Synergistic inhibition of influenza A virus replication by a plant polyphenol-rich extract and epsilon-aminocaproic acid in vitro and in vivo.

UNLABELLED: A combined antiviral effect of a polyphenol-rich extract of the medicinal plant Geranium sanguineum L. (PC) and a protease inhibitor, epsilon-aminocaproic acid (ACA) was examined in Influenza A virus (IAV)-infected MDCK cell cultures and mice. Synergistic, antagonistic, or indifferent antiviral effects were distinguished on the basis of virus yields, namely fractional yields of individual compounds and yields of both compounds in combination. Combinations of PC and ACA in particular concentrations proved synergistic in the inhibition of virus replication in MDCK cells and in protection of mice against virus infection as determined by virus titers, lung weight, mean survival time (MST), mortality rate, and protection rate (PR). Following the application of a combination of PC and ACA to the virus-infected mice, the levels of the lung protease and protease-inhibitory activity, which were increased due to the virus infection, were brought to normal. These results demonstrate the rationale for a combined application of viral inhibitors with different modes of action to the treatment of IAV infection, in particular PC as a natural inhibitor of early viral transcription and translation and ACA as a synthetic inhibitor of cellular proteases. KEYWORDS: Influenza A virus; antiviral effect; synergism; plant polyphenol extract; epsilon-aminocaproic acid; protease inhibitors.

Protective efficacy of an aerosol preparation, obtained from Geranium sanguineum L., in experimental influenza infection.

A polyphenol-rich extract from the aerial roots of the medicinal plant Geranium sanguineum L. (PC) protected mice from mortality in the experimental influenza A/Aichi/2/68 (H3N2) virus infection. To provide evidence how a maximum therapeutic benefit can be derived of this preparation, it was inoculated by 6 different routes. It was found that the aerosol application of PC was highly effective. In the dose 5.4 mg/ml, applied according to a prophylactic-therapeutic schedule, the extract exhibited a marked protective effect. The protective index reached the value of 70.1% and the mean survival time was prolonged with 2.9-4.9 days. The lung infectious virus titres and the lung consolidation of virus-infected and PC-treated animals were all reduced in comparison with control. The application of PC according to schedules, excluding the pretreatment of mice, proved that this condition was essential for protection.

In vitro investigation on the effect of a plant preparation with antiviral activity on the functions of mice phagocyte cells.

A polyphenol extract from the aerial roots of the medicinal plant Geranium sanguineum L. (PC) inhibited the reproduction of influenza viruses type A and B in vitro and in ovo and protected mice from mortality in the experimental influenza infection. The in vivo protective effect was connected with multiple biological activities of the preparation. The present paper focuses on the in vitro effects of the polyphenol extract on the functions of peritoneal and alveolar macrophages and blood polymorphonuclear leucocytes (PMNs), isolated from healthy ICR mice. It was found that PC in doses of 12.5 and 25 microg ml(-1) stimulated the phagocytic activity of peritoneal macrophages and blood PMNs. PC in the same doses did not significantly affect the phagocytic activity of alveolar macrophages, the migration of alveolar and peritoneal macrophages or the adherent activity of PMNs. Used in concentrations of 3.1-25.0 microg ml(-1), PC suppressed spontaneous NO production from peritoneal macrophages, while inducible NO production, provoked by LPS-, Ifn-gamma and LPS + Ifn-gamma inductions was not affected. The cell-toxic concentration of 100 microg ml(-1) increased spontaneous and LPS-inducible NO production. The experimental results demonstrated a stimulating effect of PC on the phagocytic activity of murine PMNs and peritoneal macrophages as well as a beneficial effect of the preparation on spontaneous NO production.

Combined protective effect of a fungal Cu/Zn-containing superoxide dismutase and rimantadine hydrochloride in experimental murine influenza a virus infection.

The combined protective effect of a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced by the fungus Humicula lutea (HL-SOD) strain 103, and the selective anti-influenza drug rimantadine hydrochloride (Rim) was evaluated in experimental virus infection in mice, induced with influenza virus A/Aichi/2/68 (H3N2). A combined application of HL-SOD and Rim in doses, which by themselves did not protect significantly mice against the infection, resulted in a synergistically increased protection, determined on the basis of protective indices. Lung virus titers, lung weights and consolidation and mortality rates were all decreased significantly, while survival times were prolonged.

