Chie Aoki

Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species

Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti‐HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti‐HCV activity with 50%‐inhibitory concentrations (IC50) of 20.0 and 8.0 μg/mL, respectively. Through bioactivity‐guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti‐HCV compounds, their IC50 being 8.8, 7.2, 4.6 and 16.4 μg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti‐HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti‐HCV activity, their IC50 being 2.5 and 6.2 μg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti‐HCV activity, with an IC50 of 3.7 μg/mL. Time‐of‐addition analysis revealed that all Glycyrrhiza‐derived anti‐HCV compounds tested in this study act at the post‐entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.

Antiviral activities of Indonesian medicinal plants in the East Java region against hepatitis C virus

BackgroundHepatitis C virus (HCV) is a major cause of liver disease and a potential cause of substantial morbidity and mortality worldwide. The overall prevalence of HCV infection is 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only 50% of the patients infected with HCV genotype 1, and is associated with significant side effects. Therefore, it is still of importance to develop new drugs for treatment of HCV. Antiviral substances obtained from natural products, including medicinal plants, are potentially good targets to study. In this study, we evaluated Indonesian medicinal plants for their anti-HCV activities.MethodsEthanol extracts of 21 samples derived from 17 species of medicinal plants explored in the East Java region were tested. Anti-HCV activities were determined by a cell culture method using Huh7.5 cells and HCV strains of 9 different genotypes (1a to 7a, 1b and 2b).ResultsFour of the 21 samples tested showed antiviral activities against HCV: Toona sureni leaves (TSL) with 50% inhibitory concentrations (IC50) of 13.9 and 2.0 μg/ml against the HCV J6/JFH1-P47 and -P1 strains, respectively, Melicope latifolia leaves (MLL) with IC50 of 3.5 and 2.1 μg/ml, respectively, Melanolepis multiglandulosa stem (MMS) with IC50 of 17.1 and 6.2 μg/ml, respectively, and Ficus fistulosa leaves (FFL) with IC50 of 15.0 and 5.7 μg/ml, respectively. Time-of-addition experiments revealed that TSL and MLL inhibited both at the entry and post-entry steps while MMS and FFL principally at the entry step. TSL and MLL inhibited all of 11 HCV strains of all the genotypes tested to the same extent. On the other hand, FFL showed significantly weaker inhibitory activities against the HCV genotype 1a strain, and MMS against the HCV strains of genotypes 2b and 7a to a lesser extent, compared to the other HCV genotypes.ConclusionsEthanol extracts of TSL, MLL, MMS and FFL showed antiviral activities against all the HCV genotypes tested with the exception that some genotype(s) showed significant resistance to FFL and to MMS to a lesser extent. These plant extracts may be good candidates for the development of anti-HCV drugs.

Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites, pheophorbide a and pyropheophorbide a, against hepatitis C virus

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo‐Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n‐hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti‐HCV activities with 50%‐inhibitory concentrations (IC50) of 20.6, 6.1, and 6.6 μg/mL, respectively. Bioactivity‐guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti‐HCV compound present in the extracts (IC50  = 0.3 μg/mL). It was also found that pyropheophorbide a possesses anti‐HCV activity (IC50 = 0.2 μg/mL). The 50%‐cytotoxic concentrations (CC50) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti‐HCV activities. Time‐of‐addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post‐entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.

Inhibition of hepatitis C virus replication by chalepin and pseudane IX isolated from Ruta angustifolia leaves

