Antiviral activity of hydroalcoholic extract from Eupatorium perfoliatum L. against the attachment of influenza A virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Eupatorium perfoliatum have been traditionally used by American natives as a treatment for fever and infections. Also modern phytotherapy in Europe documents the use of hydroalcoholic extracts of this herbal material for the treatment of infections of the upper respiratory tract. AIM OF THE STUDY: The aim of the present study was to characterize the anti-influenza A virus (IAV) potential of extracts derived from the aerial parts of E. perfoliatum and to identify their antiviral mode of action and potential active fraction's compounds of the extract. MATERIALS AND METHODS: The inhibitory effects of extracts obtained by different organic solvents with different polarities on the cytopathic effect induced by IAV replication was determined in a Madin-Darby Canine Kidney Epithelial (MDCK II) cell-based assay measuring cell viability by MTT stain (MTTIAV assay). Plaque reduction assays were used for investigation of antiviral activity. The mode of action was investigated by different incubation and treatment cycles as well as hemagglutination inhibition assays. Influence of the test extract on tumor necrosis factor (TNF-α) and epidermal growth factor (EGF)-induced cell signaling was analyzed in human lung epithelial (A549) cells. Analytical characterization of extract and fractions obtained from the extract was performed by UHPLC-MS. RESULTS: Hydroalcoholic extracts from the aerial parts of E. perfoliatum were shown to inhibit growth of a clinical isolate of IAV(H1N1)pdm09 I1 and the influenza virus A/Puerto Rico/8/34 (PR8; H1N1) with a half-maximal inhibitory concentration (IC50) of 7µg/mL and 14µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 52 and 26, respectively. Extracts prepared with dichloromethane and methanol were inactive. At concentrations >1-10µg/mL of the hydroalcoholic extract plaque formation of IAV(H1N1)pdm09 was abrogated. The extract was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. The extract blocked attachment of IAV and interfered with virus-induced hemagglutination. TNF-α-induced signal transduction in A549 cells was not affected, while the EGF-induced signaling to phosphorylated ERK was slightly upregulated by the extract. Bioassay-guided fractionation and subsequent LC-MS analysis indicated that the antiviral activity might be due to polyphenolic compounds with higher molecular weights, which strongly interact with stationary phases of different chromatographic systems. These still unknown compounds with probably high molecular weight could not be isolated in the present study. A variety of different flavonoid glycosides and caffeoyl quinic acids obtained from E. perfoliatum did definitely not contribute to the antiviral effect of the extract and its respective fractions. CONCLUSION: Hydroalcoholic extracts from the aerial parts of E. perfoliatum and its main active polyphenolic constituents protect cells from IAV infection by inhibiting viral attachment to the host cells. The extract appears to be a promising expansion of the currently available anti-influenza agents.

3-O-galloylated procyanidins from Rumex acetosa L. inhibit the attachment of influenza A virus.

Infections by influenza A viruses (IAV) are a major health burden to mankind. The current antiviral arsenal against IAV is limited and novel drugs are urgently required. Medicinal plants are known as an abundant source for bioactive compounds, including antiviral agents. The aim of the present study was to characterize the anti-IAV potential of a proanthocyanidin-enriched extract derived from the aerial parts of Rumex acetosa (RA), and to identify active compounds of RA, their mode of action, and structural features conferring anti-IAV activity. In a modified MTT (MTTIAV) assay, RA was shown to inhibit growth of the IAV strain PR8 (H1N1) and a clinical isolate of IAV(H1N1)pdm09 with a half-maximal inhibitory concentration (IC50) of 2.5 µg/mL and 2.2 µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 32 and 36, respectively. At RA concentrations>1 µg/mL plaque formation of IAV(H1N1)pdm09 was abrogated. RA was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. TNF-α and EGF-induced signal transduction in A549 cells was not affected by RA. The dimeric proanthocyanidin epicatechin-3-O-gallate-(4ß→8)-epicatechin-3'-O-gallate (procyanidin B2-di-gallate) was identified as the main active principle of RA (IC50 approx. 15 µM, SI≥13). RA and procyanidin B2-di-gallate blocked attachment of IAV and interfered with viral penetration at higher concentrations. Galloylation of the procyanidin core structure was shown to be a prerequisite for anti-IAV activity; o-trihydroxylation in the B-ring increased the anti-IAV activity. In silico docking studies indicated that procyanidin B2-di-gallate is able to interact with the receptor binding site of IAV(H1N1)pdm09 hemagglutinin (HA). In conclusion, the proanthocyanidin-enriched extract RA and its main active constituent procyanidin B2-di-gallate protect cells from IAV infection by inhibiting viral entry into the host cell. RA and procyanidin B2-di-gallate appear to be a promising expansion of the currently available anti-influenza agents.

