Functional foods effective for hepatitis C: Identification of oligomeric proanthocyanidin and its action mechanism

Abstract

Hepatitis C virus (HCV) is a major cause of viral hepatitis and currently infects approximately 170 million people worldwide. An infection by HCV causes high rates of chronic hepatitis (> 75%) and progresses to liver cirrhosis and hepatocellular carcinoma ultimately. HCV can be eliminated by a combination of pegylated α-interferon and the broad-spectrum antiviral drug ribavirin; however, this treatment is still associated with poor efficacy and tolerability and is often accompanied by serious side-effects. While some novel direct-acting antivirals against HCV have been developed recently, high medical costs limit the access to the therapy in cost-sensitive countries. To search for new natural anti-HCV agents, we screened local agricultural products for their suppressive activities against HCV replication using the HCV replicon cell system in vitro. We found a potent inhibitor of HCV RNA expression in the extracts of blueberry leaves and then identified oligomeric proanthocyanidin as the active ingredient. Further investigations into the action mechanism of oligomeric proanthocyanidin suggested that it is an inhibitor of heterogeneous nuclear ribonucleoproteins (hnRNPs) such as hnRNP A2/B1. In this review, we presented an overview of functional foods and ingredients efficient for HCV infection, the chemical structural characteristics of oligomeric proanthocyanidin, and its action mechanism.

Full text

World Journal of
Hepatology
World J Hepatol 2014 December 27; 6(12): 830-938
Published by Baishideng Publishing Group Inc
ISSN 1948-5182 (online)

Contents Monthly Volume 6 Number 12 December 27, 2014
December 27, 2014
|
Volume 6
|
Issue 12
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WJH
|
www.wjgnet.com I
830 Role of anti-angiogenesis therapy in the management of hepatocellular
carcinoma: The jury is still out
Sun H, Zhu MS, Wu WR, Shi XD, Xu LB
836 Role of hepatectomy for recurrent or initially unresectable hepatocellular
carcinoma
Kishi Y, Shimada K, Nara S, Esaki M, Kosuge T
844 Transarterial chemoembolization for hepatocellular carcinoma: A review of
techniques
Imai N, Ishigami M, Ishizu Y, Kuzuya T, Honda T, Hayashi K, Hirooka Y, Goto H
851 How did hepatitis B virus effect the host genome in the last decade?
Ozkal-Baydin P
860 Occult hepatitis B virus infection
Kwak MS, Kim YJ
870 Functional foods effective for hepatitis C: Identification of oligomeric
proanthocyanidin and its action mechanism
Ishida Y, Takeshita M, Kataoka H
880 Involvement of the TAGE-RAGE system in non-alcoholic steatohepatitis:
Novel treatment strategies
Takeuchi M, Takino J, Sakasai-Sakai A, Takata T, Ueda T, Tsutsumi M, Hyogo H,
Yamagishi S
894 Transitions of histopathologic criteria for diagnosis of nonalcoholic fatty liver
disease during the last three decades
Ikura Y
901 Vitamin D deficiency in chronic liver disease
Iruzubieta P, Terán Á, Crespo J, Fábrega E
916 Recombinase polymerase amplification as a promising tool in hepatitis C virus
diagnosis
Zaghloul H, El-shahat M
TOPIC HIGHLIGHT
REVIEW
MINIREVIEWS

Contents World Journal of Hepatology
Volume 6 Number 12 December 27, 2014
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923 Palliative external-beam radiotherapy for bone metastases from hepatocellular
carcinoma
Hayashi S, Tanaka H, Hoshi H
930 Evaluation of hepatocellular carcinoma development in patients with chronic
hepatitis C by EOB-MRI
Nojiri S, Fujiwara K, Shinkai N, Endo M, Joh T
RETROSPECTIVE
STUDY

Contents World Journal of Hepatology
Volume 6 Number 12 December 27, 2014
FLYLEAF
EDITORS FOR
THIS ISSUE
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Responsible Electronic Editor: Su-Qing Liu Proong Editorial Ofce Director: Xiu-Xia Song
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APPENDIX
ABOUT COVER
AIM AND SCOPE
I-V Instructions to authors
Editorial Board Member of
World Journal of Hepatology
, Roberto J Carvalho-
Filho, MD, PhD, Professor, Division of Gastroenterology, Hepatology Section,
Federal University of Sao Paulo, Sao Paulo, Sao Paulo 04023-060, Brazil
World Journal of Hepatology (World J Hepatol, WJH, online ISSN 1948-5182, DOI:
10.4254), is a peer-reviewed open access academic journal that aims to guide clinical
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We encourage authors to submit their manuscripts to WJH. We will give priority
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I-IV Editorial Board
INDEXING/
ABSTRACTING

