM, respectively.
these compounds were identified before the HCVcc model was established
and to our knowledge, they have not been evaluated since then.
Therefore, they will need to be tested in cell-based assays, before
considering them as new potential anti-HCV agents. This is illustrated
by the study of Li et al. who identified four polyphenolic compounds inhibiting NS3 protease in vitro, from the Chinese mangrove plant Excoecaria agallocha L. (Euphorbiaceae) . Among them, only two, excoecariphenol D and corilagin had a significant inhibitory action in the replicon assay with an IC50 of 12.6 and 13.5 µM respectively.
6. Crude Plant Extracts from Traditional Medicines
Traditional medicines represent a broad source of natural polyphenols molecules. In 2003, Liu et al.
assessed beneficial and harmful effects of medicinal herbs against HCV
infection. Thirteen randomized trials have evaluated fourteen medicinal
herbs. Only four trials had an adequate evaluation method .
Even if traditional medicine represents an attractive source of new
natural antivirals, studies with herbs need to be standardized in order
to clearly evaluate the effects due to the plant extracts on HCV
infection, and should provide all methodological details. Compounds
isolated from these herbs could be used for designing and developing
drugs for treatment of hepatitis C.
A study, which evaluated the benefice of a Far-Eastern traditional herbal formulation for patients with chronic hepatitis C ,
found some improvement of circulating aminotransferases, with no effect
on HCV RNA levels. Several other studies evaluated the effect of plant
extracts from traditional medicine using various assays, which found
extracts with anti-NS3 protease activity [106,107], anti-NS5B activity [16,107] and with anti-replicative effect in the replicon model [107,108,109].
None of these studies tried to evaluate other steps of the HCV life
cycle. In two studies, additive or synergistic actions in combination
with interferon [107,108], telaprevir or 2'-C-methylcytidine [108,109] were reported. Future studies on these plant extracts may lead to the identification of new anti-HCV molecules.
7. Concluding Remarks
this review, we discussed the diverse and broad actions of natural
molecules issued from plants as potential anti-HCV antivirals. It is
important to note that some other natural compounds, even if they do not
target the virus directly, might also be used to improve HCV therapy.
Glycyrrhizin, for instance, a component of licorice roots extract, has
been shown to prevent the development of hepatocellular carcinoma in
patients with chronic hepatitis .
a number of natural compounds with anti-HCV activities were identified
in recent years, many aspects concerning their mechanisms of action
remain unknown. Very often, the replication was the only step of the
viral life cycle that was investigated and, for older reports, the
conclusions are only based on in vitro models, mostly NS3
protease assays. Yet, ladanein and EGCG proved to be potent entry
inhibitors, and reports on quercetin anti-HCV activity suggest that it
may in fact be more active on the assembly, than on the replication
step. The HCVcc model now allows to identify anti-HCV molecules in the
context of a complete life cycle and then to pinpoint the inhibited
step, with additional and more specific assays. Importantly, we should
keep in mind that the in vitro inhibition of the enzymatic
activity of a viral protein by a natural compound does not conclusively
demonstrate that this viral protein is the bona fide target of
the compound. Additional binding and crystallization studies are
required to prove the point. For example, in the case of
silymarin/silibinin, high concentrations are required to inhibit NS5B in vitro.
It is possible instead that a cellular protein target could
mechanistically be involved in the antiviral action of these compounds.
More investigations are clearly needed to validate numbers of these
molecules in vivo. As illustrated by silymarin, the
bioavailability is an important point to consider, even for molecules
extracted from ancient herbal medicines. In conclusion, even if we do
not expect that natural molecules may replace the current anti-HCV
therapy, treatments could be more likely supplemented, and perhaps
lightened by adapted diet, limiting their cost.