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.

Cratoxylum sumatranum stem bark exhibited antimalarial activity by Lactate Dehydrogenase (LDH) assay.

OBJECTIVES: The antimalarial drug resistance is an obstacle in the effort to overcome malaria. The new alternative antimalarial drug became in great attention of urgent need. Current antimalarial drugs were derived from plants. Therefore, the plant is considering a potential source of new drugs. Cratoxylum sumatranum belongs to the Hypericaceae family contain xanthones and phenolic compounds, which was reported for their antimalarial activities. This study aims to determine the antimalarial activities of C. sumatranum extracts and fractions. METHODS: Cratoxylum  sumatranum stem bark (BP14-SB) collected from Balikpapan Botanical Garden in East Kalimantan, Indonesia, was extracted gradually with n-hexane, dichloromethane, and methanol by ultrasonic-assisted extraction method. All extracts were tested against Plasmodium falciparum 3D7 by lactate dehydrogenase (LDH) assay and followed by IC50 determination. The most active extract was further separated and tested for their antimalarial activities. RESULTS: The results showed that dichloromethane stem bark extract (BP14-SB-D) had the strongest inhibition of parasite growth with the IC50 value of 0.44 ± 0.05 µg/mL and moderately toxic with the CC50 value of 29.09 ± 0.05 µg/mL. Further fractionation of BP14-SB-D by open column chromatography using silica gel and gradient hexane-ethyl acetate obtained 12 fractions. LDH assay for these 12 fractions of BP14-SB-D showed that Fraction-6 (IC50 value of 0.19 ± 0.03 µg/mL) was performed the strongest inhibition of parasite growth, compared to other fractions. TLC identification showed that BP14-SB-D contains xanthone. CONCLUSIONS: The dichloromethane extract of C. sumatranum stem bark (BP14-SB-D) and Fraction-6 from this extract exhibited antimalarial activity and the potential to be developed an antimalarial substance.

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.

Anticancer Activity of Methanol Extract of Ficus carica Leaves and Fruits Against Proliferation, Apoptosis, and Necrosis in Huh7it Cells.

The polyphenol plant extracts have previously been demonstrated to act as chemopreventive and anticancer agents. Ficus carica is a rich source of polyphenols, yet its antioxidant and anticancer activities remain poorly characterized. This study aimed to determine the anticancer activity of F carica leaf and fruit extracts by investigating their impact on proliferation, apoptosis, and Huh7it cell necrosis. Leaves and fruits were extracted using methanol, and the phytochemical contents were analyzed using Fourier-transform infrared spectroscopy. The antioxidant activity was measured using the 2,2-diphenyl-1-picrylhydrazyl method. Anticancer activities were examined through MTT (3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay on Huh7it liver cancer cells. The apoptosis and necrosis conditions were examined using Annexin biomarkers V-PI and later analyzed in flow cytometry. F carica leaves and fruit examined were found to have strong antioxidant activities with IC50 values of 7.9875 µg/mL and 13.402 µg/mL, respectively. MTT assay results indicated F carica leaves and fruit had IC50 values >653 µg/mL and >2000 µg/mL, respectively. The flow cytometry analysis indicated a higher percentage of Huh7it apoptosis and necrosis in leaf extracts compared with fruit extracts. The difference in anticancer activity was attributed to differing compounds present in each extract.

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.