Proanthocyanidin from Blueberry LeavesSuppresses Expression of Subgenomic Hepatitis C Virus RNA*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 Affiliations1.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 1.1 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. AbstractHepatitis 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 virushnRNP ~ heterogeneous nuclear ribonucleoproteinHPLC ~ high-performance liquid chromatographyPDA ~ photodiode arrayEPMA ~ electron probe micro-analysisLC/MS-IT-TOF ~ liquid chromatography/mass spectrometry-ion trap-time of flightAPCI ~ atmospheric pressure chemical ionizationmDP ~ mean degree of polymerizationIC50 ~ concentration required for 50% inhibitionCC50 ~ concentration required for 50% cytotoxicityeIF3 ~ eukaryotic translation initiation factor 3CHAPS ~ 3-[(3-cholamidopropryl)dimethylammonio]-1-propane sulfonateIRES ~ internal ribosome entry siteDIGE ~ differential gel electrophoresis.Received April 6, 2009. Revision received June 12, 2009. Lloyd's Blueberry Extract contains 45% Proanthocyanidin