Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Hepatitis C Virus RNA

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. 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. Hepatitis C virus (HCV) 2The abbreviations used are: HCVhepatitis C virushnRNPheterogeneous nuclear ribonucleoproteinHPLChigh-performance liquid chromatographyPDAphotodiode arrayEPMAelectron probe micro-analysisLC/MS-IT-TOFliquid chromatography/mass spectrometry-ion trap-time of flightAPCIatmospheric pressure chemical ionizationmDPmean degree of polymerizationIC50concentration required for 50% inhibitionCC50concentration required for 50% cytotoxicityeIF3eukaryotic translation initiation factor 3CHAPS3-(3-cholamidopropryl)dimethylammonio-1-propane sulfonateIRESinternal ribosome entry siteDIGEdifferential gel electrophoresis. is often associated with the development of chronic liver diseases. Infection by HCV causes chronic hepatitis at high rates and finally results in liver cirrhosis and subsequent occurrence of hepatocellular carcinoma (1Choo Q.L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Science. 1989; 244: 359-362Crossref PubMed Scopus (6271) Google Scholar, 2Seeff L.B. Am. J. Med. 1999; 107: 10S-15SAbstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 3Di Bisceglie A.M. Order S.E. Klein J.L. Waggoner J.G. Sjogren M.H. Kuo G. Houghton M. Choo Q.L. Hoofnagle J.H. Am. J. Gastroenterol. 1991; 86: 335-338PubMed Google Scholar). The number of people worldwide who are infected by HCV is estimated to be over 200 million with 2 million infections in Japan (4Myles D.C. Curr. Opin. Drug Discov. Devel. 2001; 4: 411-416PubMed Google Scholar). The South Kyushu area of Japan, including Miyazaki prefecture, has a high prevalence of this virus, and it is now recognized as a social problem. There is no vaccine effective for HCV at present. The elimination of HCV may be achieved by a combination of pegylated α-interferon and ribavirin, a broad spectrum antiviral drug (4Myles D.C. Curr. Opin. Drug Discov. Devel. 2001; 4: 411-416PubMed Google Scholar, 5Dillon J.F. J. Viral. Hepat. 2004; 11 (Suppl. 1): 23-27Crossref PubMed Scopus (22) Google Scholar, 6Ni Z.J. Wagman A.S. Curr. Opin. Drug Discov. Devel. 2004; 7: 446-459PubMed Google Scholar). However, virological response to this combination therapy has been reported to be 80% for genotypes 2 and 3, but less than 50% for genotype 1 (7Manns M.P. McHutchison J.G. Gordon S.C. Rustgi V.K. Shiffman M. Reindollar R. Goodman Z.D. Koury K. Ling M. Albrecht J.K. Lancet. 2001; 358: 958-965Abstract Full Text Full Text PDF PubMed Scopus (5915) Google Scholar, 8Fried M.W. Shiffman M.L. Reddy K.R. Smith C. Marinos G. Gonçales Jr., F.L. Häussinger D. Diago M. Carosi G. Dhumeaux D. Craxi A. Lin A. Hoffman J. Yu J. N. Engl. J. Med. 2002; 347: 975-982Crossref PubMed Scopus (5899) Google Scholar). Moreover, α-interferon is associated with severe side-effects, including leucopenia, thrombocytopenia, depression, fatigue, and flu-like symptoms, and ribavirin is associated with side-effects such as hemolytic anemia (9De Franceschi L. Fattovich G. Turrini F. Ayi K. Brugnara C. Manzato F. Noventa F. Stanzial A.M. Solero P. Corrocher R. Hepatology. 2000; 31: 997-1004Crossref PubMed Scopus (411) Google Scholar). Therefore, establishment of a new modality of treatment without serious adverse effects is still required. hepatitis C virus heterogeneous nuclear ribonucleoprotein high-performance liquid chromatography photodiode array electron probe micro-analysis liquid chromatography/mass spectrometry-ion trap-time of flight atmospheric pressure chemical ionization mean degree of polymerization concentration required for 50% inhibition concentration required for 50% cytotoxicity eukaryotic translation initiation factor 3 3-(3-cholamidopropryl)dimethylammonio-1-propane sulfonate internal ribosome entry site differential gel electrophoresis. Considering the prolonged period (20–30 years) required for development of liver cirrhosis and hepatocellular carcinoma in individuals infected with HCV, we speculated that progression of the disease might be influenced by daily diet. Our research project focuses on the daily use of agricultural products that could cure or reduce the risk of disease progression by HCV. Thus, we screened local agricultural products (1700 samples from 283 species) for their suppressive activity against HCV subgenome expression using an HCV replicon cell system. We found a significant suppressive activity in extracts of blueberry leaves. Blueberries are classified in the genus Vaccinium, and the species are native only to North America. Blueberry leaves have high quinic acid and chlorogenic acid contents and also significant flavonol glycosides such as rutin. Thus, they are high in antioxidant activity. In our subsequent screening studies using various kinds of blueberry species, the most potent activity was observed in the leaf of rabbit-eye blueberry (Vaccinium virgatum Aiton), which is