Yo-ichi Ishida, Masahiko Takeshita, Hiroaki Kataoka
Yo-ichi Ishida, Department of Molecular and Cellular Biochem-
istry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588,
Japan
Masahiko Takeshita, Research Division, Minami Nippon Dairy
Co-op., Ltd., Miyakonojo, Miyazaki 885-0003, Japan
Hiroaki Kataoka, Section of Oncopathology and Regenerative
Biology, Department of Pathology, Faculty of Medicine, Univer-
sity of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan
Author contributions: Ishida Y contributed to the investigation
of the action mechanism of oligomeric proanthocyanidin, drafting
of the manuscript, and literature review; Takeshita M contributed
to the discovery of oligomeric proanthocyanidin as an anti-HCV
agent and approved the final version of this manuscript; Kataoka
H contributed to the management of the study, critically reviewed
and approved the final version of this manuscript.
Supported by The Collaboration of Regional Entities for the
Advancement of Technological Excellence from Japan Science
and Technology Agency
Correspondence to: Hiroaki Kataoka, MD, PhD, Professor,
Section of Oncopathology and Regenerative Biology, Department
of Pathology, Faculty of Medicine, University of Miyazaki, 5200
Kihara, Kiyotake, Miyazaki 889-1692,
Japan. mejina@med.miyazaki-u.ac.jp
Telephone: +81-985-852809 Fax: +81-985-856003
Received: August 26, 2014 Revised: October 3, 2014
Accepted: October 23, 2014
Published online: December 27, 2014
Abstract
Hepatitis C virus (HCV) is a major cause of viral hepa-
titis and currently infects approximately 170 million
people worldwide. An infection by HCV causes high
rates of chronic hepatitis (> 75%) and progresses to
liver cirrhosis and hepatocellular carcinoma ultimately.
HCV can be eliminated by a combination of pegylated
α-interferon and the broad-spectrum antiviral drug rib-
avirin; however, this treatment is still associated with
poor efficacy and tolerability and is often accompanied
by serious side-effects. While some novel direct-acting
antivirals against HCV have been developed recently,
high medical costs limit the access to the therapy in
cost-sensitive countries. To search for new natural an-
ti-HCV agents, we screened local agricultural products
for their suppressive activities against HCV replication
using the HCV replicon cell system
in vitro
. We found a
potent inhibitor of HCV RNA expression in the extracts
of blueberry leaves and then identified oligomeric
proanthocyanidin as the active ingredient. Further in-
vestigations into the action mechanism of oligomeric
proanthocyanidin suggested that it is an inhibitor of
heterogeneous nuclear ribonucleoproteins (hnRNPs)
such as hnRNP A2/B1. In this review, we presented an
overview of functional foods and ingredients efficient
for HCV infection, the chemical structural character-
istics of oligomeric proanthocyanidin, and its action
mechanism.
© 2014 Baishideng Publishing Group Inc. All rights reserved.
Key words: Hepatitis C virus; Blueberry leaves; Func-
tional foods; Oligomeric proanthocyanidin; Heteroge-
neous nuclear ribonucleoproteins
Core tip: An infection by hepatitis C virus (HCV) causes
chronic hepatitis, liver cirrhosis, and hepatocellu-
lar carcinoma. While the combination of pegylated
α-interferon and ribavirin is used for the elimination of
HCV, a new anti-HCV drug is required due to the poor
efficacy and serious side-effects associated with this
combination therapy. We searched for new anti-HCV
agents from natural products and then identified oligo-
meric proanthocyanidin from blueberry leaves. Further
investigations suggested that several heterogeneous
nuclear ribonucleoproteins may be the candidate pro-
teins involved in the proanthocyanidin-mediated inhibi-
tion of HCV subgenomic expression. Oligomeric proan-
thocyanidin isolated from blueberry leaves may have
potential usefulness as an anti-HCV compound.
TOPIC HIGHLIGHT
Submit a Manuscript: http://www.wjgnet.com/esps/
Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx
DOI: 10.4254/wjh.v6.i12.870
870 December 27, 2014
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World J Hepatol 2014 December 27; 6(12): 870-879
ISSN 1948-5182 (online)
© 2014 Baishideng Publishing Group Inc. All rights reserved.
Functional foods effective for hepatitis C: Identification of
oligomeric proanthocyanidin and its action mechanism
WJH 6th Anniversary Special Issues (5): Hepatitis C virus