Influenza virus variants with reduced susceptibility to inhibition by a polyphenol extract from Geranium sanguineum L.

A polyphenol extract obtained from the Bulgarian medicinal plant Geranium sanguineum L. (PC) inhibited influenza virus reproduction in vitro, in ovo and in vivo. The inhibitory activity of the plant preparation was proved to be selective and specific. Two variants of the virus A/chicken/Germany/34, strain Rostock (H7N1) (A/Rostock) with reduced drug sensitivity were selected by following passages in chicken embryo fibroblast (CEF) cells in the presence of inhibitory concentrations of the substance and by subsequent purification of resistant plaques (A/Rostock-R). PC affected to a lesser extent the biological activity and the infectivity of the resistant variants compared to the sensitive parent. The lower susceptibility of the variants was consistently observed in contact assay, ELISA, cytopathogenic effect reduction, plaque reduction and infectious virus yield reduction assays. Virus-specific protein synthesis in CEF cells infected with the resistant variants, determined by 35S-labelling and following SDS-PAGE, was also less sensitive to inhibition with PC.

Antiherpes virus effect of the red marine alga Polysiphonia denudata.

The water extract from the red marine alga Polysiphonia denudata (Dillwyn) Kutz. from the Bulgarian Black Sea coast selectively inhibited the reproduction of herpes virus type 1 and type 2 in cell cultures (EC50=8.7 to 47.7 mg/ml) as shown by the reduction of virus-induced cytopathic effect and viral infectivity. The virus-inhibitory effect was dose-related, strain-specific and depended on virus inoculum. The inhibition affected adsorption as well as the intracellular stages of viral replication. The presence of the extract throughout the whole replicative cycle was necessary for the full expression of the antiviral effect. In higher concentrations (MIC90=6.5 mg/ml) the extract exhibited strong extracellular virus inactivating activity.

Combined antiinfluenza virus activity of Flos verbasci infusion and amantadine derivatives.

The infusion prepared from flowers of Verbascum thapsiforme Schrad. (Scrophulariaceae) (FVI) reduced the infectious and haemagglutination yields of a range of influenza viruses in tissue cultures. Amantadine hydrochloride is an accepted and well studied selective inhibitor of influenza virus reproduction. The combined application of the plant preparation FVI and three amantadine derivatives resulted in a marked enhancement of the inhibitory effect of FVI on the reproduction of influenza virus A/chicken/Germany/27, strain Weybridge (H7N7) in cell cultures of chicken embryo fibroblasts. The antiviral activity was determined by the difference in the infectious titres of control and treated viruses. The combined effect was defined on the basis of infectious viral yields. The most pronounced enhancement was shown for the combination of FVI and adamantanamine glucuronide.

Antiinfluenza virus effect of extracts from marine algae and invertebrates.

Sixty products, derived from marine organisms, typical of the Bulgarian Black Sea coast, were examined for inhibitory activity on the reproduction of influenza viruses in tissue cultures. The antiviral effect was investigated by the reduction of virus infectivity. Using representative strains of influenza virus it was shown that apparently the inhibitory effect was strain-specific. The most effective products were further studied in fertile hen's eggs and in experimental influenza infection in white mice.

Antiherpes virus activity of extracts from the medicinal plant Geranium sanguineum L.

The herpes virus inhibitory effect of five extracts from the Bulgarian medicinal plant Geranium sanguineum L. (Geraniaceae) was investigated. The water extract (WE) from the aerial roots of the plant was the least toxic for cell cultures and inhibited significantly the replication of herpes simplex virus type 1 and type 2 (EC50 = 3.6-6.2 microg/ml) as shown by the reduction of virus induced cytopathogenic effect (CPE) and the protection of cells in MTT assay. The inhibition was dose-related, strain-specific and depended on virus inoculum. In higher concentrations (MIC90 = 120 microg/ml) the preparation exhibited strong extracellular virus inactivating activity. The presence of WE throughout the whole replicative cycle was necessary for the full expression of the antiviral effect. In a preliminary experiment in albino guinea pigs the extract delayed the development of herpetic vesicles following primary infection with HSV-1, strain Kupka. Phytochemical investigation of the plant preparation revealed the presence of flavonoids, catechins, a polyphenolic acid and condensed tannins. The inhibitory effect of the extract on herpes simplex virus replication is related to the rich content of polyphenol compounds.