cultivated in southern areas of Japan. In this study, extracts of rabbit-eye blueberry leaves were used in an effort to purify and identify the compound responsible for inhibition of the expression of subgenomic HCV RNA. We identified oligomeric proanthocyanidin with mean degree of polymerization (mDP) around eight as an inhibitor of HCV subgenome expression. We also analyzed cellular that have affinity to the oligomeric proanthocyanidin in HCV replicon and identified heterogeneous nuclear ribonucleoprotein A2/B1 as of in the inhibition of HCV subgenome expression. from leaves of rabbit-eye blueberry virgatum was by of the was with of methanol at with for and the was The methanol extract was with of by for and the and were The was in in and The was with of and methanol to a and the was and with of to the The was and The was also and HCV activity was in the the in the fraction activity against HCV RNA expression, we of to the of HCV expression suppressive was on a reversed-phase 3 at with at consisting of and was at a of as from to from to by from to the was on a reversed-phase 3 consisting of and was at a of as from to from to by with from to The fraction in of methanol was and the to were The for the was from to from to by with from to of the was the as the In the the was by methanol and with from to and from to from to were In suppressive activity of fraction against HCV RNA expression was using replicon cells. The cell an was used K. M. A. A. N. M. M. M. PubMed Scopus Google Scholar). The was from by with subgenomic RNA in which the HCV structural were by the the internal ribosome entry site of the virus and the this HCV the of subgenomic HCV expression could be estimated by activity in the replicon cells. The HCV replicon were in with and were at in a the HCV subgenome expression the replicon in with and were in and for the were with various of samples for of the activity was using the to the and the was by The activity was as the concentration required for 50% inhibition activity was as a number of activity was by by specific activity. 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J. 2002; PubMed Scopus Google with of purified samples in was with of methanol with and of in The was at for and at for 3 or in was also to the to the of and in the The was with methanol and analyzed by 3 was used at as was at The was from to from to by from to and of the to initial the of thiolysis and to was also in a and their by thiolysis of were used as a The was by the of of in which is of by proanthocyanidin from blueberry was with of for and the was This was repeated to the from the by in of for The were with The of leaves was with of methanol for and the was This was repeated and the methanol extracts were by at and finally in of The methanol extract was in of methanol and on a was using the of fraction of methanol fraction of methanol fraction of The of fraction indicated that fraction was composed of quinic chlorogenic and flavonol glycosides such as rutin. of proanthocyanidin from to as analyzed by of proanthocyanidin that were or In the was from replicon were using were at for on and by gel and to a The was with a probe of the of the were with and the was by internal RNA probe was replicon were and cellular were with inhibitor The samples were by using gel The were to an The was with a for used were and and and eukaryotic translation initiation factor 3 and anti-HCV NS-3 K. M. A. A. N. M. M. M. PubMed Scopus Google Scholar). The were with or or and by using as Purified blueberry leaf-derived proanthocyanidin or was with to the HCV replicon were with and inhibitor The protein extract was to the and at with The were for 1 and the was with the The were by in with at for The was with an and the was by the concentration was by the using as the was using and with a of the reported R. J. R. J. F. M. 2001; PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar). of gel were using a and in of 2 and of of or were at for The was by with for by of an of the with and gel was to the The samples were to for in the The was by using an was on a were with and were with and In this study, from or were with a of of samples was with and used as a for of as R. PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar). of was by and with of were from the gel and with extract was a of acid and to for which the were with were using in and The data was in an to the with in the data from the of and were from of A2/B1 was by The replicon were with using to the the were We screened 283 species of local agricultural products for their suppressive activity against the expression of subgenomic HCV RNA using an HCV replicon cell and found significant suppressive activity in the leaves of the blueberry (Vaccinium virgatum Aiton), of of and of of extracts of blueberry leaves the Therefore, we purified a compound from blueberry leaves that inhibited expression of subgenomic HCV RNA in replicon cells. is in and a of the is in of from the of