A fungal Cu/Zn-containing superoxide dismutase enhances the therapeutic efficacy of a plant polyphenol extract in experimental influenza virus infection.

The combined protective effect of a polyphenol-rich extract, isolated from Geranium sanguineum L. (PC), and a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced from the fungal strain Humicula lutea 103 (HL-SOD), in the experimental influenza A virus infection (EIVI) in mice, induced with the virus A/Aichi/2/68 (H3N2), was investigated. The combined application of HL-SOD and PC in doses, which by themselves do not defend significantly mice in EIVI, resulted in a synergistically increased protection, determined on the basis of protective indices and amelioration of lung injury. Lung weights and consolidation as well as infectious lung virus titers were all decreased significantly parallel to the reduction of the mortality rates; lung indices were raised. The excessive production of reactive oxygen species (ROS) by alveolar macrophages (aMphi) as well as the elevated levels of the lung antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), induced by EIVI, were brought to normal. For comparative reasons the combined protective effect of PC and vitamin C was investigated. The obtained results support the combined use of antioxidants for the treatment of influenza virus infection and in general indicate the beneficial protective role of combinations of viral inhibitors of natural origin with diverse modes of action.

Antioxidant and prooxidant properties of a polyphenol-rich extract from Geranium sanguineum L. in vitro and in vivo.

A polyphenol-rich extract from Geranium sanguineum L. (PC) protected biological membranes due to its antioxidant capacity. PC caused a dose-dependent decrease of the osmotic hemolysis of human erythrocytes and increased their resistance against the toxic effect of H(2)O(2); no effect on catalase activity was observed. While PC reduced the accumulation of TBA-reactive products in rat liver microsomes in inducible lipid peroxidation (LPO), the non-induced LPO was not affected. Further the effect of PC on the products of LPO was investigated in the lungs, livers and sera of intact and influenza virus-infected mice (VIM). The infection enhanced LPO in the lungs and livers. In the group of PC-treated VIM, malondialdehyde (MDA) in the lungs and livers was brought to control levels. PC-treatment caused a significant increase of MDA in the lungs of intact mice, a slight one in the livers and did not affect MDA in the sera. Thus the extract exhibited prooxidant characteristics in intact animals as well as antioxidant properties in VIM. The reducing ability of PC on LPO could be an alternative mechanism of its protective effect in experimental influenza infection.

Antimicrobial activity and protective properties of vaginal lactobacilli from healthy Bulgarian women.

The role of vaginal Lactobacillus as an efficient barrier against invading pathogens is of considerable interest. The purpose of the present study was to assess in vitro the ability of 20 recently identified vaginal lactobacilli to protect the vagina. In order to evaluate their significance, the antimicrobial, hemagglutination (HA) and aggregation (Agg) activities, as well as acid and hydrogen peroxide (H(2)O(2)) production, were estimated. The cell-free cultures of eight strains showed a stable antimicrobial activity after elimination of the putative effects of lactic acid and H(2)O(2). Three of the isolated vaginal lactobacilli expressed a broad spectrum of anti-bacterial activity including Gram-negative pathogens. Strains with anti-Gardnerella and anti-herpes simplex virus type 2 activities were found. All tested isolates were H(2)O(2) producers, actively acidifying the growth media to pH 3.92+/-0.04, which is presumed to neutralize sexually transmitted infection pathogens. The major part (75%) expressed an HA activity and different Agg phenotypes, estimated as important properties in the competition with invading pathogens and in host defense. These results are encouraging and prompt further research of the characterized active strains and their possible application in prophylaxis of vaginal disorders.

Effect of a plant polyphenol-rich extract on the lung protease activities of influenza-virus-infected mice.

Influenza infection was induced in white mice by intranasal inoculation of the virus A/Aichi/2/68 (H3N2). The lung protease and the protease-inhibitory activities were followed for 9 days after infection. The intranasal application of a polyphenol-rich extract (PC) isolated from Geranium sanguineum L. induced a continuous rise in the anti-protease activity but did not cause substantial changes in the lung protease activity of healthy mice. Influenza virus infection triggered a slight reduction in protease activity in the lungs at 5 and 48 h post infection (p.i.) and a marked increase at 24 h and 6 day p.i.. Protease inhibition in the lungs was reduced at 24 and 48 h p.i. and an increase was observed at 5 h and 6 and 9 days p.i.. PC treatment brought both activities to normal levels. The restoration of the examined parameters was consistent with a prolongation of mean survival time and reduction of mortality rate, infectious virus titre and lung consolidation. PC reinstated superoxide production by alveolar macrophages and increased their number in virus-infected mice. The favourable effect on the protease and the protease-inhibitory activities in the lungs of influenza-virus-infected mice apparently contributes to the overall protective effect of PC in the murine experimental influenza A/Aichi infection. The antiviral effect of the individual constituents was evaluated.