Proanthocyanidin-enriched extract from Myrothamnus flabellifolia Welw. exerts antiviral activity against herpes simplex virus type 1 by inhibition of viral adsorption and penetration.

AIM OF THE STUDY: Extracts from the aerial parts of the South African resurrection plant Myrothamnus flabellifolia Welw. have been used traditionally against infections of the upper respiratory tract and skin diseases. A polyphenol-enriched extract was investigated for potential antiviral effects against herpes simplex virus type 1 (HSV-1) and adenovirus, and the underlying mode of action was to be studied. MATERIALS AND METHODS: Antiviral effects of an acetone-water extract (MF) from Myrothamnus flabellifolia on HSV-1 and adenovirus type 3 were tested in infected Vero cells by plaque reduction assay, MTT test and immunofluorescence. The influence of the extract on the HSV-1 envelope glycoprotein D was shown by Western blot. Organotypic full thickness skin models consisting of multilayer skin equivalents were used for the investigation of MF effects on HSV-1 replication. RESULTS: MF exhibited strong antiviral activity against HSV-1. The HSV-1-specific inhibitory concentration (IC(50)) was determined as 0.4 µg/mL and the cytotoxic concentration (CC(50)) against Vero cells as 50 µg/mL. A selectivity index (SI) (ratio of CC(50) to IC(50)) of approximately 120 was calculated when MF was added to the virus inoculum for 1h at 37°C prior to infection. The replication of adenovirus 3 was not affected by MF. MF abolished virus entry into the host cell by blocking viral attachment to the cell surface. When added after attachment at a concentration of >6 µg/mL, the extract also inhibited penetration of HSV-1 into the host cell. Polyphenolic compounds from MF directly interacted with viral particles, leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein D (gD). Using organotypic full thickness tissue cultures, it was shown that treatment of HSV-1 infected cultures with the MF resulted in reduced viral spread. CONCLUSIONS: A polyphenol-enriched extract from Myrothamnus flabellifolia strongly acts against HSV-1 by blocking viral entry into the cells.

Inhibition of viral adsorption and penetration by an aqueous extract from Rhododendron ferrugineum L. as antiviral principle against herpes simplex virus type-1.

The polyphenol-enriched aqueous extract RF from the aerial parts of Rhododendron ferrugineum exhibited strong antiviral activity against herpes simplex virus type-1 while adenovirus 3 was not affected. RF exhibited an IC(50) of 7.4 µg/mL and a selectivity index of 64 when added to the virus inoculum prior to infection. RF abolished virus entry into the host cell by blocking attachment to the cell surface. When added after attachment at a concentration of >25 µg/mL, RF inhibited also penetration of HSV-1 into the host cell. RF directly interacts with viral envelope proteins as demonstrated for the viral glycoprotein gD.

Oligomeric proanthocyanidins from Rumex acetosa L. inhibit the attachment of herpes simplex virus type-1.

The polyphenole-enriched acetone-water extract R2 from the aerial parts of Rumex acetosa L. containing high amounts of oligomeric and polymeric proanthocyanidins and flavonoids was tested for antiviral activity. R2 exhibited strong antiviral activity against herpes simplex virus type-1 (HSV-1) while the replication of adenovirus 3 was not affected. By plaque reduction test and MTT assay on Vero cells, the HSV-1-specific inhibitory concentration (IC(50)) and cytotoxic concentration (CC(50)) were determined. R2 exibited an IC(50) of 0.8 µg/mL and a selectivity index (SI) (ratio of IC(50) to CC(50)) of approximately 100 when added to the virus inoculum for 1h at 37°C prior to infection. The antiviral activity was due to the presence of flavan-3-ols and oligomeric proanthocyanidins in the extract. Structure-activity analyses indicated that flavan-3-ols and proanthocyanidins with galloylation at position O-3 are highly potent compounds (SI>40), while ungalloylated compounds did not exhibit antiviral effects (SI<1). R2 and a major proanthocyanidin from R2, epicatechin-3-O-gallate-(4ß→8)-epicatechin-3-O-gallate abolished virus entry into the host cell by blocking attachment to the cell surface. When added after attachment at a concentration of ≥ 12.5 µg/mL, R2 inhibited also penetration of HSV-1 into the host cell. R2 and epicatechin-3-O-gallate-(4ß→8)-epicatechin-3-O-gallate were shown to directly interact with viral particles leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein gD. Using raft cultures with three-dimensional organotypic human skin equivalents it was shown that treatment of cultures with R2 after infection with HSV-1 resulted in a reduced viral spread.