methanol extracts was The of the methanol extracts was The activity was in the fraction in which the was The specific activity of was than that of the initial methanol extracts and the of the activity that an been of HCV subgenome expression activity in blueberry expression, in a new The fraction was to a subsequent in which a of was The data indicated that a activity around of with results the of HCV subgenome expression in the fraction purify the most we the at and the fraction at repeated we of fraction from of methanol The of this fraction for HCV RNA expression was a specific activity than that of the initial methanol extracts In the we as from to by of from to fraction was from to and with an In the we and with methanol of The fraction was from to and finally of with a The for HCV RNA expression of was with a 63-fold increase in specific activity to the initial methanol extracts We also the on replicon cells. 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The structural analysis of the HCV inhibitor proanthocyanidin from blueberry leaves is in The of proanthocyanidin in this fraction was estimated to be the of was is indicated as in results of purified fraction from blueberry consisting of and consisting of and consisting of and in a new the purified HCV proanthocyanidin of blueberry leaf was with 7.7, we the polymerization was required for activity. the of such as and of which were of and also of the were by HCV replicon These and the of activity of of proanthocyanidin on expression of HCV subgenome in replicon or expression, proanthocyanidin from blueberry leaf in a new We the degree of polymerization of proanthocyanidin the The fraction of was by the of as The of HCV RNA expression of this fraction was activity than the methanol on a was analyzed by and thiolysis to the and of proanthocyanidin the blueberry leaf-derived with were for HCV activity. The activity of blueberry leaf proanthocyanidin was on the polymerization and the activity was observed at a polymerization of to 9 In our HCV RNA expression in replicon was as activity. Thus, the observed activity may have from inhibition of by proanthocyanidin. Therefore, we examined the of the purified proanthocyanidin on the expression of the neomycin-resistant gene and the protein of which were in the HCV subgenome of replicon cells. The purified blueberry proanthocyanidin the expression of the neomycin-resistant gene and also the of protein in a that the proanthocyanidin purified from blueberry leaves in the expression of HCV subgenome in the replicon the mechanism the of HCV RNA expression by we identified affinity to the purified proanthocyanidin from blueberry leaves. 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PubMed Scopus Google Scholar). Moreover, identified have been reported to be associated with HCV RNA such as and J.H. J. PubMed Google Scholar, D. D.C. J. 2004; PubMed Scopus Google Scholar, J.H. K. 2004; PubMed Scopus Google Scholar, D. Full Text Full Text PDF PubMed Scopus Google Scholar, J.H. J. PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar, J.H. PubMed Scopus Google Scholar). These results that proanthocyanidin may cellular such as and the and HCV subgenome expression, we examined the effects of of we and which are to be in of M. A.M. G. D. PubMed Scopus Google Scholar). However, of the activity in replicon cells. we A2/B1 the activity of HCV subgenomic replicon and this was using kinds of suppressive were also by such as and The HCV infection is a major cause of chronic liver which results in liver such as cirrhosis and hepatocellular carcinoma. extract from rabbit-eye blueberry virgatum leaves significant activity against HCV RNA expression when analyzed in HCV subgenomic replicon cells. In this study, we to purify a compound that HCV subgenome expression from the blueberry leaves. The final purified was identified as and it was effective at that are of the in replicon cells. The of the proanthocyanidin in purified anti-HCV expression fraction was with a high of as the analysis indicated that the blueberry leaf-derived proanthocyanidin with a degree of polymerization of the activity. the purified proanthocyanidin from blueberry leaf extracts the expression of the gene and the NS-3 protein gene in HCV subgenome replicon in a dose-dependent These data suggest the potential of blueberry leaf proanthocyanidin for the treatment of HCV Proanthocyanidin is a that polymerization of than of such as and There are in in which the has of or and the has of and The and Google Scholar). were as and are in various and to such as and Am. J. Scholar, F. J. 2001; 7: Scopus Google Scholar). There are a number of and that with and their has been in the and proanthocyanidin in the of the PubMed Scopus Google Scholar). from is a natural in the and and Med. PubMed Scopus Google Scholar, Med. 2001; Google which have been to against and virus J.H. PubMed Scopus Google Scholar, L.R. 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