Eight extremophile plants from Tunisia were screened to find natural products with benefits in human health.These plants were collected in different areas in Tunisia. Their methanolic extracts were evaluated for their total phenolic content and for their antiradical (DPPH), antimicrobial (on 35 bacteria and one yeast), antiviral (hepatitis C virus, HCV) and cytotoxic activity (against WI38 and J774 cell lines). The most active species were subjected to a bioguided fractionation.The screening revealed promising activity for four plants, but two species have both antiradical and antimicrobial activity: Juncus maritimus and Limonium virgatum. The rhizomes extract of J. maritimus showed the highest activity against HCV, a selective antibacterial activity against Streptococcus dysgalactiae, and a moderate antiradical activity which is due to luteolin isolated in one step by centrifugal partition chromatography. The stems' and leaves' extracts of L. virgatum were rich in polyphenols responsible for the antiradical activity. Also, Limonium extracts showed an antibacterial activity with a broad spectrum.Extremophile plants have proven to be a promising source for bioactive metabolites. They have a powerful antioxidant system highly influenced by biotic and abiotic factors and the ability to produce secondary metabolites with antimicrobial activity.
The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3'-monogallate (TF2), and theaflavin-3-3'-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.
Abstract Recent emergence of direct acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, search for new inhibitors and with lower cost of production should be pursued. In this context, crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed isolating and identifying the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (EC 50 = 1.35 µM). This antiviral activity was confirmed with a HCV subgenomic replicon and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit replication of two frequent daclatasvir resistant mutants (L31M or Y93H in NS5A). Finally, resistant mutants to dehydrojuncusol were obtained and showed that HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus , inhibits infection of different HCV genotypes by targeting NS5A protein and is active against HCV resistant variants frequently found in patients with treatment failure. Importance Tens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of the treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to appearance of viral resistance. Here we report on the identification of a new HCV inhibitor, dehydrojuncusol that targets HCV NS5A and is able to inhibit replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus , a halophilic plant species very common in the coastlines worldwide. This molecule might serve as a lead for the development of new therapy more accessible to hepatitis C patients in the future.
According to some recent studies, Cameroon is one of the sub-Saharan African countries most affected by hepatitis C, with low access to the standard therapy based on the combination of pegylated interferon and ribavirin. A first ethnobotanical survey, conducted in the Western region of Cameroon, reported the use of several medicinal plants in traditional medicine for the healing of liver-related disorders. Crude organic extracts of five plants surveyed were prepared and their effect against hepatitis C virus (HCV) infection investigated. The HCV JFH1 strain cell culture system HCVcc was used. The antiviral activity was quantified by immunofluorescent labeling of HCV E1 envelope protein at 30 h post-infection in the presence of the plant extracts. Active compounds were then tested in time course infection experiments. Dose-response and cellular toxicity assays were also determined. Three extracts, methanol extracts from roots of Trichilia dregeana, stems of Detarium microcarpum and leaves of Phragmanthera capitata, showed anti-HCV activity, with half-maximal inhibitory concentration of 16.16, 1.42, and 13.17 μg/mL, respectively. Huh-7 cells were incubated with the extracts for 72 h and it appears that T. dregeana extract is not toxic up to 200 μg/mL, D. microcarpum up to 100 μg/mL and P. capitata up to 800 μg/mL. All the three extracts showed a strong inhibition of HCV entry and no effect on replication or secretion. Taken together, these results showed that extracts from Cameroonian medicinal plants are promising sources of anti-HCV agents.
L’hepatite C est une maladie affectant aujourd’hui 170 millions d’individus dans le monde. Cette maladie est causee par le virus de l'hepatite C (VHC). Les nouveaux traitements recemment mis sur le marche, visant a soigner l’hepatite C, sont tres onereux et donc inaccessibles pour les pays du Sud. De plus, l’utilisation de ces nouvelles molecules engendre l’apparition de mutation de resistance virale responsable de l’echec des traitements pour 5 a 10% des patients. La decouverte de nouveaux antiviraux est donc toujours indispensable. Les plantes utilisees en medecine traditionnelle depuis des siecles sont une source de composes bio-actifs tres interessante. L’utilisation de ces plantes ou de composes naturels en complement des molecules utilisees en therapie classique, permettrait de reduire significativement le cout de ces traitements et de les rendre accessibles a davantage de patients.L’epigallocatechine-3-gallate (EGCG) et la delphinidine sont des molecules naturelles issues respectivement du the vert et des fruits rouges. Ces deux molecules inhibent l’etape d’entree du VHC dans les cellules hepatocytaires. Nous avons montre que l'EGCG et la delphinidine inhibent cette etape virale dans les hepatocytes en deformant les particules et bloquant ainsi leur attachement a la surface cellulaire. Nous nous sommes ensuite interesses au processus engendrant cette deformation qui est probablement lie a l’agregation des glycoproteines d’enveloppe virale.Nous avons procede au criblage d’extraits bruts de plantes extremophiles tunisiennes et mis en evidence que l’extrait brut de rhizome de Juncus maritimus inhibait l’etape de replication du VHC. Le J. maritimus est une plante fortement presente dans les sols arides en Tunisie mais egalement sur les cotes francaises. En collaboration avec le laboratoire de Pharmacognosie de Lille et grâce a un fractionnement bioguide, le principe actif a pu etre isole. Il s’agit du dehydrojuncusol qui inhibe la replication virale avec une concentration effective mediane de 1,31 µM. De plus, nous avons montre que le dehydrojuncusol pouvait inhiber la replication de virus presentant des mutations de resistance aux traitements actuels ciblant la proteine virale NS5A. Nous avons cherche a identifier la cible virale du dehydrojuncusol et il semblerait que ce soit egalement la proteine NS5A.Les resultats obtenus dans cette these confortent l’hypothese que des molecules naturelles pourraient etre utilisees dans le traitement de l’hepatite C.
Abstract The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3’-monogallate (TF2), and theaflavin-3-3’-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.
Recent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.
