Antibacterial and anti-coronavirus investigation of selected Senegalese plant species according to an ethnobotanical survey.

ETHNOPHARMACOLOGICAL RELEVANCE: In Senegal, upper and lower respiratory tract infections constitute a real health problem. To manage these disorders, most people rely on the use of local medicinal plants. This is particularly the case for species belonging to the botanical families, Combretaceae, Fabaceae, Myrtaceae and Rubiaceae, which are widely used to treat various respiratory problems such as colds, flu, rhinitis, sinusitis, otitis, angina, bronchitis, bronchiolitis and also pneumonia. AIM OF THE STUDY: The aim of this study was to identify medicinal plants traditionally used for the management of infectious diseases, in particular those of the respiratory tract. On the basis of these ethnopharmacological uses, this study made it possible to highlight the antibacterial, antiviral and cytotoxic activities of selected plant species. MATERIALS AND METHODS: An ethnobotanical survey was conducted in Senegal among informants, including herbalists, traditional healers, and households, using medicinal plants in the management of infectious diseases, with a focus on respiratory tract infections. The most cited plant species were evaluated in vitro on a panel of 18 human pathogenic bacteria may be involved in respiratory infections and against the human coronavirus HCoV-229E in Huh-7 cells. The antiviral activity of the most active extracts against HCoV-229E was also evaluated on COVID-19 causing agent, SARS-CoV-2 in Vero-81 cells. In parallel, cytotoxic activities were evaluated on Huh-7 cells. RESULTS: A total of 127 informants, including 100 men (78.74%) and 27 women (21.26%) participated in this study. The ethnobotanical survey led to the inventory of 41 plant species belonging to 19 botanical families used by herbalists and/or traditional healers and some households to treat infectious diseases, with a specific focus on upper respiratory tract disorders. Among the 41 plant species, the most frequently mentioned in the survey were Guiera senegalensis J.F. Gmel. (95.2%), Combretum glutinosum Perr. Ex DC. (93.9%) and Eucalyptus spp. (82.8%). Combretaceae (30.2%) represented the most cited botanical family with six species, followed by Fabaceae (29.3%, 12 species). A total of 33 crude methanolic extracts of the 24 plant species selected for their number of citations were evaluated in vitro for their antimicrobial and cytotoxic activities. Guiera senegalensis, Combretum glutinosum, Vachellia nilotica subsp. tomentosa (Benth.) Kyal. & Boatwr, Eucalyptus camaldulensis Dehnh., and Terminalia avicennioides Guill. & Perr., showed antibacterial activities. The most active plants against HCoV-229E were: Ficus sycomorus L., Mitragyna inermis (Willd.) Kuntze, Pterocarpus erinaceus Poir., and Spermacoce verticillata L. One of these plants, Mitragyna inermis, was also active against SARS-CoV-2. CONCLUSION: This work confirmed the anti-infective properties of plant species traditionally used in Senegal. Overall, the most frequently cited plant species showed the best antibacterial activities. Moreover, some of the selected plant species could be considered as a potential source for the management of coronavirus infections. This new scientific data justified the use of these plants in the management of some infectious pathologies, especially those of the respiratory tract.

A Photoactivable Natural Product with Broad Antiviral Activity against Enveloped Viruses, Including Highly Pathogenic Coronaviruses.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against human CoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane, as demonstrated by cryoelectron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2-contaminated surfaces.

Anti-HCV Tannins From Plants Traditionally Used in West Africa and Extracted With Green Solvents.