Ishida Y, Takeshita M, Kataoka H. Functional foods effective for
hepatitis C: Identification of oligomeric proanthocyanidin and its
action mechanism. World J Hepatol
2014; 6(12): 870-879 Avail-
able from: URL: http://www.wjgnet.com/1948-5182/full/v6/
i12/870.htm DOI: http://dx.doi.org/10.4254/wjh.v6.i12.870
INTRODUCTION
Hepatitis C virus (HCV) is a major cause of viral hepati-
tis and currently infects approximately 170 million people
worldwide[1,2]. An infection by HCV causes high rates of
chronic hepatitis (> 75%) and progresses to liver cirrhosis
and hepatocellular carcinoma ultimately[3]. A total of 27%
and 25% of individuals that develop liver cirrhosis and
hepatocellular carcinoma worldwide, respectively, arise in
HCV-infected people[4]. The World Health Organization
reported that between 350000 and 500000 people die
from HCV-related diseases each year. However, there is
no effective vaccine against HCV infection at present.
Currently, the combination of pegylated α-interferon
and a broad spectrum antiviral drug, ribavirin, is used
as the standard therapy for chronic HCV infection[2,5,6] .
However, its option is unfortunately limited by efcacy,
tolerability, and signicant side-effects. Therefore, it had
been required to establish a new therapeutic modality
without serious adverse effects. Recently, direct-acting
antivirals (DAAs) that inhibit HCV-specic proteins have
be clinically investigated[7,8]. For example, boceprevir and
telaprevir are new DAAs that were rst approved by the
United States Food and Drug Administration (FDA) in
2011[9]. DAAs are expected to provide new promising
treatment options in hepatitis C patients; however, at
present, they face difficulties to disseminate worldwide
due to high costs. Therefore, new anti-HCV agents that
are safe, economical, and complementary with present
therapies, are still required.
Since the development of HCV-related liver cirrhosis
and hepatocellular carcinoma requires a prolonged period
(20-30 years), the progression of this disease may be inu-
enced by a diet including dairy products. Interest in func-
tional foods and their ingredients as natural resources for
cancer prevention and treatment is increasing[10,11]. Eating
habits, foods, nutrients contained in them, and other food
constituents play important roles on the development
of several types of cancer and 35% of cancer deaths are
estimated to be possibly related to dietary factors[12]. Poly-
phenols derived from various fruits and vegetables have
recently been suggested to be effective in the prevention
of cancer. The South Kyushu region of Japan, including
the prefecture of Miyazaki, has been recognized as a high
prevalence area of HCV and it emerges as a social issue.
Therefore, attempts were made to identify functional food
ingredients having suppressive activities against HCV rep-
lication as an industry-academia-government collabora-
tion study[13]. By screening of 1700 samples from 283 ag-
ricultural products in Miyazaki prefecture, we found that
oligomeric proanthocyanidin, a polyphenolic ingredient
abundantly contained in the leaves of the blueberry plant,
suppressed the expression of HCV subgenomic RNA in
an HCV replicon cell system[13].
In this review, we presented an overview of func-
tional foods and ingredients efcient for HCV infection,
the chemical structural characteristics of oligomeric pro-
anthocyanidin, and its action mechanism.
HCV LIFE CYCLE AND ANALYTICAL
TOOL
HCV belongs to Hepacivirus genus of the Flaviviridae fam-
ily and has a positive-sense single stranded RNA of 9.6
kb wrapped with enveloped membrane[14]. After their ad-
sorption on the surface of host cells, HCV particles are
internalized into endocytic compartments and viral ge-
nomic RNA is then released into the cytoplasm by fusion
of the viral envelope and cellular membrane. Genomic
RNA serves as mRNA for viral proteins and is translated
into a single polyprotein (3011 amino acids), resulting in
4 structural proteins (Core, E1, E2, and p7) and 6 non-
structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A,
and NS5B) by post-translational processing (Figure 1A).
It also serves as a template for viral genome replication.
Non-translated regions (NTRs), 5’NTR and 3’NTR, are
connected with the HCV polyprotein-coding region, and
modulate viral protein synthesis and genome replication.
The assembly of these viral components occurs on the
endoplasmic reticulum (ER) membrane. Viral proteins
and genomic RNA assemble on the cytoplasmic side of
the membrane and then progeny virions bud into the ER
lumen, followed by their release to the extracellular space.
In the life cycle of HCV, each viral protein functions as
described below[14]. Core is a highly basic protein that
encapsidates HCV genomic RNA. E1 and E2 are glyco-
proteins integrated into the viral envelope. p7 functions
as an ion channel and an antiviral drug, amantadine, is the
p7 ion channel blocker[15]. Importantly, several steps of
HCV infectious process are coordinated by NS proteins.
NS2 and NS3 are a cysteine protease and serine prote-
ase, respectively, that play roles in the post-translational
processing of viral proteins. NS3 serine protease activity
requires NS4A as a cofactor. NS4B and NS5A have been
suggested to serve in viral assembly on the ER membrane
and NS5B is an RNA-dependent RNA polymerase. Many
studies to date have reported that these viral proteins are
associated not only with viral replication, but also patho-
genicity via interactions with various host proteins. The
identication of host proteins associated with the HCV
life cycle is very important for anti-HCV drugs, and the
HCV replicon cell system has contributed significantly
to the development of these drugs[16,17]. This system con-
sists of the human hepatocellular carcinoma line Huh-7
in which the transfected luciferase gene connected with
HCV subgenomic RNA including the downstream cod-
ing regions of NS3 and the expression of HCV subge-
nomic RNA can be quantied by luciferase activity (Figure
1B). It provides a useful tool for HCV drug development
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Ishida Y
et al
. Oligomeric proanthocyanidin suppresses HCV subgenomic replication

