Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Virus RNA^*

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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. 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.