Hepatitis C virus (HCV) infection is highly prevalent among global populations, with an estimated number of infected patients being 170 million. Approximately 70-80% of patients acutely infected with HCV will progress to chronic liver disease, such as liver cirrhosis and hepatocellular carcinoma, which is a substantial cause of morbidity and mortality worldwide. New therapies for HCV infection have been developed, however, the therapeutic efficacies still need to be improved. Medicinal plants are promising sources for antivirals against HCV. A variety of plants have been tested and proven to be beneficial as antiviral drug candidates against HCV. In this study, we examined extracts, their subfractions and isolated compounds of Ruta angustifolia leaves for antiviral activities against HCV in cell culture. We isolated six compounds, chalepin, scopoletin, γ-fagarine, arborinine, kokusaginine and pseudane IX. Among them, chalepin and pseudane IX showed strong anti-HCV activities with 50% inhibitory concentration (IC₅₀) of 1.7 ± 0.5 and 1.4 ± 0.2 μg/ml, respectively, without apparent cytotoxicity. Their anti-HCV activities were stronger than that of ribavirin (2.8 ± 0.4 μg/ml), which has been widely used for the treatment of HCV infection. Mode-of-action analyses revealed that chalepin and pseudane IX inhibited HCV at the post-entry step and decreased the levels of HCV RNA replication and viral protein synthesis. We also observed that arborinine, kokusaginine and γ-fagarine possessed moderate levels of anti-HCV activities with IC₅₀ values being 6.4 ± 0.7, 6.4 ± 1.6 and 20.4 ± 0.4 μg/ml, respectively, whereas scopoletin did not exert significant anti-HCV activities at 30 μg/ml.

Induction of cell-mediated immune responses in mice by DNA vaccines that express hepatitis C virus NS3 mutants lacking serine protease and NTPase/RNA helicase activities.

Effective therapeutic vaccines against virus infection must induce sufficient levels of cell-mediated immune responses against the target viral epitopes and also must avoid concomitant risk factors, such as potential carcinogenic properties. The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) carries a variety of CD4+ and CD8+ T cell epitopes, and induces strong HCV-specific T cell responses, which are correlated with viral clearance and resolution of acute HCV infection. On the other hand, NS3 possesses serine protease and nucleoside triphosphatase (NTPase)/RNA helicase activities, which not only play important roles in viral life cycle but also concomitantly interfere with host defense mechanisms by deregulating normal cellular functions. In this study, we constructed a series of DNA vaccines that express NS3 of HCV. To avoid the potential harm of NS3, we introduced mutations to the catalytic triad of the serine protease (H57A, D81A and S139A) and the NTPase/RNA helicase domain (K210N, F444A, R461Q and W501A) to eliminate the enzymatic activities. Immunization of BALB/c mice with each of the DNA vaccine candidates (pNS3[S139A/K210N], pNS3[S139A/F444A], pNS3[S139A/R461Q] and pNS3[S139A/W501A]) that expresses an NS3 mutant lacking both serine protease and NTPase/helicase activities induced T cell immune responses to the degree comparable to that induced by the wild type NS3 and the NS3/4A complex, as demonstrated by interferon-γ production and cytotoxic T lymphocytes activities against NS3. The present study has demonstrated that plasmids expressing NS3 mutants, NS3(S139A/K210N), NS3(S139A/F444A), NS3(S139A/R461Q) and NS3(S139A/W501A), which lack both serine protease and NTPase/RNA helicase activities, would be good candidates for safe and efficient therapeutic DNA vaccines against HCV infection.

Generation of a recombinant reporter hepatitis C virus useful for the analyses of virus entry, intra-cellular replication and virion production

The lack of a culture system that efficiently produces progeny virus has hampered hepatitis C virus (HCV) research. Recently, the discovery of a novel HCV isolate JFH1 and its chimeric derivative J6/JFH1 has led to the development of an efficient virus productive culture system. To construct an easy monitoring system for the viral life cycle of HCV, we generated bicistronic luciferase reporter virus genomes based on the JFH1 and J6/JFH1 isolates, respectively. Transfection of the J6/JFH1-based reporter genome to Huh7.5 cells produced significantly greater levels of progeny virus than transfection of the JFH1 genome. Furthermore, the expression of dominant-negative Vps4, a key molecule of the endosomal sorting complex required for transport machinery, inhibited the virus production of JFH1, but not that of J6/JFH1. These results may account for the different abilities to produce progeny virus between JFH1 and J6/JFH1. Using the J6/JFH1/Luc system, we showed that the two polyanions heparin and polyvinyl sulfate decreased the infectivity of J6/JFH1/Luc virus in a dose-dependent manner. We also analyzed the function of microRNA on HCV replication and found that miR-34b could affect the replication of HCV. The reporter virus generated in this study will be useful for investigating the nature of the HCV life cycle and for identification of HCV inhibitors.