and the elucidation of mechanisms underlying HCV
genome replication[17]. We have used this HCV replicon
system to screen functional foods with anti-HCV activity.
THERAPEUTIC OPTIONS FOR CHRONIC
HCV INFECTION
Currently, the combination of pegylated α-interferon and
a broad spectrum antiviral drug, ribavirin, is used as the
standard therapy for chronic HCV infection[2,5,6]. How-
ever, the HCV genotype is an important determinant of
its efficacy and tolerability. Whereas the virological re-
sponse to this combination therapy is more than 70% for
genotypes 2 and 3, it is less than 50% for genotype 1[18-20].
Furthermore, this therapy causes signicant side-effects
such as thrombocytopenia, u-like symptoms, fever, rash,
anorexia, and thyroid dysfunction. Depression and irrita-
bility that are expressed as neuropsychological disorders
during therapy impair quality of life universally. There-
fore, it had been required to establish a new therapeutic
modality without serious adverse effects.
Recently, DAAs that inhibit HCV-specific proteins
have been clinically investigated[7,8]. Two DAAs, bocepre-
vir and telaprevir first came to the HCV drug market
and were approved by FDA in May 2011. Boceprevir
or telaprevir was used as triple therapy with pegylated
α-interferon and ribavirin for hepatitis C patients with
genotype 1[9]. These DAAs are inhibitors against HCV
NS3/4A serine protease and bind covalently with active
site of the enzyme[21-23]. The triple therapy using bocepre-
vir or telaprevir significantly increased the rate of sus-
tained virological response (SVR) for naive or previous
treated hepatitis C patients with HCV genotype 1[24-29].
After that, next generation DAAs, ABT-450/r, simepre-
vir, and faldaprevir, which are also NS3/4A protease
inhibitors, have been reported to have advantages of
their convenience and improved side effects prole[30-32].
Further, daclatasvir and sofosbuvir, which are an NS5A
replication complex inhibitor and a nucleotide analogue
NS5B polymerase inhibitor, respectively, also increased
SVR rate[33-35]. Notably, the combination of these DAAs
only was the highly effective treatment for patients with
HCV genotype 1[36,37] and it is feasible to treat HCV with-
out interferon and ribavirin.
While patients with hepatitis C can be treated by
above mentioned DAAs without signicant side-effects,
it requires high medical costs and limits access to the
therapy in cost-sensitive countries[38]. Of the 20 countries
with the high prevalence of HCV, 12 are categorized as
low or lower-middle income countries[39]. Therefore, new
anti-HCV agents that are safe, economical, and comple-
mentary with present therapies, are still required and we
focus attention on functional foods and their ingredients.
FUNCTIONAL FOOD INGREDIENTS
EFFECTIVE FOR HCV
The development of HCV-related liver cirrhosis and
hepatocellular carcinoma requires a prolonged period
(20-30 years). Therefore, the progression of the disease
and HCV infectivity may be inuenced by a diet includ-
ing dairy products. Functional foods and their ingredients
are known to be capable of modulating various biologi-
cal processes such as apoptosis and have been attracting
interest as natural resources for the prevention and treat-
ment of cancer[10,11,40]. Dietary polyphenols derived from
various fruits and vegetables have been suggested to be
effective in cancer prevention. Although the importance
of functional food ingredients as DAAs against HCV is
not fully recognized, these ndings suggest that they con-
tribute to the elimination of the virus.
Several functional food ingredients have been re-
ported to interfere with different steps of the HCV life
cycle. Epigallocatechin-3-gallate (EGCG) (Figure 2A) and
curcumin (Figure 2B), which are ingredients of green tea
(Camellia sinensis) and the Indian spice turmeric (Cur-
cuma longa), respectively, inhibit the entry of HCV into
host cells[41,42]. Quercetin (Figure 2C), a flavonoid that
is abundantly contained in onions, apples, berries, and
red wine, has been shown to inhibit NS3 protease activ-
ity[43]. Punicalagin (Figure 2D) and its related substance
punicalin from the pomegranate (Punica granatum L.) re-
duced the replication of HCV[44]. Naringenin (Figure 2E)
from the grapefruit (Citrus X paradisi Macfady.) has been
identified as an ingredient that interferes with viral as-
sembly[45,46]. Diosgenin (Figure 2F) and epicatechin (Figure
2G), which are contained in yams (Dioscorea spp.) and
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HCV genomic RNA
5’NTR 3’NTR
Polyprotein-coding region
Translation
Polyprotein (3011 amino acids)
Structural proteins
Post-translational processing
Nonstructural proteins
Core p7 NS4BNS4A
NS2 NS3 NS5A NS5BE1 E2
5’NTR 3’NTR
Luciferase NS3
NS4A NS4B
NS5A NS5B
B
A
Figure 1 Structure of the hepatitis C virus genome and cell system for an-
ti-hepatitis C virus drug discovery. A: HCV genomic RNA and viral proteins.
HCV genomic RNA encodes a single polyprotein of 3011 amino acids. After
being translated, the polyprotein is processed into 4 structural proteins (Core,
E1, E2, and p7) and 6 non-structural (NS) proteins (NS2, NS3, NS4A, NS4B,
NS5A, and NS5B). The polyprotein-coding region is anked by 5’ and 3’NTRs.
Viral RNA also serves as a template for viral genome replication and both NTRs
modulate viral protein synthesis and genome replication; B: The HCV replicon
cell system. Huh-7 cells were transfected with the luciferase gene connected
with HCV subgenomic RNA including the downstream coding regions of NS3.
The expression of HCV subgenomic RNA could be quantied by luciferase ac-
tivity. HCV: Hepatitis C virus; NTRs: Non-translated regions.
Ishida Y
et al
. Oligomeric proanthocyanidin suppresses HCV subgenomic replication

