Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Virus RNA*
UNcensored at the SOURCE ~ See Also
1. Masahiko Takeshita‡, 2. Yo-ichi Ishida§, 3. Ena Akamatsu§, 4. Yusuke Ohmori¶,
5. Masayuki Sudoh¶, 6. Hirofumi Uto?, 7. Hirohito Tsubouchi? and 8. Hiroaki Kataoka**,1
+ Author Affiliations
1. From the ‡Research Division, Minami Nippon Dairy Co-op Co., Ltd., Miyazaki 885-0073,
2. the §Miyazaki Prefectural Industrial Support Foundation, Miyazaki 880-0303,
3. the ¶Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., Kanagawa 247-8530,
4. the ?Department of Digestive Disease and
Life-style Related Disease, Health Research Human and Environmental
Sciences, Kagoshima University, Graduate School of Medicine and Dental
Sciences, Kagoshima 890-8520, and
5. the **Section of Oncopathology and Regenerative Biology,
Department of Pathology, Faculty of Medicine, University of Miyazaki,
Miyazaki 889-1692, Japan
- 1 To whom correspondence should be addressed:
Section of Oncopathology and Regenerative Biology, Dept. of
Pathology, Faculty of Medicine, University of Miyazaki, 5200 Kihara,
Kiyotake, Miyazaki 889-1692, Japan.
Tel.: 81-985-85-2809; Fax: 81-985-85-6003; E-mail: mejina@fc.miyazaki-u.ac.jp.
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic liver
disease such as chronic hepatitis, cirrhosis, and hepatocellular
carcinoma. While searching for new natural anti-HCV agents in
agricultural products, we found a potent inhibitor of HCV RNA
expression in extracts of blueberry leaves when examined in an HCV
subgenomic replicon cell culture system. This activity was observed in
a methanol extract fraction of blueberry leaves and was purified by
repeated fractionations in reversed-phase high-performance liquid
chromatography. The final purified fraction showed a 63-fold increase
in specific activity compared with the initial methanol extracts and
was composed only of carbon, hydrogen, and oxygen. Liquid
chromatography/mass-ion trap-time of flight analysis and butanol-HCl
hydrolysis analysis of the purified fraction revealed that the
blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore,
structural analysis using acid thiolysis indicated that the mean degree
of polymerization of the purified proanthocyanidin was 7.7, consisting
predominantly of epicatechin. Proanthocyanidin with a polymerization
degree of 8 to 9 showed the greatest potency at inhibiting the
expression of subgenomic HCV RNA. Purified proanthocyanidin showed
dose-dependent inhibition of expression of the neomycin-resistant gene
and the NS-3 protein gene in the HCV subgenome in replicon cells. While
characterizing the mechanism by which proanthocyanidin inhibited HCV
subgenome expression, we found that heterogeneous nuclear
ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived
proanthocyanidin and was indispensable for HCV subgenome expression in
replicon cells. These data suggest that proanthocyanidin isolated from
blueberry leaves may have potential usefulness as an anti-HCV compound
by inhibiting viral replication.
Footnotes
- ?*
This study was supported by a grant from the Collaboration of Regional
Entities for the Advancement of Technological Excellence (CREATE) from
Japan Science and Technology Agency.
- ? The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.
- ?2 The abbreviations used are:
HCV - hepatitis C virus
hnRNP - heterogeneous nuclear ribonucleoprotein
HPLC - high-performance liquid chromatography
PDA - photodiode array
EPMA - electron probe micro-analysis
LC/MS-IT-TOF - liquid chromatography / mass spectrometry-ion trap-time of flight
APCI - atmospheric pressure chemical ionization
mDP - mean degree of polymerization
IC50 - concentration required for 50% inhibition
CC50 - concentration required for 50% cytotoxicity
eIF3 - eukaryotic translation initiation factor 3
CHAPS - 3-[(3-cholamidopropryl)dimethylammonio]-1-propane sulfonate
IRES - internal ribosome entry site
DIGE - differential gel electrophoresis.
- Received April 6, 2009.
- Revision received June 12, 2009.