Identification of an Oligostilbene, Vaticanol B, from Dryobalanops aromatica Leaves as an Antiviral Compound against the Hepatitis C Virus.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Although current medications using direct-acting antivirals (DAAs) are highly effective and well-tolerated for treating patients with chronic HCV, high prices and the existence of DAA-resistant variants hamper treatment. There is thus a need for easily accessible antivirals with different mechanisms of action. During the screening of Indonesian medicinal plants for anti-HCV activity, we found that a crude extract of Dryobalanops aromatica leaves possessed strong antiviral activity against HCV. Bioassay-guided purification identified an oligostilbene, vaticanol B, as an active compound responsible for the anti-HCV activity. Vaticanol B inhibited HCV infection in a dose-dependent manner with 50% effective and cytotoxic concentrations of 3.6 and 559.5 µg/mL, respectively (Selectivity Index: 155.4). A time-of-addition study revealed that the infectivity of HCV virions was largely lost upon vaticanol B pretreatment. Also, the addition of vaticanol B following viral entry slightly but significantly suppressed HCV replication and HCV protein expression in HCV-infected and a subgenomic HCV replicon cells. Thus, the results clearly demonstrated that vaticanol B acted mainly on the viral entry step, while acting weakly on the post-entry step as well. Furthermore, co-treatment of the HCV NS5A inhibitor daclatasvir with vaticanol B increased the anti-HCV effect. Collectively, the present study has identified a plant-derived oligostilbene, vaticanol B, as a novel anti-HCV compound.

Acacia mangium: A promising plant for isolating anti-hepatitis C virus agents.

Background: Medicinal plants are potential resources for isolating drug candidates. Various plants have been reported to possess pharmacological effects including anti-hepatitis C activities. The current study examined the anti-hepatitis C virus (HCV) activities of Acacia mangium extracts in solvents with various polarities and further evaluated the mechanism of action of the extracts using Western blotting and combination treatment models. Methods: The leaves of A. mangium were extracted in two phases, first in ethanol and then in solvents with different polarities (n-hexane, dichloromethane, and methanol). HCV-infected Huh7it-1 cells were treated with the extracts at concentrations of 0.01, 0.1, 1, 10, 50, and 100 µg/mL. Results: The results revealed the strong anti-HCV activities of the extracts. The 50% inhibition concentrations (IC 50s) of the ethanol, n-hexane, dichloromethane and methanol extracts were of 4.6 ± 0.3, 2.9 ± 0.2, 0.2 ± 0.3, and 2.8 ± 0.2 µg/mL, respectively, and no cytotoxic effect was detected. These extracts displayed stronger effects than the positive control ribavirin. The mode of action of the ethanol extract was evaluated at 30 µg/mL, revealing that the inhibitory effect was stronger on the post-entry step than on the entry step. Western blotting revealed that the extracts decreased NS3 protein expression, indicating that virus replication was suppressed. Further evaluation illustrated that combined treatment with the ethanol extract enhanced the anti-viral activity of simeprevir. Conclusions: These results indicated that A. mangium leaves could represent sources of anti-HCV agents.

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 Cananga odorata Against Hepatitis B Virus.

Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Current therapeutic drugs for chronic hepatitis B using pegylated interferons and nucleos(t)ide analogs have limited efficacy. Therefore, the development of novel and safe antivirals is required. Natural products including medicinal plants produce complex and structurally diverse compounds, some of which offer suitable targets for antiviral screening studies. In the present study, we screened various crude extracts from Indonesian plants for anti-HBV activity by determining their effects on the production of extracellular HBV DNA in Hep38.7-Tet cells and HBV entry onto a HBV-susceptible cell line, HepG2-NTCP, with the following results: (1) In Hep38.7-Tet cells, Cananga odorata exhibited the highest anti-HBV activity with a 50% inhibitory concentration (IC50) of 56.5 µg/ml and 50% cytotoxic concentration (CC50) of 540.2 µg/ml (Selectivity Index: 9.6). (2) The treatment of HepG2-NTCP cells with Cassia fistula, C. odorata, and Melastoma malabathricum at concentrations of 100 µg/ml lowered the levels of HBsAg production to 51.2%, 58.0%, and 40.1%, respectively, compared to untreated controls, and IC50 and CC50 values of C. odorata were 142.9 µg/ml and >400 µg/ml. In conclusion, the C. odorata extract could be a good candidate for the development of anti-HBV drugs.