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anum) and consists of at least 7 flavonoid compounds,
was also found to interfere with several steps of HCV
infectious process, such as NS5B polymerase activity and
virus entry and transmission[51]. As shown in Figure 2,
most ingredients are polyphenol compounds and, EGCG
(A), quercetin (C), naringenin (E), and epicatechin (G)
have similar chemical structures. There may be a charac-
teristic structure modulating viral proteins and their asso-
ciations with host proteins.
green tea, respectively, also affect the signal transduction
pathways of host cells and inhibit HCV replication via
the signal transducer and activator of transcription 3 and
cycloxygenase-2 pathways, respectively[47,48]. The finding
that curcumin and quercetin also inhibited HCV replica-
tion by associating with sterol regulatory element binding
protein-1 and heat shock proteins, respectively, indicated
the existence of multifunctional ingredients[49,50]. Silyma-
rin, which is an extract from milk thistle (Silybum mari-
Figure 2 Chemical structure of functional food ingredients with anti-hepatitis C virus activities. A: Epigallocatechin-3-gallate; B: Curcumin; C: Quercetin; D:
Punicalagin; E: Naringenin; F: Diosgenin; G: (-)-epicatechin.
Ishida Y
et al
. Oligomeric proanthocyanidin suppresses HCV subgenomic replication
CH3
CH3
O
O
O
O
HO OH
OH
OH
OH
OH
HO O
O
HO
HO
HO
HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
OH
OH
O
O
OH
OH
OH
OH
HO O
HO
CH3
CH3
O
O
CH3
H
H
H
H
H
A B
C
D E
F G
HO
OH
O
OH
OH
OH
OH
OH
OH
O
O