Millions of people are still infected with hepatitis C virus (HCV) nowadays. Although recent antivirals targeting HCV proteins are very efficient, they are not affordable for many people infected with this virus. Therefore, new and more accessible treatments are needed. Several Ivorian medicinal plants are traditionally used to treat "yellow malaria", a nosological category including illness characterized by symptomatic jaundice such as hepatitis. Therefore, some of these plants might be active against HCV. An ethnobotanical survey in Côte d'Ivoire allowed us to select such medicinal plants. Those were first extracted with methanol and tested for their anti-HCV activity. The most active ones were further studied to specify their IC50 and to evaluate their toxicity in vitro. Greener solvents were tested to obtain extracts with similar activities. Following a phytochemical screening, tannins of the most active plants were removed before re-testing on HCV. Some of these tannins were identified by UPLC-MS and pure molecules were tested against HCV. Out of the fifteen Ivorian medicinal plants selected for their putative antiviral activities, Carapa procera DC. and Pericopsis laxiflora (Benth. ex Baker) Meeuwen were the most active against HCV (IC50: 0.71 and 0.23 µg/ml respectively) and not toxic for hepatic cells. Their crude extracts were rich in polyphenols, including tannins such as procyanidins A2 which is active against HCV. The same extracts without tannin lost their anti-HCV activity. Replacing methanol by hydro-ethanolic solvent led to tannins-rich extracts with similar antiviral activities, and higher than that of aqueous extracts.

Viral hepatitis in the Peruvian Amazon: Ethnomedical context and phytomedical resource.

ETHNOPHARMACOLOGICAL RELEVANCE: An extensive ethnopharmacological survey was carried out in the Peruvian Amazonian district of Loreto with informants of various cultural origins from the surroundings of Iquitos (capital city of Loreto) and from 15 isolated riverine Quechua communities of the Pastaza River. A close attention was paid to the medical context and plant therapy, leading to the selection of 35 plant species (45 extracts). The extracts were tested for antiviral activity against HCV with counting of Huh-7 cellular death in case of toxicity, and cytotoxicity was evaluated in HepG2 cells. AIM OF THE STUDY: The aim of the study was to inventory the plants used against hepatitis in Loreto, then to evaluate their antiviral activity and to suggest a way to improve local therapeutic strategy against viral hepatitis, which is a fatal disease that is still increasing in this area. MATERIALS AND METHODS: An ethnographic survey was carried out using "participant-observation" methodology and focusing on plant therapy against hepatitis including associated remedies. 45 parts of plant were extracted with methanol and tested in vitro for anti-HCV activity in 96-well plate, using HCV cell culture system with immunofluorescent detection assisted by automated confocal microscopy. Toxicity of plant extracts was also evaluated in microplates on hepatic cells by immunofluorescent detection, for the Huh-7 nuclei viability, and by UV-absorbance measurement of MTT formazan for cytotoxicity in HepG2 cells. RESULTS: In vitro assay revealed interesting activity of 18 extracts (50% infection inhibition at 25 µg/mL) with low cytotoxicity for 15 of them. Result analysis showed that at least 30% of HCV virus were inhibited at 25 µg/mL for 60% of the plant extracts. Moreover, the ethnomedical survey showed that remedies used with low and accurate dosing as targeted therapy against hepatitis are usually more active than species indicated with more flexible dosing to alleviate symptoms of hepatic diseases. CONCLUSION: Together with bibliographic data analysis, this study supported the traditional medicinal uses of many plants and contributed to a better understanding of the local medical system. It also permitted to refine the therapeutic plant indications regarding patients' liver injuries and vulnerability. Only 2 of the 15 most active plant species have already been studied for antiviral activity against hepatitis suggesting new avenues to be followed for the 13 other species.

Dehydrojuncusol, a Natural Phenanthrene Compound Extracted from Juncus maritimus, Is a New Inhibitor of Hepatitis C Virus RNA Replication.

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.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 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 the 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 RNA 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 that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.

Theaflavins, polyphenols of black tea, inhibit entry of hepatitis C virus in cell culture.

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.

An ecological approach to discover new bioactive extracts and products: the case of extremophile plants.

OBJECTIVES: Eight extremophile plants from Tunisia were screened to find natural products with benefits in human health. METHODS: 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. KEY FINDINGS: 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. CONCLUSIONS: 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.

Identification of a New Benzimidazole Derivative as an Antiviral against Hepatitis C Virus.

UNLABELLED: Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle. IMPORTANCE: In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle.

Khaya grandifoliola C.DC: a potential source of active ingredients against hepatitis C virus in vitro.