Proteinase inhibitors from Streptomyces with antiviral activity.

An extensive screening study for the production of proteolytic inhibitors has been carried out on 75 Streptomyces strains. It was found that 18 of the strains and/or their variants (24%) produced proteinaceous substances, which belonged to the group of typical serine protease inhibitors. 23 samples were tested for inhibitory activity on the replication of influenza virus A/Germany/34, strain Rostock (H7N1) (A/Rostock) in chicken embryonic fibroblast (CEF) cells. Eleven of the tested samples (52.2%) significantly inhibited viral growth. Further the specific inhibitory effect on the replication of influenza virus A/Aichi/2/68 (H3N2) (A/Aichi) in Madin-Darby canine kidney (MDCK) cells and on the growth of herpes simplex virus type 1, strain DA (HSV-1) in Madin-Darby bovine kidney (MDBK) cells was tested. Nine samples significantly inhibited A/Aichi and four - HSV-1. The most effective inhibitors, produced by Streptomyces sp. 225b (SS 225b) and Streptomyces chromofuscus 34-1 (SS 34-1) protected mice from mortality in the experimental influenza A/Aichi virus infection.

Biologically active constituents of a polyphenol extract from Geranium sanguineum L. with anti-influenza activity.

From the aerial roots of the medicinal plant Geranium sanguineum L. a polyphenol-rich extract with strong anti-influenza activity has been isolated. To investigate its active fractions, the extract was partitioned by solvents with increasing polarity. The n-BuOH fraction contained the majority of the in vitro antiviral activity; the EtOAc fraction was the most effective one in vivo. A bioassay-directed fractionation of the n-BuOH and EtOAc fractions was performed to obtain information about the nature of the chemical components of the plant extract, responsible for the antiviral effect. The individual constituents were identified by spectroscopic methods and comparison with authentic samples and by HPLC. The cell-toxic and virus-inhibitory effects of the fractions and some individual polyphenol compounds, found in Geranium sanguineum L., were studied using the replication of representative influenza viruses in cell cultures. This study showed that the presence of a variety of biologically active compounds as well as the possible synergistic interactions between them seem to be decisive for the overall antiviral effect.

Purification and characterisation of a protease inhibitor from Streptomyces chromofuscus 34-1 with an antiviral activity.

The aim of this study was to purify and characterise a novel protease inhibitor (PISC-2002) isolated from culture supernatants of Streptomyces chromofuscus. PISC-2002 was purified by anion-exchange chromatography, and RP-HPLC analysis. PISC-2002 had a molecular mass of 11.2 kDa and a high content of hydrophobic amino acids and proline. N-terminal sequence gave two sequences differing by one residue. The main sequence is ASLPAVSALVLTV and the shorter sequence is SLPAVSALVLTV. This shows its homology to Streptomyces subtilisin inhibitor family. Besides its large spectrum of powerful inhibitory activities against various serine proteases, PISC-2002 displayed significant antiviral effect against influenza virus A/Rostock/34 (H7N7).

In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L.

Recent evidence shows that plant polyphenols exhibit antioxidant and radical scavenging properties. By three separate and complementary methods--DPPH assay, beta-carotene-linoleic acid assay and NBT-reduction assay it was established that a polyphenol-rich extract from the medicinal plant Geranium sanguineum L. with strong anti-influenza virus activity, possessed antioxidant and radical scavenging capacities. For comparative reasons caffeic acid and the synthetic antioxidant BHT were used. Total soluble phenolic constituents of the MeOH extract measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). Further it was demonstrated that the EtOAc fraction, retaining the majority of the in vivo protective effect exhibited a strong O2-scavenging activity while the n-BuOH fraction, containing the majority of the in vitro antiviral activity provoked generation of O2-. The O2- scavenging activity of all three preparations correlated with the rate of the protective effect shown in the murine model of experimental influenza virus infection. The present results are in accordance with our intensive studies on the mode of the protective effect of the plant extract which showed positively that the protection may possibly be attributed to the combination of more than one biological activities and that the use of antioxidants might be an useful approach in the treatment of influenza infection.

A plant polyphenol-rich extract restores the suppressed functions of phagocytes in influenza virus-infected mice.