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Clinically, the supplementation of vitamin group has
been reported to increase SVR rates in chronic hepatitis
C patients who underwent the standard therapy with
pegylated α-interferon and ribavirin[52-54]. Regarding sig-
nificant side-effects of the standard therapy, a tomato-
based functional food abundant in natural antioxidants
alleviated the severity of anemia caused by ribavirin and
improved the tolerance to the drug[55].
OLIGOMERIC PROANTHOCYANIDIN
FROM BLUEBERRY LEAVES HAS
SUPPRESSIVE ACTIVITY AGAINST HCV
SUBGENOME REPLICATION IN VITRO
To identify functional food ingredients effective for
hepatitis C, we comprehensively screened the extracts of
commonly ingested agricultural products (1700 samples
from 283 species) grown in Miyazaki prefecture, Japan
using an HCV replicon cell system[13 ]. Samples having
high antioxidative activities were rst selected irrespective
of edible part or non-edible part, and then the inhibitory
activities against HCV subgenomic RNA replication were
examined using the system. We found that extracts of
blueberry leaves signicantly suppressed the replication.
Furthermore, by comparing the inhibitory activities us-
ing leaves from various kinds of blueberry species, it was
found that the leaves of rabbit-eye blueberry (Vaccinium
virgatum Aiton) had the highest activity[13]. Rabbit-eye
blueberry is cultivated in a region with a warm climate,
such as the southern areas of Japan, including Miyazaki
prefecture. Its leaves have been also reported to be good
sources of polyphenols and natural antioxidants[56].
We identified oligomeric proanthocyanidin as the
blueberry leaf-derived inhibitor of HCV subgenomic
RNA replication[13]. Proanthocyanidin is a polyphenol
and has polymerized structures in which more than two
avan-3-ol units such as catechin (Figure 3A) and epicat-
echin (Figure 2G) are covalently linked. Figure 3B shows
an example of the chemical structure of proanthocyani-
din. Proanthocyanidin possesses two interflavan bonds,
in which the A-type and B-type have two bond linkages
(C4→C8 and O7→C2) and one linkage (C4→C8 or C4
→C6), respectively[57], and both types co-exist in proan-
thocyanidin from the rabbit-eye blueberry plant[13]. While
catechin, epicatechin, EGCG, and dimers such as procy-
anidin B2 did not exhibit inhibitory activity against HCV
subgenomic expression in our experimental system, pro-
anthocyanidin oligomer having polymerization degree of
8 to 9 markedly inhibited this expression[13]. This nding
suggested that the HCV inhibitory activity of oligomeric
proanthocyanidin in the replicon assay may require an
oligomerized structure.
Proanthocyanidins are abundantly contained in vari-
ous plants and foods[58] and contribute to organoleptic
properties such as bitterness and astringency[59]. Proan-
thocyanidin-containing foods and nutritional supplements
are known to have benets in health promotion. United
States Department of Agriculture Database reported
proanthocyanidin contents of various foods, showing
that apple peel, red kidney beans, pinto beans, cacao
beans, cocoa, grape seeds, several nuts (almonds, hazel-
nuts, pecans, and pistachios), sorghum, and cinnamon are
proanthocyanidin-rich[60]. Blueberry fruits are also rela-
tively proanthocyanidin-rich; however, the fruits did not
show signicant HCV inhibitory activity compared to the
leaves (unpublished data). In the fruits, proanthocyanidin
contents of monomer, dimer, trimer, 4-6mer, 7-10mer,
and polymer with degrees of polymerization greater than
10mer are 3.46, 5.71, 4.15, 19.57, 14.55, and 129.05 mg
per 100 g edible portion, respectively[60]. As the inhibitory
activity required the oligomeric structure of proanthocy-
anidin having a polymerization degree of 8 to 9 but not
polymer and fresh blueberry leaf contained 3000-4000
mg proanthocyanidins per 100 g total extracts[13], leaves
but not fruits from blueberry are likely suitable for the
prevention of HCV-related diseases. With regard to the
oral uptake, oligomeric proanthocyanidin seems to elute
off by boiling for cooking as shown with pint beans[60].
Therefore, oligomeric proanthocyanidin from blueberry
HO
OH
OH
OH
OH
O
HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
O
O
O
1
2
3
4
5
6
7
8
9
10
8
n
A
B
Figure 3 Chemical structures of a avan-3-ol and proanthocyanidin. A:
(+)-catechin; B: An example of a procyanidin B-type polymer with an (-)-epicat-
echin based structure.
Ishida Y
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. Oligomeric proanthocyanidin suppresses HCV subgenomic replication