In this study, we examined the antiviral properties of Khaya grandifoliola C.DC (Meliaceae) on the hepatitis C virus (HCV) life cycle in vitro and identified some of the chemical constituents contained in the fraction with the most antiviral activity. Dried bark powder was extracted by maceration in a methylene chloride/methanol (MCM) system (50:50; v/v) and separated on silica gel by flash chromatography. Infection and replication rates in Huh-7 cells were investigated by luciferase reporter assay and indirect immunofluorescence assay using subgenomic replicons, HCV pseudotyped particles, and cell-culture-derived HCV (HCVcc), respectively. Cell viability was assessed by MTT assay, and cellular gene expression was analysed by qRT-PCR. The chemical composition of the fraction with the most antiviral activity was analysed by coupled gas chromatography and mass spectrometry (GC-MS). Five fractions of different polarities (F0-F100) were obtained from the MCM extract. One fraction (KgF25) showed the strongest antiviral effect on LucUbiNeoET replicons at nontoxic concentrations. Tested at 100 µg/mL, KgF25 had a high inhibitory effect on HCV replication, comparable to that of 0.01 µM daclatasvir or 1 µM telaprevir. This fraction also inhibited HCVcc infection by mostly targeting the entry step. KgF25 inhibited HCV entry in a pan-genotypic manner by directly inactivating free viral particles. Its antiviral effects were mediated by the transcriptional upregulation of the haem oxygenase-1 gene and interferon antiviral response. Three constituents, namely, benzene, 1,1'-(oxydiethylidene)bis (1), carbamic acid, (4-methylphenyl)-, 1-phenyl (2), and 6-phenyl, 4-(1'-oxyethylphenyl) hexene (3), were identified from the active fraction KgF25 by GC-MS. Khaya grandifoliola contains ingredients capable of acting on different steps of the HCV life cycle.

Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action.

UNLABELLED: Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface. IMPORTANCE: In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors.

Plant extracts from Cameroonian medicinal plants strongly inhibit hepatitis C virus infection in vitro.

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.

Hepatitis C virus and natural compounds: a new antiviral approach?

Hepatitis C is a major global health burden with an estimated 160 million infected individuals worldwide. This long-term disease evolves slowly, often leading to chronicity and potentially to liver failure. There is no anti-HCV vaccine, and, until recently, the only treatment available, based on pegylated interferon and ribavirin, was partially effective, and had considerable side effects. With recent advances in the understanding of the HCV life cycle, the development of promising direct acting antivirals (DAAs) has been achieved. Their use in combination with the current treatment has led to encouraging results for HCV genotype 1 patients. However, this therapy is quite expensive and will probably not be accessible for all patients worldwide. For this reason, constant efforts are being made to identify new antiviral molecules. Recent reports about natural compounds highlight their antiviral activity against HCV. Here, we aim to review the natural molecules that interfere with the HCV life cycle and discuss their potential use in HCV therapy.

(-)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry.

UNLABELLED: Here, we identify (-)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 µM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 4°C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell-free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell-to-cell spread. Finally, by successive inoculation of naïve cells with supernatant of HCV-infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. CONCLUSION: EGCG is a new, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte.

Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity.

Obesity is a major public health problem and is often associated with type 2 diabetes mellitus, cardiovascular disease, and metabolic syndrome. Leptin is the crucial adipostatic hormone that controls food intake and body weight through the activation of specific leptin receptors (OB-R) in the hypothalamic arcuate nucleus (ARC). However, in most obese patients, high circulating levels of leptin fail to bring about weight loss. The prevention of this "leptin resistance" is a major goal for obesity research. We report here a successful prevention of diet-induced obesity (DIO) by silencing a negative regulator of OB-R function, the OB-R gene-related protein (OB-RGRP), whose transcript is genetically linked to the OB-R transcript. We provide in vitro evidence that OB-RGRP controls OB-R function by negatively regulating its cell surface expression. In the DIO mouse model, obesity was prevented by silencing OB-RGRP through stereotactic injection of a lentiviral vector encoding a shRNA directed against OB-RGRP in the ARC. This work demonstrates that OB-RGRP is a potential target for obesity treatment. Indeed, regulators of the receptor could be more appropriate targets than the receptor itself. This finding could serve as the basis for an approach to identifying potential new therapeutic targets for a variety of diseases, including obesity.