Anti-hepatitis C virus activity of a crude extract from longan ( Dimocarpus longan Lour.) leaves

Infection with hepatitis C virus (HCV) results in hepatitis C, a disease characterized by chronic infection, cirrhosis, and hepatocellular carcinoma. Currently, the standard therapy is a combination of pegylated interferon-α plus ribavirin with NS3 protease inhibitors. Addition of NS3 protease inhibitors to the standard therapy improves response rates; however, use of NS3 protease inhibitors is also associated with significant adverse effects and an increase in the overall cost of treatment. Therefore, there is a need to develop safe and inexpensive drugs for the treatment of HCV infections. In this study, we examined the antiviral activity of a crude extract from Dimocarpus longan leaves against HCV (genotype 2a strain JFH1). The D. longan crude extract (DL-CE) exhibited anti-HCV activity with a 50% effective concentration (EC50) of 19.4 μg/ml without cytotoxicity. A time-of-addition study demonstrated that DL-CE has anti-HCV activity at both the entry and post-entry steps and markedly blocks the viral entry step through direct virucidal activity with marginal inhibition of virion assembly. Co-treatment of DL-CE with cyclosporine A, an immunosuppressant or telaprevir, an NS3 protease inhibitor, resulted in additive and synergistic antiviral effects, respectively. Our findings suggest that DL-CE may be useful as an add-on therapy candidate for treating HCV infections.

A point mutation at Asn-534 that disrupts a conserved N-glycosylation motif of the E2 glycoprotein of hepatitis C virus markedly enhances the sensitivity to antibody neutralization

The molecular basis of antibody neutralization against hepatitis C virus (HCV) is poorly understood. The E2 glycoprotein of HCV is critically involved in viral infectivity through specific binding to the principal virus receptor component CD81, and is targeted by anti‐HCV neutralizing antibodies. A previous study showed that a mutation at position 534 (N534H) within the sixth N‐glycosylation motif of E2 of the J6/JFH1 strain of HCV genotype 2a (HCV‐2a) was responsible for more efficient access of E2 to CD81 so that the mutant virus could infect the target cells more efficiently. The purpose of this study was to analyze the sensitivity of the parental J6/JFH1, its cell culture‐adapted variant P‐47 possessing 10 amino acid mutations and recombinant viruses with the adaptive mutations to neutralization by anti‐HCV antibodies in sera of HCV‐infected patients. The J6/JFH1 virus was neutralized by antibodies in sera of patients infected with HCV‐2a and ‐1b, with mean 50% neutralization titers being 1:670 and 1:200, respectively (P < 0.00001). On the other hand, the P‐47 variant showed 50‐ to 200‐times higher sensitivity to antibody neutralization than the parental J6/JFH1 without genotype specificity. The N534H mutation, and another one at position 416 (T416A) near the first N‐glycosylation motif to a lesser extent, were shown to be responsible for the enhanced sensitivity to antibody neutralization. The present results suggest that the residues 534, and 416 to a lesser extent, of the E2 glycoprotein are critically involved in the HCV infectivity and antibody neutralization. J. Med. Virol. 84:229–234, 2012.

Antiviral Activity of Indonesian Plants from East Java Region Againts Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. The most recent WHO estimate of the prevalence of HCV infection is 2%, representing 120 million people. Current standard of care is effective in only 50% of the patients, poorly tolerated, and associated with significant side effects and viral resistance. Therefore, a new drugs is needed for the development of complementary and alternative treatment strategies for HCV infection. A variety of medicinal plants have demonstrated antiviral efficacies and some of them possess broad spectrum antiviral activities. In this study, some of Indonesian medicinal plants were evaluated for their anti-HCV activities. Ethanol extracts of 21 samples derived from 19 species of plants that were explored from East Java Region were tested. Anti HCV activities were determined by cell culture method using Huh 7.5 cells and HCV J6/JFH1. The results showed that 6 of 21 samples have potential activity against HCV: Eucalyptus globulus stem (IC5 : 15.1 μg/ml), Toona sureni leaves (IC5 : 11 μg/ml), Melicope vitiflora leaves and stem (IC5 : 9.6 and 15.7 ug/ml, respectively), Melanolepis multiglandulosa stem (IC5 : 15.5 μg/ml) and Ficus fistulosa leaves (IC5 5 : 23.0 μg/ml). These plant extracts may be good candidates for the development of anti-HCV drugs.