Influenza infection was induced in white ICR mice by intranasal (i.n.) inoculation of the virus A/Aichi/2/68 (H3N2). The number, migration and phagocyte indices of alveolar and peritoneal macrophages (pMØ) and of blood polymorphonuclear leukocytes (PMNs), as well as the inhibition of the PMN adherence in the presence of a specific antigen were followed for 9 days after infection. The effect of the i.n. application of a polyphenol-rich extract, designated as polyphenolic complex (PC), isolated from the medicinal plant Geranium sanguineum L., on the inspected immune parameters was studied in parallel with the virological parameters of the infection, e.g. rate of mortality, mean survival time (MST), infectious lung virus titre and consolidation of the lungs. It was found that the application of PC induced a continuous 2- to 2.5-fold rise in the number of both peritoneal and alveolar macrophages (aMØ) in the infected and healthy controls. The migration of both peritoneal and aMØ increased 1.5- to 2-fold in the group of infected PC-treated animals and four to fivefold in the control group, the maximum being on day 9. PC stimulated phagocyte activities of blood PMNs in both infected and healthy mice. The leukocyte adherence inhibition (LAI) index decreased in the infected and PC-treated animals. The restoration of the suppressed functions of phagocytes in influenza virus-infected mice (VIM) was consistent with a prolongation of MST and reduction in mortality rate, infectious virus titre and lung consolidation. The immunoenhancing properties of PC apparently contribute to the overall protective effect of the plant preparation in the lethal murine experimental influenza A/Aichi infection.

Antiviral activity of the red marine alga Ceramium rubrum.

An extract from the red marine alga Ceramium rubrum (Huds.) Ag. from the Bulgarian Black Sea seacoast considerably inhibited the reproduction of influenza viruses type A and B in vitro and in ovo. The virus-induced cytopathogenic effect (CPE), infectious virus yields and the production of hemagglutinin were all reduced at non-toxic concentrations of the extract. The virus-inhibitory effect was selective, dose-related and strain-specific; selectivity indices ranged 9.5-68.3. The inhibition affected adsorption as well as the intracellular stages of viral replication. The extract inhibited also the reproduction of herpes simplex virus (HSV) type 1 and type 2 in cell cultures. The preparation exhibited a strong HSV-inactivating activity.

In vitro antioxidant, antimicrobial, and antiviral activities of the essential oil and various extracts from herbal parts and callus cultures of Origanum acutidens.

The essential oil and various extracts obtained from Origanum acutidens and methanol extracts (MeOH) from callus cultures have been evaluated for their antioxidative, antimicrobial, and antiviral properties. The essential oil exhibited strong antimicrobial activity with a significant inhibitory effect against 27 (77%) of the 35 bacteria, 12 (67%) of the 18 fungi, and a yeast tested and moderate antioxidative capacity in DPPH and beta-carotene/linoleic acid assays. GC and GC-MS analyses of the oil resulted in the identification of 38 constituents, carvacrol being the main component. The MeOH extracts obtained from herbal parts showed better antioxidative effect than that of butylated hydroxytoluene (BHT), whereas callus cultures also exhibited interesting antioxidative patterns. Concerning antiviral activity, none of the extracts inhibited the reproduction of influenza A/Aichi virus in MDCK cells. The MeOH extracts from herbal parts inhibited the reproduction of HSV-1, and also callus cultures exerted slight antiherpetic effect.

Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103.

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu2+ (from 1 to 750 microg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74-85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn23-Glu-Ser. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.

In vitro anti-influenza virus activity of the pavine alkaloid (-)-thalimonine isolated from Thalictrum simplex L.

The pavine alkaloid (-)-thalimonine (Thl), isolated from the Mongolian plant Thalictrum simplex inhibited markedly the reproduction of influenza virus A/Germany/27, str. Weybridge (H7N7) and A/Germany/34, str. Rostock (H7N1) in cell cultures of chicken embryo fibroblasts. In a number of assays at a non-toxic concentration range of 0.1-6.4 microM the alkaloid inhibited viral reproduction in a selective and specific way (selectivity index = 640, 106.6, respectively). Expression of viral glycoproteins haemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) on the surface of infected cells, virus-induced cytopathic effect, infectious virus yields, HA production and virus-specific protein synthesis were all reduced. The inhibition was dose-related and depended on virus inoculum. The time of addition experiments indicated that viral reproduction was markedly inhibited when Thl was added at 4-5 h of infection. No inactivating effect on extracellular virus was found.

In vitro anti-influenza virus activity of isoquinoline alkaloids from thalictrum species.

The in vitro anti-influenza virus effects of some isoquinoline alkaloids, isolated from Thalictrum species (Ranunculaceae), growing in Mongolia and Sweden have been studied. (-)-thalimonine (Thl) and (-)-thalimonine N-oxide, isolated from the Mongolian plant T. simplex, inhibited markedly the influenza virus reproduction in vitro; thalictuberine N-oxide was less effective. The synthetic analogues of Thl as well as the rest of the tested alkaloids, isolated from T. foetidum and T. flavum, were not active. At a concentration range between 0.1 - 6.4 microM Thl inhibited viral reproduction in a selective and specific way.

A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect.

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD. To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined. Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture. Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1). The novel enzyme was purified to electrophoretic homogeneity. The presence of Cu and Zn were confirmed by atomic absorption spectrometry. The molecular mass of H. lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits. The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources. H. lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters. Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters. Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice. The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86%. The H. lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD. In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.