In vitro anti-influenza virus activity of a plant preparation from Geranium sanguineum L.

A polyphenolic complex (PC), isolated from the Bulgarian medicinal plant Geranium sanguineum L., was shown to have selective anti-influenza activity in vitro. Expression of HA on the surface of cells infected with A/chicken/Rostock/34, virus-induced cytopathic effect, infectious virus yield and plaque formation were all reduced at non-toxic concentrations of PC. Synthesis of virus proteins was also selectively inhibited. High concentrations of PC (> 200 microg/ml) exhibited a strong virucidal effect. Although the action was directed against an early stage of infection (within 3 h of infection), the process directly affected was not identified. The selectivity of antiviral action was confirmed by the variation in sensitivity of different influenza viruses to PC and the selection of variants with reduced drug sensitivity.

Antiinfective activity of a plant preparation from Geranium sanguineum L.

A methanol extract (PC) has been obtained from the Bulgarian medicinal plant Geranium sanguineum L. The antiinfective activity of the plant preparation was studied. The extract inhibited the reproduction of a range of viruses (influenza, herpes simplex, vaccinia, HIV-I) in cell cultures. Its antiinfluenza effect was most pronounced; PC reduced the infectivity of various influenza virus strains in vitro and protected mice in experimental influenza infection. The polyphenol extract inhibited the in vitro growth of Staphylococcus aureus and Candida albicans.

Combined protective effect of an immunostimulatory bacterial preparation and rimantadine in experimental influenza A virus infection.

The protective effect of an immunostimulatory bacterial preparation, cytoplasmic membranes of Escherichia coli WF stable protoplast type L-forms (CM) alone and in combination with the selective antiviral drug rimantadine was evaluated in experimental influenza A/Aichi/2/68 (H3N2) virus infection in mice. In sublethal infection, CM administered intraperitoneally (i.p.) 7 days before virus exposure in a single dose of 25 mg/kg did not reduce significantly the virus lung titers. In lethal infection, CM applied in the same way weakly reduced the mortality rate. The combined application of CM with rimantadine resulted in synergistically increased protection, determined on the basis of virus lung titers, lung consolidation, mortality rates, protective indices, and survival times.

Inhibition of influenza virus protein synthesis by a plant preparation from Geranium sanguineum L.

A polyphenolic complex (PC) with antiviral properties has been isolated from the Bulgarian medicinal plant Geranium sanguineum L. A study was undertaken to investigate the effect of PC on virus-specific protein synthesis in influenza virus-infected cells. The expression of viral glycoproteins on the surface of chick embryo fibroblasts infected with virus A/FPV, strain Rostock (H7N1) was suppressed. Virus protein synthesis was selectively inhibited as shown by SDS polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins and proteins immunoprecipitated with monoclonal antibodies. The inhibitory effect was dose-dependent and better pronounced when PC was applied after virus infection. Two variants of influenza virus FPV/Rostock with reduced drug susceptibility were selected. PC affected to a lesser extent the synthesis of viral proteins in cells infected with the variants as compared to the sensitive parental virus.

Isolation of a polysaccharide with antiviral effect from Ulva lactuca.

A polysaccharide from the green marine algae Ulva lactuca has been isolated. The substance has been investigated after acid hydrolysis by thin-layer and gas chromatography. The following carbohydrate components have been found: arabinose-xylose-rhamnose-galactose-mannose-glucose in ratio 1:1:9:5:2.5:16 respectively. One unidentified sugar has been demonstrated too. The polysaccharide has been studied for antiviral activity in vitro against a number of human and avian influenza viruses. A considerable inhibition of the viral reproduction was found. The effect was dose-dependent, strain-specific and selective.

Combined antiinfluenza activity of a plant preparation SHS-174 and amantadine derivatives.

