An in vitro study of an Artocarpus heterophyllus substance as a hepatitis C antiviral and its combination with current anti-HCV drugs.

BACKGROUND: Current therapy of chronic hepatitis C virus (HCV) with direct-acting antivirals (DAAs) has dramatically improved the sustained virologic response (SVR) of affected patients; however, treatment with DAAs remains expensive, and drug-resistant HCV variants remain a threat. As a result, there is still a need to continue to develop affordable and effective drugs for the treatment of HCV. Previously, we have demonstrated that a crude extract from Artocarpus heterophyllus leaves is a potential anti-HCV candidate. In this study, we have further purified this crude extract, examined which sub-fraction possesses the highest antiviral activity, and then explored its efficacy at different HCV life cycle stages. We also assessed synergistic antiviral effects between the A. heterophyllus extract and commercially available anti-HCV drugs. METHODS: We used vacuum liquid chromatography (VLC) and high-performance liquid chromatography (HPLC) to fractionate a dichloromethane extract of A. heterophyllus leaves. We then examined the anti-HCV activity of the fractions using HCV genotype 2a, JFH1a; the antiviral mode of action was determined by exploring adding the treatments at different times. We examined the antiviral effects on the viral entry stage through a virucidal activity test, viral adsorption examination, and pretreatment of cells with the drug. The effects on the post-viral entry stage were determined by the levels of HCV protein expression and HCV RNA expression in infected cells. RESULTS: Through activity guided purification, we identified the sub-fraction FR3T3 as possessing the most robust anti-HCV activity with an IC50 value of 4.7 ± 1.0 µg/mL. Mode-of-action analysis revealed that FR3T3 inhibited post-viral entry stages such as HCV NS3 protein expression and HCV RNA replication with marginal effects on the viral entry stage. Thin-layer Chromatography (TLC) indicated that FR3T3 contained terpenoids and chlorophyll-related compounds. We also found a synergistic antiviral activity when the DCM extract of A. heterohyllus was used in combination therapy with commercial anti-HCV drugs; Ribavirin, Simeprevir, Cyclosporin A. CONCLUSIONS: The extract of A. heterophyllus and its sub-fraction, FR3T3, presented here have anti-HCV activities and could be candidate drugs for add-on-therapy for treatment of chronic HCV infections.

Alkaloid and benzopyran compounds of Melicope latifolia fruit exhibit anti-hepatitis C virus activities.

BACKGROUND: New agents for developing alternative or complementary medicine to treat the hepatitis C virus (HCV) are still needed due to high rates of HCV infection globally and the current limitations of available treatments. Treatment of HCV with a combination of direct acting antivirals have been shown to be approximately 90% effective but will be limited in the future due to the emergence of drug resistance and high cost. The leaves of Melicope latifolia have previously been reported to have anti-HCV activity and are a potential source of bioactive compounds for future novel drug development. This study aimed to evaluate the efficacy of the extract of M. latifolia fruit to treat HCV and to isolate its active compounds. METHOD: M. latifolia fruit was extracted using methanol and purified using vacuum liquid chromatography (VLC) and Radial Chromatography. The anti-HCV activity was analyzed using cell culture lines Huh7it-1 and JFH1 (genotype 2a). Time-of-addition and immunoblotting studies were performed to identify the mode of action of the isolated active compounds. The structures of the active compounds were determined using nuclear magnetic resonance (NMR) spectra, UV, IR, and Mass Spectra. RESULTS: Six known compounds were isolated from M. latifolia fruit: O-methyloktadrenolon, alloevodionol, isopimpinellin, alloxanthoxyletin, methylevodionol, and N-methylflindersine. N-methylflidersine was the most active compound with IC50 value of 3.8 µg/ml while methylevodionol, isopimpinellin, and alloevodionol were less active. O-methyloktadrenolon and alloxanthoxyletin were moderately active with IC50 values of 10.9 and 21.72 µg/ml, respectively. N-methylflidersine decreased level of HCV NS3 protein expression in the cells. CONCLUSION: The alkaloid compound, N-methylflindersine which was isolated from M. latifolia possesses anti-HCV activity through post-entry inhibition and suppressed NS3 protein expression.

Antiviral activity of the dichloromethane extracts from Artocarpus heterophyllus leaves against hepatitis C virus

Objective: To determine anti-viral activities of three Artocarpus species: Artocarpus altilis, Artocarpus camansi, and Artocarpus heterophyllus (A. heterophyllus) against Hepatitis C Virus (HCV). Methods: Antiviral activities of the crude extracts were examined by cell culture method using Huh7it-1 cells and HCV genotype 2a strain JFH1. The mode of action for anti-HCV activities was determined by time-of-addition experiments. The effect on HCV RNA replication and HCV accumulation in cells were analyzed by quantitative reverse transcription-PCR and western blotting, respectively. Results: The dichloromethane (DCM) extract of A. heterophyllus exhibited strong anti-HCV activity with an inhibitory concentration (IC50) value of (1.5 ± 0.6)μg/mL without obvious toxicity. The DCM extracts from Artocarpus altilis and Artocarpus camansi showed moderate anti-HCV activities with IC50 values being (6.5 ± 0.3) μg/mL and (9.7 ± 1.1) μg/mL, respectively. A time-of-addition studies showed that DCM extract from A. heterophyllus inhibited viral entry process though a direct virucidal activity and targeting host cells. HCV RNA replication and HCV protein expression were slightly reduced by the DCM treatment at high concentration. Conclusions: The DCM extract from A. heterophyllus is a good candidate to develop an antiviral agent to prevent HCV grant reinfection following liver transplantation.

High rate of seronegative HCV infection in HIV-positive patients.

Co-infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) is a significant global health problem. The two viruses are transmitted with high efficacy via blood-to-blood contact, mainly intravenous drug use (IVDU), whereas HCV is less easily transmitted sexually. Antibody testing is the main screening method for HCV infection, although it may not be the optimal option for HIV infection. The aim of this study was to investigate HCV infection in HIV-positive patients, with and without a detectable anti-HCV antibody response. A total of 187 plasma samples were obtained from HIV-positive patients in Surabaya, Indonesia and examined for anti-HCV [HCV enzyme immunoassay (EIA) 3.0], HCV genotype/subtype [reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting a part of NS5B/5'UTR followed by sequencing] and HCV viral load (quantitative RT-PCR). A total of 119 patients (63.6%) were found to be anti-HCV-positive and, among these, HCV RNA was detected in 73 (61.3%), with HCV-1a as the predominant subtype (31.5%). Of the 68 anti-HCV-negative samples, HCV RNA was detected in 26/68 (38.2%) mostly as the HCV-3a subtype (50%). High HCV viral loads were more common among the HCV-seropositive patients. The HCV-seropositive samples with detected HCV RNA were mostly obtained from HIV-positive patients with parenteral transmission (IVDU) (76.7%); however, the HCV-seronegative samples with detected HCV RNA were mostly from patients who had acquired HCV through heterosexual transmission (61.5%). In conclusion, HIV-positive patients were at high risk of becoming co-infected with HCV and several remained HCV-seronegative. Furthermore, there may exist differences in HCV seropositivity and subtypes between HIV-positive patients who acquired HCV sexually and those who acquired HCV parenterally.

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 (IC(50)) 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 IC(50) 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 IC(50) being 2.5 and 6.2 µg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti-HCV activity, with an IC(50) 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.

BACKGROUND: Hepatitis 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. METHODS: Ethanol 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). RESULTS: Four 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. CONCLUSIONS: Ethanol 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.

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.