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leaves might be ingested as a hot water extract such as
herbal tea. However, absorption efciency of oligomeric
proanthocyanidin in the intestine may be very low.
Proanthocyanidin has also been reported to possess
anti-viral activity against other viruses, herpes simplex
virus and human immunodeciency virus type 1[61-65]. To
the best of our knowledge, we rst reported that the pro-
anthocyanidin oligomer inhibited the expression of HCV
subgenomic RNA[13]. However, the effects of oligomeric
proanthocyanidin on HCV replication in hepatocytes in
vivo currently remain unknown.
ACTION MECHANISM OF OLIGOMERIC
PROANTHOCYANIDIN IN HCV REPLICON
CELLS
The suppression of HCV subgenomic RNA replica-
tion by oligomeric proanthocyanidin has been attracting
increasing attention. Polyphenolic compounds gener-
ally have high antioxidant activities[10,11,58]. Therefore,
the nonspecic antioxidant activity of polyphenols may
contribute to the suppression of HCV subgenomic RNA
replication by oligomeric proanthocyanidin. However, we
examined other polyphenolic compounds in our HCV
replicon assay, and found that constitutional units such
as catechin and epicatechin did not display suppressive
activity, which requires the oligomerized structure of
proanthocyanidin[13]. While it currently remains unknown
whether proanthocyanidin oligomer can be translocated
within the cells in spite of the structure, the ingredient
has been reported to be absorbed from the digestive
tract[66,67], implying the internalization into cells. Oligo-
meric proanthocyanidin appears to suppress HCV sub-
genomic RNA replication via a specic association with
certain intracellular molecules.
Proteomic approach using two-dimensional differen-
tial gel electrophoresis combined with mass spectrometry
provides a powerful tool to determine the cellular re-
sponse to functional foods[40]. To clarify the action mech-
anism of oligomeric proanthocyanidin in HCV replicon
cells, we performed proteomic analysis of proanthocy-
anidin-binding proteins puried by afnity chromatogra-
phy[13]. Then, cellular proteins from replicon cells having
higher affinity to proanthocyanidin than catechin were
identied by a mass spectrometric analysis, and whether
the proteins identied were associated with HCV RNA
expression was further examined using a siRNA-based
replicon assay (Figure 4). Four heterogeneous nuclear
ribonucleoproteins (hnRNPs), hnRNP A/B, A2/B1, K,
and L, were suggested to be possible cellular binding
proteins of oligomeric proanthocyanidin. While siRNA
targeting hnRNP A/B, K, and L showed weak inhibitory
activities, the knockdown of hnRNP A2/B1 signicantly
suppressed HCV subgenomic replication[13].
HnRNPs comprise a family of RNA-binding proteins
that are involved in diverse RNA-related biological pro-
cesses[68]. They are multifunctional proteins composed
of major and minor hnRNP proteins, and hnRNP A/B,
A2/B1, K, and L that we identified belonged to the
major hnRNPs[69]. Previous studies demonstrated that
these hnRNPs regulated the metabolism of RNA such as
pre-mRNA splicing and transcription[70-76]. For example,
hnRNP A2/B1 was shown to affect the alternative splic-
ing of several tumor suppressors and oncogenes in glio-
blastoma cells[72]. Furthermore, several studies reported
interactions and cooperation between these hnRNPs[77-79].
hnRNP A2 and hnRNP L have also been shown to ex-
ist as a complex and regulate the expression of glucose
transporter-1 by binding to mRNA 3’NTR[80,81].
In the HCV life cycle, hnRNPs are associated with
HCV genome RNA and regulate its replication. hnRNP
A1, which exhibits high homology with hnRNP A2/B1,
was shown to facilitate HCV replication via binding to
the HCV 5’ and 3’NTRs (Figure 1), and the replication
was significantly suppressed by the double knockdown
of hnRNP A1 and hnRNP A2[82]. hnRNP K and hnRNP
L are also NTR-binding proteins[83-85]. Furthermore, all
Affinity chromatography
Two-dimensional
electrophoresis
vs
Differential analysis
Mass spectrometric analysis
siRNA-based replicon assay
Total proteins extracted from HCV replicon cells
Proanthocyanidin-
binding proteins
Catechin-
binding proteins
Proteins having higher affinity to
proanthocyanidin than catechin
hnRNP A2/B1
hnRNP A/B
hnRNP K
hnRNP L
Figure 4 Identification strategy of candidate proteins involved in the
proanthocyanidin-mediated inhibition of hepatitis C virus subgenomic ex-
pression[13]. Total proteins were extracted from hepatitis C virus (HCV) replicon
cells and then proanthocyanidin-binding and catechin-binding proteins were
puried by afnity chromatography using sepharose beads coupled with pro-
anthocyanidin and catechin, respectively. Puried proteins were separated by
two-dimensional electrophoresis followed by detecting spots of proteins having
higher afnity to proanthocyanidin than catechin (arrows). Mass spectrometric
analysis and further screening by a siRNA-based replicon assay showed that
hnRNP A2/B1, A/B, K, and L are candidate proteins involved in the oligomeric
proanthocyanidin-mediated inhibition of HCV subgenomic expression. hnRNP:
Heterogeneous nuclear ribonucleoprotein.
Ishida Y
et al
. Oligomeric proanthocyanidin suppresses HCV subgenomic replication