The results of the study on the combined antiviral activity of the SHS-174 preparation (a lyophylized infusion from three higher plants) and three amantadine derivatives (rimantadine, amantadine glucuronide and its derivative) are presented. The antiviral effect of the drugs on the reproduction of influenza virus strains A/H1N1 and A/H3N2 in vitro was studied. The combined antiviral effect was evaluated on the basis of viral yields and in many cases a synergism was found. The most synergistic effect was shown for the combination of SHS-174 with the derivative of amantadine glucuronide.

Anti-influenza virus effect of some propolis constituents and their analogues (esters of substituted cinnamic acids).

The antiviral activity of six synthetic substances, esters of substituted cinnamic acids, identical with or analogous to some of the constituents of the Et2O fraction of propolis was studied in vitro. One of them, isopentyl ferulate, inhibited significantly the infectious activity of influenza virus A/Hong Kong (H3N2) in vitro and the production of hemagglutinins in ovo. By the use of diverse experimental patterns, it was found that the maximal inhibition of viral reproduction was observed when test substances were present in the medium during the whole infectious process.

Polyphenols from Bulgarian medicinal plants with anti-infectious activity.

Three Bulgarian medicinal plants--Geranium macrorrhizum L. and G. sanguineum L. (Geraniaceae), and Epilobium hirsutum L. (Onagraceae) were analyzed phytochemically. Different polyphenols like flavonoids and tannis have been found to be principal constituents of the plants. A series of water or alcohol extracts was obtained, and their anti-infectious activity was tested. A significant inhibitory effect of water-alcohol extract and of four fractions from the polyphenolic mixture of E. hirsutum on the reproduction of influenza viruses in vitro, in ovo, and in vivo was established. Four extracts from G. macrorrhizum and three extracts from G. sanguineum were studied for in vitro inhibition of the growth of some Gram-negative bacteria (Klebsiella pneumoniae, Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa), Gram-positive bacterium (Staphylococcus aureus), and fungus (Candida albicans). Some geranium extracts caused a strong increase of the survival rate in an infection with K. pneumoniae in mice. Augmentation of the nonspecific host resistance in relation to the influence of the extracts on the classical complement activation pathway was also studied.

Plant polyphenolic complex inhibits the reproduction of influenza and herpes simplex viruses.

A polyphenolic complex (PC), isolated from the Bulgarian medicinal plant Germanium sanguineum L., inhibited the reproduction of influenza virus types A and B in vitro and in ovo. It protected white mice in an experimental lethal influenza infection. When influenza viruses were treated with a high dose (1 mg/mL), their hemagglutination, neuraminidase and infections activities were reduced completely. It was assumed that the PC-specific inhibitory effect on influenza virus reproduction in tissue cultures (50 micrograms/mL) was mediated by partial inactivation of viral glycoproteins which prevented the initial stages of reproduction. The PC also affected the intracellular stages of viral replication. The effect was dependent on the mode of application, the viral strain, and the biological test system. The PC also inhibited the reproduction of herpes simplex virus in vitro. Infectious titers were reduced and newly synthesized virion with damaged protein envelopes were observed by electron microscopy.

Antiviral activity of Flos verbasci infusion against influenza and Herpes simplex viruses.

The lyophilized infusion from flowers of Verbascum thapsiforme Schrad. (FVI) showed antiviral activity in in vitro studies against Fowl plague virus, several influenza A strains, influenza B strain as well as Herpes simplex virus. Influenza viruses titer decreased by 1-3 log units, while of H. simplex virus by 2.3 log. FVI has shown virucidal activity on H. simplex virus at 300 micrograms/ml, but did not inactivate influenza viruses. Phytochemical investigations of FVI have shown the presence of flavonoids, iridoids, phenolic acids, saponins, amino acids and free sugars.

A study on the antiviral action of a polyphenolic complex isolated from the medicinal plant Geranium sanguineum L. VIII. Inhibitory effect on the reproduction of herpes simplex virus type 1.

The antiviral activity of a polyphenolic complex, isolated from a Bulgarian medicinal plant Geranium sanguineum L., on the reproduction of herpes simplex virus type 1 was studied. The substance inhibited the intracellular stages of viral replication and exhibited a virucidal effect.