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the hnRNPs we identied as the target protein candidates
of oligomeric proanthocyanidin were included in HCV
3’NTR-binding proteins[86]. Collectively, these findings
suggested that a complex composed of hnRNP A2/B1,
A/B, K, and L may serve in HCV genome replication
by binding to NTRs and oligomeric proanthocyanidin is
an inhibitor of the replication complex. This possibility
should be addressed in a further study.
CONCLUSION
Currently, a combination of pegylated recombinant in-
terferons and ribavirin is used as the standard therapy
for hepatitis C patients. Recently emerged DAAs are
expected to provide new promising treatment options in
hepatitis C patients. However, their high medical costs
may make difcult to disseminate worldwide. We demon-
strated that extracts of blueberry leaves suppressed HCV
subgenome replication in vitro, and their active ingredient
was oligomeric proanthocyanidin[13]. Investigations into
the underlying action mechanism suggested that proan-
thocyanidin may be an inhibitor of several hnRNPs such
as hnRNP A2/B1[13]. On the other hand, it currently re-
mains unknown whether the oligomeric form of proan-
thocyanidin, which is required for the inhibition of HCV
replication, can be efciently absorbed from the digestive
tract to maintain effective plasma concentrations in vivo.
However, further basic research on the action mechanism
of oligomeric proanthocyanidin against HCV replication
may open ways to develop novel anti-HCV drugs and
supplements for hepatitis C patients worldwide.
ACKNOWLEDGMENTS
We dedicate this work to Mr. Fumiaki Mieno (deceased,
March 19, 2013), who inspired our work in the protec-
tion and exploitation of intellectual property. We thank
Sachiko Tomiyama, Tokoyo Imai, Toshiro Morishita, and
Makoto Kodama (Miyazaki Prefectural Industrial Sup-
port Foundation) for coordinating our study.
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P- Reviewer: Ampuero J, Chuang WL, Conti B,
Hernanda PY, Tijera MFH, Qin JM
S- Editor: Tian YL L- Editor: A E- Editor: Liu SQ
Ishida Y
et al
. Oligomeric proanthocyanidin suppresses HCV subgenomic replication

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